足细胞免疫-代谢之间的动态相互作用，以及促进足细胞损伤的机制

Podocytes, specialized epithelial cells that envelop the glomerular capillaries, play a pivotal role in maintaining renal health. The current description and quantification of features on pathology slides are limited, prompting the need for innovative solutions to comprehensively assess diverse phenotypic attributes within Whole Slide Images (WSIs). In particular, understanding the morphological characteristics of podocytes, terminally differentiated glomerular epithelial cells, is crucial for studying glomerular injury. This paper introduces the Spatial Pathomics Toolkit (SPT) and applies it to podocyte pathomics. The SPT consists of three main components: (1) instance object segmentation, enabling precise identification of podocyte nuclei; (2) pathomics feature generation, extracting a comprehensive array of quantitative features from the identified nuclei; and (3) robust statistical analyses, facilitating a comprehensive exploration of spatial relationships between morphological and spatial transcriptomics features.The SPT successfully extracted and analyzed morphological and textural features from podocyte nuclei, revealing a multitude of podocyte morphomic features through statistical analysis. Additionally, we demonstrated the SPT's ability to unravel spatial information inherent to podocyte distribution, shedding light on spatial patterns associated with glomerular injury. By disseminating the SPT, our goal is to provide the research community with a powerful and user-friendly resource that advances cellular spatial pathomics in renal pathology. The implementation and its complete source code of the toolkit are made openly accessible at https://github.com/hrlblab/spatial_pathomics.

Background: Automated podocyte foot process quantification is vital for kidney research, but the established "Automatic Morphological Analysis of Podocytes" (AMAP) method is hindered by high computational demands, a lack of a user interface, and Linux dependency. We developed AMAP-APP, a cross-platform desktop application designed to overcome these barriers. Methods: AMAP-APP optimizes efficiency by replacing intensive instance segmentation with classic image processing while retaining the original semantic segmentation model. It introduces a refined Region of Interest (ROI) algorithm to improve precision. Validation involved 365 mouse and human images (STED and confocal), benchmarking performance against the original AMAP via Pearson correlation and Two One-Sided T-tests (TOST). Results: AMAP-APP achieved a 147-fold increase in processing speed on consumer hardware. Morphometric outputs (area, perimeter, circularity, and slit diaphragm density) showed high correlation (r>0.90) and statistical equivalence (TOST P<0.05) to the original method. Additionally, the new ROI algorithm demonstrated superior accuracy compared to the original, showing reduced deviation from manual delineations. Conclusion: AMAP-APP democratizes deep learning-based podocyte morphometry. By eliminating the need for high-performance computing clusters and providing a user-friendly interface for Windows, macOS, and Linux, it enables widespread adoption in nephrology research and potential clinical diagnostics.

Generating high-fidelity and biologically plausible synthetic single-cell RNA sequencing (scRNA-seq) data, especially with conditional control, is challenging due to its high dimensionality, sparsity, and complex biological variations. Existing generative models often struggle to capture these unique characteristics and ensure robustness to structural noise in cellular networks. We introduce LapDDPM, a novel conditional Graph Diffusion Probabilistic Model for robust and high-fidelity scRNA-seq generation. LapDDPM uniquely integrates graph-based representations with a score-based diffusion model, enhanced by a novel spectral adversarial perturbation mechanism on graph edge weights. Our contributions are threefold: we leverage Laplacian Positional Encodings (LPEs) to enrich the latent space with crucial cellular relationship information; we develop a conditional score-based diffusion model for effective learning and generation from complex scRNA-seq distributions; and we employ a unique spectral adversarial training scheme on graph edge weights, boosting robustness against structural variations. Extensive experiments on diverse scRNA-seq datasets demonstrate LapDDPM's superior performance, achieving high fidelity and generating biologically-plausible, cell-type-specific samples. LapDDPM sets a new benchmark for conditional scRNA-seq data generation, offering a robust tool for various downstream biological applications.

Accurate cell type annotation across datasets is a key challenge in single-cell analysis. snRNA-seq enables profiling of frozen or difficult-to-dissociate tissues, complementing scRNA-seq by capturing fragile or rare cell types. However, cross-annotation between these two datasets remains largely unexplored, as existing methods treat them independently. We introduce ScNucAdapt, a method designed for cross-annotation between paired and unpaired scRNA-seq and snRNA-seq datasets. To address distributional and cell composition differences, ScNucAdapt employs partial domain adaptation. Experiments across both unpaired and paired scRNA-seq and snRNA-seq show that ScNucAdapt achieves robust and accurate cell type annotation, outperforming existing approaches. Therefore, ScNucAdapt provides a practical framework for the cross-domain cell type annotation between scRNA-seq and snRNA seq data.

scRNA-seq clustering is a critical task for analyzing single-cell RNA sequencing (scRNA-seq) data, as it groups cells with similar gene expression profiles. Transformers, as powerful foundational models, have been applied to scRNA-seq clustering. Their self-attention mechanism automatically assigns higher attention weights to cells within the same cluster, enhancing the distinction between clusters. Existing methods for scRNA-seq clustering, such as graph transformer-based models, treat each cell as a token in a sequence. Their computational and space complexities are $\mathcal{O}(n^2)$ with respect to the number of cells, limiting their applicability to large-scale scRNA-seq datasets.To address this challenge, we propose a Bipartite Graph Transformer-based clustering model (BGFormer) for scRNA-seq data. We introduce a set of learnable anchor tokens as shared reference points to represent the entire dataset. A bipartite graph attention mechanism is introduced to learn the similarity between cells and anchor tokens, bringing cells of the same class closer together in the embedding space. BGFormer achieves linear computational complexity with respect to the number of cells, making it scalable to large datasets. Experimental results on multiple large-scale scRNA-seq datasets demonstrate the effectiveness and scalability of BGFormer.

Single-cell RNA sequencing (scRNA-seq) technology has profiled hundreds of millions of human cells across organs, diseases, development and perturbations to date. However, the high-dimensional sparsity, batch effect noise, category imbalance, and ever-increasing data scale of the original sequencing data pose significant challenges for multi-center knowledge transfer, data fusion, and cross-validation between scRNA-seq datasets. To address these barriers, (1) we first propose a latent codes-based scRNA-seq dataset distillation framework named scDD, which transfers and distills foundation model knowledge and original dataset information into a compact latent space and generates synthetic scRNA-seq dataset by a generator to replace the original dataset. Then, (2) we propose a single-step conditional diffusion generator named SCDG, which perform single-step gradient back-propagation to help scDD optimize distillation quality and avoid gradient decay caused by multi-step back-propagation. Meanwhile, SCDG ensures the scRNA-seq data characteristics and inter-class discriminability of the synthetic dataset through flexible conditional control and generation quality assurance. Finally, we propose a comprehensive benchmark to evaluate the performance of scRNA-seq dataset distillation in different data analysis tasks. It is validated that our proposed method can achieve 7.61% absolute and 15.70% relative improvement over previous state-of-the-art methods on average task.

Single-cell RNA sequencing (scRNA-seq) enables researchers to analyze gene expression at single-cell level. One important task in scRNA-seq data analysis is unsupervised clustering, which helps identify distinct cell types, laying down the foundation for other downstream analysis tasks. In this paper, we propose a novel method called Cluster-aware Iterative Contrastive Learning (CICL in short) for scRNA-seq data clustering, which utilizes an iterative representation learning and clustering framework to progressively learn the clustering structure of scRNA-seq data with a cluster-aware contrastive loss. CICL consists of a Transformer encoder, a clustering head, a projection head and a contrastive loss module. First, CICL extracts the feature vectors of the original and augmented data by the Transformer encoder. Then, it computes the clustering centroids by K-means and employs the student t-distribution to assign pseudo-labels to all cells in the clustering head. The projection-head uses a Multi-Layer Perceptron (MLP) to obtain projections of the augmented data. At last, both pseudo-labels and projections are used in the contrastive loss to guide the model training. Such a process goes iteratively so that the clustering result becomes better and better. Extensive experiments on 25 real world scRNA-seq datasets show that CICL outperforms the SOTA methods. Concretely, CICL surpasses the existing methods by from 14% to 280%, and from 5% to 133% on average in terms of performance metrics ARI and NMI respectively.

Single-cell RNA sequencing (scRNA-seq) is widely used to reveal heterogeneity in cells, which has given us insights into cell-cell communication, cell differentiation, and differential gene expression. However, analyzing scRNA-seq data is a challenge due to sparsity and the large number of genes involved. Therefore, dimensionality reduction and feature selection are important for removing spurious signals and enhancing downstream analysis. Correlated clustering and projection (CCP) was recently introduced as an effective method for preprocessing scRNA-seq data. CCP utilizes gene-gene correlations to partition the genes and, based on the partition, employs cell-cell interactions to obtain super-genes. Because CCP is a data-domain approach that does not require matrix diagonalization, it can be used in many downstream machine learning tasks. In this work, we utilize CCP as an initialization tool for uniform manifold approximation and projection (UMAP) and t-distributed stochastic neighbor embedding (t-SNE). By using eight publicly available datasets, we have found that CCP significantly improves UMAP and t-SNE visualization and dramatically improve their accuracy.

Motivation: Single cell RNA sequencing (scRNA-seq) data makes studying the development of cells possible at unparalleled resolution. Given that many cellular differentiation processes are hierarchical, their scRNA-seq data is expected to be approximately tree-shaped in gene expression space. Inference and representation of this tree-structure in two dimensions is highly desirable for biological interpretation and exploratory analysis.Results:Our two contributions are an approach for identifying a meaningful tree structure from high-dimensional scRNA-seq data, and a visualization method respecting the tree-structure. We extract the tree structure by means of a density based minimum spanning tree on a vector quantization of the data and show that it captures biological information well. We then introduce DTAE, a tree-biased autoencoder that emphasizes the tree structure of the data in low dimensional space. We compare to other dimension reduction methods and demonstrate the success of our method both qualitatively and quantitatively on real and toy data.Availability: Our implementation relying on PyTorch and Higra is available at https://github.com/hci-unihd/DTAE.

Analysis of single-cell RNA sequencing data is often conducted through network projections such as coexpression networks, primarily due to the abundant availability of network analysis tools for downstream tasks. However, this approach has several limitations: loss of higher-order information, inefficient data representation caused by converting a sparse dataset to a fully connected network, and overestimation of coexpression due to zero-inflation. To address these limitations, we propose conceptualizing scRNA-seq expression data as hypergraphs, which are generalized graphs in which the hyperedges can connect more than two vertices. In the context of scRNA-seq data, the hypergraph nodes represent cells and the edges represent genes. Each hyperedge connects all cells where its corresponding gene is actively expressed and records the expression of the gene across different cells. This hypergraph conceptualization enables us to explore multi-way relationships beyond the pairwise interactions in coexpression networks without loss of information. We propose two novel clustering methods: (1) the Dual-Importance Preference Hypergraph Walk (DIPHW) and (2) the Coexpression and Memory-Integrated Dual-Importance Preference Hypergraph Walk (CoMem-DIPHW). They outperform established methods on both simulated and real scRNA-seq datasets. The improvement brought by our proposed methods is especially significant when data modularity is weak. Furthermore, CoMem-DIPHW incorporates the gene coexpression network, cell coexpression network, and the cell-gene expression hypergraph from the single-cell abundance counts data altogether for embedding computation. This approach accounts for both the local level information from single-cell level gene expression and the global level information from the pairwise similarity in the two coexpression networks.

Single-cell RNA sequencing (scRNA-seq) determines RNA expression at single-cell resolution. It provides a powerful tool for studying immunity, regulation, and other life activities of cells. However, due to the limitations of the sequencing technique, the scRNA-seq data are represented with sparsity, whichcontains missing gene values, i.e., zero values, called dropout. Therefore, it is necessary to impute missing values before analyzing scRNA-seq data. However, existing imputation computation methods often only focus on the identification of technical zeros or imputing all zeros based on cell similarity. This study proposes a new method (SFAG) to reconstruct the gene expression relationship matrix by usinggraph regularization technology to preserve the high-dimensional manifold information of the data, andto mine the relationship between genes and cells in the data, and then uses a method of averaging the clustering results to fill in the identified technical zeros. Experimental results show that SFAGcan helpimprove downstream analysis and reconstruct cell trajectory

Spatial studies of transcriptome provide biologists with gene expression maps of heterogeneous and complex tissues. However, most experimental protocols for spatial transcriptomics suffer from the need to select beforehand a small fraction of genes to be quantified over the entire transcriptome. Standard single-cell RNA sequencing (scRNA-seq) is more prevalent, easier to implement and can in principle capture any gene but cannot recover the spatial location of the cells. In this manuscript, we focus on the problem of imputation of missing genes in spatial transcriptomic data based on (unpaired) standard scRNA-seq data from the same biological tissue. Building upon domain adaptation work, we propose gimVI, a deep generative model for the integration of spatial transcriptomic data and scRNA-seq data that can be used to impute missing genes. After describing our generative model and an inference procedure for it, we compare gimVI to alternative methods from computational biology or domain adaptation on real datasets and outperform Seurat Anchors, Liger and CORAL to impute held-out genes.

Time-series single-cell RNA-sequencing (scRNA-seq) datasets offer unprecedented insights into the dynamics and heterogeneity of cellular systems. These systems exhibit multiscale collective behaviors driven by intricate intracellular gene regulatory networks and intercellular interactions of molecules. However, inferring interacting cell population dynamics from time-series scRNA-seq data remains a significant challenge, as cells are isolated and destroyed during sequencing. To address this, we introduce scIMF, a single-cell deep generative Interacting Mean Field model, designed to learn collective multi-cellular dynamics. Our approach leverages a transformer-enhanced stochastic differential equation network to simultaneously capture cell-intrinsic dynamics and intercellular interactions. Through extensive benchmarking on multiple scRNA-seq datasets, scIMF outperforms existing methods in reconstructing gene expression at held-out time points, demonstrating that modeling cell-cell communication enhances the accuracy of multicellular dynamics characterization.Additionally, our model provides biologically interpretable insights into cell-cell interactions during dynamic processes, offering a powerful tool for understanding complex cellular systems.

Modeling cellular dynamics from single-cell RNA sequencing (scRNA-seq) data is critical for understanding cell development and underlying gene regulatory relationships. Many current methods rely on single-cell velocity to obtain pseudotime, which can lead to inconsistencies between pseudotime and velocity. It is challenging to simultaneously infer cell pseudotime and gene interaction networks, especially in multi-branch differentiation scenarios. We present single-cell Piecewise Network (scPN), a novel high-dimensional dynamical modeling approach that iteratively extracts temporal patterns and inter-gene relationships from scRNA-seq data. To tackle multi-branch differentiation challenges, scPN models gene regulatory dynamics using piecewise gene-gene interaction networks, offering an interpretable framework for deciphering complex gene regulation patterns over time. Results on synthetic data and multiple scRNA-seq datasets demonstrate the superior performance of scPN in reconstructing cellular dynamics and identifying key transcription factors involved in development compared to existing methods. To the best of our knowledge, scPN is the first attempt at modeling that can recover pseudotime, velocity fields, and gene interactions all at once on multi-branch datasets.

The rapid development of spatial transcriptomics (ST) technologies is revolutionizing our understanding of the spatial organization of biological tissues. Current ST methods, categorized into next-generation sequencing-based (seq-based) and fluorescence in situ hybridization-based (image-based) methods, offer innovative insights into the functional dynamics of biological tissues. However, these methods are limited by their cellular resolution and the quantity of genes they can detect. To address these limitations, we propose SpaDiT, a deep learning method that utilizes a diffusion generative model to integrate scRNA-seq and ST data for the prediction of undetected genes. By employing a Transformer-based diffusion model, SpaDiT not only accurately predicts unknown genes but also effectively generates the spatial structure of ST genes. We have demonstrated the effectiveness of SpaDiT through extensive experiments on both seq-based and image-based ST data. SpaDiT significantly contributes to ST gene prediction methods with its innovative approach. Compared to eight leading baseline methods, SpaDiT achieved state-of-the-art performance across multiple metrics, highlighting its substantial bioinformatics contribution.

Single-cell RNA sequencing (scRNA-seq) enables single-cell transcriptomic profiling, revealing cellular heterogeneity and rare populations. Recent deep learning models like Geneformer and Mouse-Geneformer perform well on tasks such as cell-type classification and in silico perturbation. However, their species-specific design limits cross-species generalization and translational applications, which are crucial for advancing translational research and drug discovery. We present Mix-Geneformer, a novel Transformer-based model that integrates human and mouse scRNA-seq data into a unified representation via a hybrid self-supervised approach combining Masked Language Modeling (MLM) and SimCSE-based contrastive loss to capture both shared and species-specific gene patterns. A rank-value encoding scheme further emphasizes high-variance gene signals during training. Trained on about 50 million cells from diverse human and mouse organs, Mix-Geneformer matched or outperformed state-of-the-art baselines in cell-type classification and in silico perturbation tasks, achieving 95.8% accuracy on mouse kidney data versus 94.9% from the best existing model. It also successfully identified key regulatory genes validated by in vivo studies. By enabling scalable cross-species transcriptomic modeling, Mix-Geneformer offers a powerful tool for comparative transcriptomics and translational applications. While our results demonstrate strong performance, we also acknowledge limitations, such as the computational cost and variability in zero-shot transfer.

Motivation: Single-cell RNA sequencing (scRNA-seq) is a groundbreaking technology extensively utilized in biological research, facilitating the examination of gene expression at the individual cell level within a given tissue sample. While numerous tools have been developed for scRNA-seq data analysis, the challenge persists in capturing the distinct features of such data and replicating virtual datasets that share analogous statistical properties. Results: Our study introduces a generative approach termed scRNA-seq Diffusion Transformer (scRDiT). This method generates virtual scRNA-seq data by leveraging a real dataset. The method is a neural network constructed based on Denoising Diffusion Probabilistic Models (DDPMs) and Diffusion Transformers (DiTs). This involves subjecting Gaussian noises to the real dataset through iterative noise-adding steps and ultimately restoring the noises to form scRNA-seq samples. This scheme allows us to learn data features from actual scRNA-seq samples during model training. Our experiments, conducted on two distinct scRNA-seq datasets, demonstrate superior performance. Additionally, the model sampling process is expedited by incorporating Denoising Diffusion Implicit Models (DDIM). scRDiT presents a unified methodology empowering users to train neural network models with their unique scRNA-seq datasets, enabling the generation of numerous high-quality scRNA-seq samples. Availability and implementation: https://github.com/DongShengze/scRDiT

Due to the sequential sample arrival, changing experiment conditions, and evolution of knowledge, the demand to continually visualize evolving structures of sequential and diverse single-cell RNA-sequencing (scRNA-seq) data becomes indispensable. However, as one of the state-of-the-art visualization and analysis methods for scRNA-seq, t-distributed stochastic neighbor embedding (t-SNE) merely visualizes static scRNA-seq data offline and fails to meet the demand well. To address these challenges, we introduce online t-SNE to seamlessly integrate sequential scRNA-seq data. Online t-SNE achieves this by leveraging the embedding space of old samples, exploring the embedding space of new samples, and aligning the two embedding spaces on the fly. Consequently, online t-SNE dramatically enables the continual discovery of new structures and high-quality visualization of new scRNA-seq data without retraining from scratch. We showcase the formidable visualization capabilities of online t-SNE across diverse sequential scRNA-seq datasets.

Single-cell RNA sequencing (scRNA-seq) is a fast growing approach to measure the genome-wide transcriptome of many individual cells in parallel, but results in noisy data with many dropout events. Existing methods to learn molecular signatures from bulk transcriptomic data may therefore not be adapted to scRNA-seq data, in order to automatically classify individual cells into predefined classes. We propose a new method called DropLasso to learn a molecular signature from scRNA-seq data. DropLasso extends the dropout regularisation technique, popular in neural network training, to esti- mate sparse linear models. It is well adapted to data corrupted by dropout noise, such as scRNA-seq data, and we clarify how it relates to elastic net regularisation. We provide promising results on simulated and real scRNA-seq data, suggesting that DropLasso may be better adapted than standard regularisa- tions to infer molecular signatures from scRNA-seq data.

The single-cell RNA sequencing (scRNA-seq) technology enables researchers to study complex biological systems and diseases with high resolution. The central challenge is synthesizing enough scRNA-seq samples; insufficient samples can impede downstream analysis and reproducibility. While various methods have been attempted in past research, the resulting scRNA-seq samples were often of poor quality or limited in terms of useful specific cell subpopulations. To address these issues, we propose a novel method called Single-Cell Latent Diffusion (SCLD) based on the Diffusion Model. This method is capable of synthesizing large-scale, high-quality scRNA-seq samples, including both 'holistic' or targeted specific cellular subpopulations within a unified framework. A pre-guidance mechanism is designed for synthesizing specific cellular subpopulations, while a post-guidance mechanism aims to enhance the quality of scRNA-seq samples. The SCLD can synthesize large-scale and high-quality scRNA-seq samples for various downstream tasks. Our experimental results demonstrate state-of-the-art performance in cell classification and data distribution distances when evaluated on two scRNA-seq benchmarks. Additionally, visualization experiments show the SCLD's capability in synthesizing specific cellular subpopulations.

Single-cell RNA sequencing (scRNA-seq) has transformed our ability to explore biological systems. Nevertheless, proficient expertise is essential for handling and interpreting the data. In this paper, we present scX, an R package built on the Shiny framework that streamlines the analysis, exploration, and visualization of single-cell experiments. With an interactive graphic interface, implemented as a web application, scX provides easy access to key scRNAseq analyses, including marker identification, gene expression profiling, and differential gene expression analysis. Additionally, scX seamlessly integrates with commonly used single-cell Seurat and SingleCellExperiment R objects, resulting in efficient processing and visualization of varied datasets. Overall, scX serves as a valuable and user-friendly tool for effortless exploration and sharing of single-cell data, simplifying some of the complexities inherent in scRNAseq analysis.

In this study, we analyze discordance between the transcriptome and proteome using paired scRNA-Seq and multiplexed spatial proteomics data from HuBMAP. Our findings highlight persistent transcripts in key immune markers, including CD45-RO, Ki67, CD45, CD20, and HLA-DR. CD45-RO is consistently expressed in memory T cells, while Ki67, associated with cell proliferation, also displays sustained expression. Furthermore, HLA-DR, part of the MHC class II molecules, demonstrates continuous expression, possibly crucial for APCs to trigger an effective immune response. This investigation provides novel insights into the complexity of gene expression regulation and protein function.

Single-cell RNA sequencing (scRNA-seq) enables transcriptomic profiling at cellular resolution but suffers from pervasive dropout events that obscure biological signals. We present SCR-MF, a modular two-stage workflow that combines principled dropout detection using scRecover with robust non-parametric imputation via missForest. Across public and simulated datasets, SCR-MF achieves robust and interpretable performance comparable to or exceeding existing imputation methods in most cases, while preserving biological fidelity and transparency. Runtime analysis demonstrates that SCR-MF provides a competitive balance between accuracy and computational efficiency, making it suitable for mid-scale single-cell datasets.

The utilization of glycolysis in aerobic conditions have been a subject of debate for more than a century. A hypothesis supported by previous data is that glycolysis has a higher rate of ATP production per protein mass and per occupied volume than oxidative phosphorylation (OxPhos). However, a recent work by Shen et al14 challenges previous estimates, reporting that OxPhos has a higher rate of ATP production per protein mass than glycolysis. Here I show that Shen et al14 make a key assumption that is a subject of debate: that the proteomic cost of OxPhos is limited to proteins in enzymes of OxPhos and the TCA cycle. I argue that an intact mitochondria is required for functional OxPhos and therefore the whole mitochondrial protein content should be included for the cost estimate of OxPhos. After doing so, glycolysis is the most efficient pathway per protein mass or per volume fraction.

Studies have reported "dysbiotic" changes to gut microbiota, such as depletion of gut bacteria that produce short-chain fatty acids (SCFAs) through gut fermentation of fiber, in CKD and diabetes. Dietary fiber is associated with decreased inflammation and mortality in CKD, and SCFAs have been proposed to mediate this effect. To explore dietary fiber's effect on development of experimental diabetic nephropathy, we used streptozotocin to induce diabetes in wild-type C57BL/6 and knockout mice lacking the genes encoding G protein-coupled receptors GPR43 or GPR109A. Diabetic mice were randomized to high-fiber, normal chow, or zero-fiber diets, or SCFAs in drinking water. We used proton nuclear magnetic resonance spectroscopy for metabolic profiling and 16S ribosomal RNA sequencing to assess the gut microbiome. Diabetic mice fed a high-fiber diet were significantly less likely to develop diabetic nephropathy, exhibiting less albuminuria, glomerular hypertrophy, podocyte injury, and interstitial fibrosis compared with diabetic controls fed normal chow or a zero-fiber diet. Fiber beneficially reshaped gut microbial ecology and improved dysbiosis, promoting expansion of SCFA-producing bacteria of the genera Dietary fiber protects against diabetic nephropathy through modulation of the gut microbiota, enrichment of SCFA-producing bacteria, and increased SCFA production. GPR43 and GPR109A are critical to SCFA-mediated protection against this condition. Interventions targeting the gut microbiota warrant further investigation as a novel renoprotective therapy in diabetic nephropathy.

The ageing global population faces a rising prevalence of chronic kidney disease (CKD), now recognized as a state of accelerated ageing driven by chronic low-grade inflammation-inflammaging. This review synthesizes current evidence positioning the kidney not merely as a passive target but as an active participant in systemic inflammaging, a process fueled by immunosenescence, metabolic reprogramming, and cellular senescence. We explore how resident renal cells, including tubular epithelial cells, podocytes, and mesangial cells, adopt a senescence-associated secretory phenotype (SASP) that perpetuates inflammation, fibrosis, and functional decline. Key mechanisms (NLRP

Globally, IgA nephropathy (IgAN) is the most common primary glomerulonephritis that can progress to renal failure. The exact pathogenesis of IgAN is not well defined, but current biochemical and genetic data implicate overproduction of aberrantly glycosylated IgA1. These aberrant immunoglobulins are characterized by galactose deficiency of some hinge-region O-linked glycans. However, aberrant glycosylation alone is insufficient to induce renal injury: the participation of glycan-specific IgA and IgG autoantibodies that recognize the undergalactosylated IgA1 molecule is required. Glomerular deposits of immune complexes containing undergalactosylated IgA1 activate mesangial cells, leading to the local overproduction of cytokines, chemokines and complement. Emerging data indicate that mesangial-derived mediators that are released following mesangial deposition of IgA1 lead to podocyte and tubulointerstitial injury via humoral crosstalk. Patients can present with a range of signs and symptoms, from asymptomatic microscopic haematuria to macroscopic haematuria. The clinical progression varies, with 30-40% of patients reaching end-stage renal disease 20-30 years after the first clinical presentation. Currently, no IgAN-specific therapies are available and patients are managed with the aim of controlling blood pressure and maintaining renal function. However, new therapeutic approaches are being developed, building upon our ever-improving understanding of disease pathogenesis.

Diabetic nephropathy (DN) is the most common chronic kidney disease. Accumulation of glucose and metabolites activates resident macrophages in kidneys. Resident macrophages play diverse roles on diabetic kidney injuries by releasing cytokines/chemokines, recruiting peripheral monocytes/macrophages, enhancing renal cell injuries (podocytes, mesangial cells, endothelial cells and tubular epithelial cells), and macrophage-myofibroblast transition. The differentiation and cross-talks of macrophages ultimately result renal inflammation and fibrosis in DN. Emerging evidence shows that targeting macrophages by suppressing macrophage activation/transition, and macrophages-cell interactions may be a promising approach to attenuate DN. In the review, we summarized the diverse roles of macrophages and the cross-talks to other cells in DN, and highlighted the therapeutic potentials by targeting macrophages.

Diabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 μM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1β as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg

Doxorubicin (DOX) is a widely used anticancer drug with a marked nephrotoxic effect. The Chinese medicine Tangshen Formula (TSF) has a kidney-protective effect in diabetic kidney disease patients and has been shown to attenuate kidney damage in rodents. Our study explored the putative targets and regulatory mechanisms of TSF in attenuating DOX-induced nephrotoxicity. Protein factor microarray, bioinformatics analysis, MPC5 cell line were used in this work. Through the comprehensive means of biological information analysis, in vitro experiments, in vivo experiments and molecular docking, the pathological relationship between TSF and DOX induced nephrotoxic was revealed, and the potential small-molecule inhibitor were identified. TSF administration significantly alleviated albuminuria and histopathologic lesions. Rat renal cytokine array and GO analysis revealed that signaling pathways of TSF were closely related to apoptosis and pyroptosis. In vivo results verified that TSF inhibited the apoptosis and pyroptosis of DOX induced nephrotoxic rat podocyte. TSF increased levels of WT1, and nephrin, which are podocyte markers, and reduced levels of GSDMD, which is a pyroptosis marker protein in the rat kidney. Furthermore, immunofluorescence co-localization confirmed that TSF could decrease the level of BAX and increase the level of BCL2 in podocytes of DOX induced nephrotoxic rat. In vitro, BTSA1, which is a BAX activator, could mitigate the beneficial effects of TSF. In addition, we identified the compounds with high binding efficiency to BAX from TSF in plasma by CDOCKER, and the top three were selected for verification by western blot, ginsenoside having the best effect to MPC pyroptosis. This study demonstrated a novel action of TSF in attenuating podocyte death by regulating BAX-mediated crosstalk between pyroptosis and apoptosis. TSF is a potential therapeutic herbal therapy against AN, and ginsenoside is the most effective inhibition of BAX levels. Ginsenoside Rb1 (PubChem CID: 9898279); Notoginsenoside R1 (PubChem CID: 441934); Naringin (PubChem CID: 442428).

Increased podocyte detachment begins immediately after kidney transplantation and is associated with long-term allograft failure. We hypothesized that cell-specific transcriptional changes in podocytes and glomerular endothelial cells after transplantation would offer mechanistic insights into the podocyte detachment process. To test this, we evaluated cell-specific transcriptional profiles of glomerular endothelial cells and podocytes from 14 patients of their first-year surveillance biopsies with normal histology from low immune risk recipients with no post-transplant complications and compared these to biopsies of 20 healthy living donor controls. Glomerular endothelial cells from these surveillance biopsies were enriched for genes related to fluid shear stress, angiogenesis, and interferon signaling. In podocytes, pathways were enriched for genes in response to growth factor signaling and actin cytoskeletal reorganization but also showed evidence of podocyte stress as indicated by reduced nephrin (adhesion protein) gene expression. In parallel, transcripts coding for proteins required to maintain podocyte adherence to the underlying glomerular basement membrane were downregulated, including the major glomerular podocyte integrin α3 and the actin cytoskeleton-related gene synaptopodin. The reduction in integrin α3 protein expression in surveillance biopsies was confirmed by immunoperoxidase staining. The combined growth and stress response of patient allografts post-transplantation paralleled similar changes in a rodent model of nephrectomy-induced glomerular hypertrophic stress that progress to develop proteinuria and glomerulosclerosis with shortened kidney life span. Thus, even among patients with apparently healthy allografts with no detectable histologic abnormality including alloimmune injury, transcriptomic changes reflecting cell stresses are already set in motion that could drive hypertrophy-associated glomerular disease progression.

RNA methylation is a widely known post-transcriptional regulation which exists in many cancer and immune system diseases. However, the potential role and crosstalk of five types RNA methylation regulators in diabetic nephropathy (DN) and immune microenvironment remain unclear. The mRNA expression of 37 RNA modification regulators and RNA modification regulators related genes were identified in 112 samples from 5 Gene Expression Omnibus datasets. Nonnegative Matrix Factorization clustering method was performed to determine RNA modification patterns. The ssGSEA algorithms and the expression of human leukocyte antigen were employed to assess the immune microenvironment characteristics. Risk model based on differentially expression genes responsible for the modification regulators was constructed to evaluate its predictive capability in DN patients. Furthermore, the results were validated by using immunofluorescence co-localizations and protein experiments in vitro. We found 24 RNA methylation regulators were significant differently expressed in glomeruli in DN group compared with control group. Four methylation-related genes and six RNA regulators were introduced into riskScore model using univariate Logistic regression and integrated LASSO regression, which could precisely distinguish the DN and healthy individuals. Group with high-risk score was associated with high immune infiltration. Three distinct RNA modification patterns were identified, which has significant differences in immune microenvironment, biological pathway and eGFR. Validation analyses showed the METTL3, ADAR1, DNMT1 were upregulated whereas YTHDC1 was downregulated in DN podocyte cell lines comparing with cells cultured by the normal glucose. Our study reveals that RNA methylation regulators and immune infiltration regulation play critical roles in the pathogenesis of DN. The bioinformatic analyses combine with verification in vitro could provide robust evidence for identification of predictive RNA methylation regulators in DN.

The C3b receptor of human erythrocytes, neutrophils, monocytes, all mature B cells, a subpopulation of T cells, and glomerular podocytes is a single chain glycoprotein that exists in two allotypic forms having Mr's of approximately 250,000 (F) and 260,000 (S). The number of receptors present on erythrocytes varies by eight-fold among different individuals and is genetically regulated by two codominant alleles that are distinct from the alleles determining the structural polymorphism. The number of receptors expressed by neutrophils is subject to rapid increases from 5000 per cell to 40,000 per cell by exposure to nanomolar concentrations of C5adesArg, in vitro, and a similar mechanism is probably the basis for observing increased receptor expression on neutrophils in patients undergoing hemodialysis. Cytoskeletal association of the C3b receptor on monocytes and neutrophils is suggested by experiments demonstrating receptor-mediated phagocytosis, adsorptive endocytosis through coated pits, and restricted lateral diffusion, and by the reciprocal co-redistribution of cross-linked C3b and Fc receptors, and the detergent-insolubility of cross-linked C3b receptors. The factor H-like cofactor activity of the C3b receptor promotes the cleavage of bound C3b to iC3b, C3c and C3d, g, reactions that may enhance the clearance of circulating immune complexes and the generation of ligands for CR2 and CR3. The inherited partial deficiency of erythrocyte C3b receptors in patients with SLE, and the absence of glomerular C3b receptors in these patients with proliferative glomerulonephritis may contribute to systemic and organ-specific abnormalities in the clearance of immune complexes that contribute to the pathogenesis of this disease.

Angiogenesis and immunosuppression are closely related pathophysiologic processes. Widely prescribed in malignant tumor and proliferative retinal lesions, VEGF signaling pathway inhibitors may cause hypertension and renal injury in some patients, presenting with proteinuria, nephrotic syndrome, renal failure and thrombotic microangiopathy. VEGF signaling pathway inhibitors block the action of both VEGF-A and VEGF-C. However, VEGF-A and VEGF-C produced by podocytes are vital to maintain the physiological function of glomerular endothelial cells and podocytes. There is still no effective treatment for kidney disease associated with VEGF signaling pathway inhibitors and some patients have progressive renal failure even after withdrawal of the drug. Recent studies reveal that blocking of VEGF-A and VEGF-C can activate CD4

Diabetic nephropathy is a major consequence of inflammation developing in type 1 diabetes, with interleukin-8 (IL-8)-CXCR1/2 axis playing a key role in kidney disease progression. In this study, we investigated the therapeutic potential of a CXCR1/2 non-competitive allosteric antagonist (Ladarixin) in preventing high glucose-mediated injury in human podocytes and epithelial cells differentiated from renal stem/progenitor cells (RSC) cultured as nephrospheres. We used human RSCs cultured as nephrospheres through a sphere-forming functional assay to investigate hyperglycemia-mediated effects on IL-8 signalling in human podocytes and tubular epithelial cells. High glucose impairs RSC self-renewal, induces an increase in IL-8 transcript expression and protein secretion and induces DNA damage in RSC-differentiated podocytes, while exerting no effect on RSC-differentiated epithelial cells. Accordingly, the supernatant from epithelial cells or podocytes cultured in high glucose was able to differentially activate leucocyte-mediated secretion of pro-inflammatory cytokines, suggesting that the crosstalk between immune and non-immune cells may be involved in disease progression in vivo. Treatment with Ladarixin during RSC differentiation prevented high glucose-mediated effects on podocytes and modulated either podocyte or epithelial cell-dependent leucocyte secretion of pro-inflammatory cytokines, suggesting CXCR1/2 antagonists as possible pharmacological approaches for the treatment of diabetic nephropathy.

To examine the T helper 17 (Th17)/regulatory T (Treg) immune balance in passive Heymann nephritis (PHN) rats with dampness syndrome (DS). Rats were divided into four groups: normal control (NC), PHN model, PHN + DS model, and DS model. The DS model was created by administering lard, a 60% cold sucrose solution, and Chinese Baijiu This study demonstrated a significant increase in proteinuria and total cholesterol levels in PHN rats with DS, along with more severe histopathological kidney damage. DS exacerbated podocyte damage in PHN rats. Additionally, the number of Treg cells was significantly reduced, while the ratio of Th17/Treg cells was significantly elevated in PHN rats with DS. In conclusion, the findings of our study indicate that the presence of DS exacerbates renal injury in PHN, a rat model used to simulate experimental membranous nephropathy. This observation may be closely linked to the exacerbation of the Th17/Treg imbalance and podocyte injury in PHN rats induced by DS.

Lupus nephritis (LN), a severe complication of systemic lupus erythematosus (SLE), is characterized by persistent immune dysregulation and glomerular injury predominantly driven by macrophage infiltration and type I interferon (IFN-I) signaling. Crosstalk between injured podocytes and infiltrating macrophages is increasingly recognized as a key contributor to LN progression; however, the molecular mediators and their corresponding receptors remain poorly defined. This study investigates whether ANGPTL3 upregulation in podocytes activates macrophages through MSR1 and drives interferon-biased inflammation in LN. We employed secretome-based protein-protein interaction screening to identify ANGPTL3 receptors. The ANGPTL3-MSR1 interaction was supported by co-immunoprecipitation. In vitro, bone marrow-derived macrophages (BMDMs) were stimulated with ANGPTL3-conditioned media, and gene expression was analyzed by RNA sequencing. MSR1 was silenced using siRNA. In vivo, a pristane-induced lupus mouse model was used to assess glomerular ANGPTL3/MSR1 expression, renal function, and macrophage infiltration. Clinical relevance was assessed using Nephroseq datasets and human LN kidney specimens. ANGPTL3 was identified as an MSR1-interacting ligand through a cell-based membrane protein screening assay and further supported by co-immunoprecipitation. In vitro, ANGPTL3-conditioned media induced an interferon-dominant transcriptional response in BMDMs, including upregulation of This study identifies the ANGPTL3-MSR1 axis as a critical pathway linking podocyte injury to macrophage-driven inflammation in LN, highlighting its therapeutic potential. Targeting this axis may disrupt maladaptive immune crosstalk, offering a novel strategy for LN management.

Previous clinical case studies revealed that patients with severe liver damage often develop kidney damage in occupational medicamentose-like dermatitis due to trichloroethylene (OMDT). However, the mechanism of this crosstalk is unknown. In this study, we extracted hepatocyte mtDNA from TCE-sensitized positive mice and co-treated it with a mouse podocyte line, MPC5. The expression of the stromal interaction molecule 1 (Stim1) and calcium release-activated calcium channel protein 1 (Orai1) proteins did not change significantly with or without interference with the Stimulator of interferon gene (STING) protein in hepatocyte mtDNA pretreated MPC5 cells. Subsequently, siRNA interference treatment of Stim1 and Orai1, respectively, was found to significantly ameliorate the damage to podocytes. Moreover, lentiviral transfection for overexpression of the key protein Orai1 was carried out, and the expression of inflammatory factors was significantly elevated. In animal experiments, the SOCE inhibitor YM-58483 was used to treat TCE-sensitized mice, and the administration of YM-58483 alleviated the reduction in the expression of podocyte injury proteins. In summary, this study explored the role of hepatocyte mtDNA-mediated podocyte injury in Ca

Deposition of immune complexes in the glomerulus leads to irreversible renal damage in lupus nephritis (LN), of which podocyte malfunction arises earlier. Fasudil, the only Rho GTPases inhibitor approved in clinical settings, possesses well-established renoprotective actions; yet, no studies addressed the amelioration derived from fasudil in LN. To clarify, we investigated whether fasudil exerted renal remission in lupus-prone mice. In this study, fasudil (20 mg/kg) was intraperitoneally administered to female MRL/lpr mice for 10 weeks. We report that fasudil administration swept antibodies (anti-dsDNA) and attenuated systemic inflammatory response in MRL/lpr mice, accompanied by preserving podocyte ultrastructure and averting immune complex deposition. Mechanistically, it repressed the expression of CaMK4 in glomerulopathy by preserving nephrin and synaptopodin expression. And fasudil further blocked cytoskeletal breakage in the Rho GTPases-dependent action. Further analyses showed that beneficial actions of fasudil on the podocytes required intra-nuclear YAP activation underlying actin dynamics. In addition, in vitro assays revealed that fasudil normalized the motile imbalance by suppressing intracellular calcium enrichment, thereby contributing to the resistance of apoptosis in podocytes. Altogether, our findings suggest that the precise manners of crosstalks between cytoskeletal assembly and YAP activation underlying the upstream CaMK4/Rho GTPases signal in podocytes is a reliable target for podocytopathies treatment, and fasudil might serve as a promising therapeutic agent to compensate for the podocyte injury in LN.

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has significant morbidity and mortality as 20-40% of patients progress to end-stage renal disease within 20 years of onset. In order to gain insight into the molecular mechanisms involved in the progression of IgAN, we systematically evaluated renal biopsies from such patients. This showed that the MAPK/ERK signaling pathway was activated in the mesangium of patients presenting with over 1 g/day proteinuria and elevated blood pressure, but absent in biopsy specimens of patients with IgAN and modest proteinuria (<1 g/day). ERK activation was not associated with elevated galactose-deficient IgA1 or IgG specific for galactose-deficient IgA1 in the serum. In human mesangial cells in vitro, ERK activation through mesangial IgA1 receptor (CD71) controlled pro-inflammatory cytokine secretion and was induced by large-molecular-mass IgA1-containing circulating immune complexes purified from patient sera. Moreover, IgA1-dependent ERK activation required renin-angiotensin system as its blockade was efficient in reducing proteinuria in those patients exhibiting substantial mesangial activation of ERK. Thus, ERK activation alters mesangial cell-podocyte crosstalk, leading to renal dysfunction in IgAN. Assessment of MAPK/ERK activation in diagnostic renal biopsies may predict the therapeutic efficacy of renin-angiotensin system blockers in IgAN.

This study evaluated endothelial cells and podocytes, both being primary components of the glomerular filtration barrier, in the progression of membranoproliferative glomerulonephritis (MPGN) using modified scanning electron microscopy (mSEM) analysis. BXSB/MpJ-Yaa model mice exhibited autoimmune-mediated MPGN characterised by elevated serum autoantibody levels, albuminuria, renal dysfunctional parameters, and decreased glomerular endothelial fenestrations (EF) and podocyte foot process (PFP) effacement with immune cell infiltration. Similar to transmission electron microscopy, mSEM revealed a series of pathological changes in basement membrane and densities of EF and PFP in BXSB/MpJ-Yaa compared with control BXSB/MpJ at different stages. Further, immunopositive area of endothelial marker (CD34), podocyte functional molecules (Nephrin, Podocin, Synaptopodin, and Wilms' tumour 1 (WT1)), and vascular endothelial growth factor A (VEGF A) significantly decreased in the glomerulus of BXSB/MpJ-Yaa compared with BXSB at final stage. The indices of glomerular endothelial injuries (EF density and immunopositive area of CD34 and VEGF A) and podocyte injuries (PEP density and immunopositive area of podocyte functional molecules) were also significantly correlated with each other and with indices of autoimmune disease and renal dysfunction. Thus, our results elucidated the pathological crosstalk between endothelial cells and podocytes in MPGN progression and the usefulness of mSEM for glomerular pathological analysis.

IgA nephropathy (IgAN) is an immune complex-mediated disease involved in the kidney disease. Recent studies have revealed that Notch signaling-related genes are aberrantly expressed in various cell types and maybe associate with inflammation-induced carcinogenesis. The aim of our study was to investigate the function of Notch1 in the inflammatory response of IgAN. The expression of Notch1, Jagged1 and NICD1 in 52 IgAN renal tissues and 20 control renal tissues was first determined using quantitative real-time PCR and Western blot. ELISA was then used to estimate the inflammatory response of human podocytes to LPS. NF-κB activity was measured using dual-luciferase reporter assay. Activation of Notch1 and NF-κB signaling pathway was assessed using Western blot. The expression of Notch1, NICD1 and Jagged1 was significantly higher in IgAN renal tissues than control renal tissues (P < 0.05). LPS treatment resulted in an obvious increase of MCP-1, IL-8 and phosphorylated NF-κB p65 in podocytes polymeric IgA (pIgA) IgAN group compared to control group (P < 0.05 for all). Activated Notch1 and its target genes, Hes1 and Hey1 were also enhanced upon LPS stimulation. Silencing of Notch1 signaling with inhibitor DAPT, NF-κB activation and LPS-induced inflammatory response were obviously attenuated, whereas Notch1 activator Jagged1 could markedly restore NF-κB activity and LPS-induced inflammatory response (P < 0.05 for all). Crosstalk between TLR4 and Notch1 signaling regulates the inflammatory response in the IgAN and maybe plays an important role in the progression of IgAN.

The signaling molecule stimulator of IFN genes (STING) was identified as a crucial regulator of the DNA-sensing cyclic GMP-AMP synthase (cGAS)-STING pathway, and this signaling pathway regulates inflammation and energy homeostasis under conditions of obesity, kidney fibrosis, and AKI. However, the role of STING in causing CKD, including diabetic kidney disease (DKD) and Alport syndrome, is unknown. To investigate whether STING activation contributes to the development and progression of glomerular diseases such as DKD and Alport syndrome, immortalized human and murine podocytes were differentiated for 14 days and treated with a STING-specific agonist. We used diabetic The activation of the STING pathway acts as a mediator of disease progression in DKD and Alport syndrome. Targeting STING may offer a therapeutic option to treat glomerular diseases of metabolic and nonmetabolic origin or prevent their development, progression, or both.

Diabetic nephropathy (DN) is one of the most serious complications of diabetes and the main cause of end-stage renal failure. Rhubarb is a widely used traditional Chinese herb, and it has exhibited efficacy in reducing proteinuria, lowering blood sugar levels and improving kidney function in patients with DN. However, the exact pharmacological mechanism by rhubarb improves DN remain unclear due to the complexity of its ingredients. Hence, we systematically explored the underlying mechanisms of rhubarb in the treatment of DN. We adopted a network pharmacology approach, focusing on the identification of active ingredients, drug target prediction, gene collection, Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment. Molecular docking technology was used to verify the binding ability between the main active compounds and central therapeutic targets, and screen out the core active ingredients in rhubarb for the treatment of DN. Finally, molecular dynamics simulation was performed for the optimal core protein-ligand obtained by molecular docking using GROMACS software. The network analysis identified 16 active compounds in rhubarb that were linked to 37 possible therapeutic targets related to DN. Through protein-protein interaction analysis, TP53, CASP8, CASP3, MYC, JUN and PTGS2 were identified as the key therapeutic targets. By validation of molecular docking, finding that the central therapeutic targets have good affinities with the main active compounds of rhubarb, and rhein, beta-sitosterol and aloe-emodin were identified as the core active ingredients in rhubarb for the treatment of DN. Results from molecular dynamics simulations showed that TP53 and aloe-emodin bound very stably with a binding free energy of - 26.98 kcal/mol between the two. The results of the gene enrichment analysis revealed that the PI3K-Akt signalling pathway, p53 signalling pathway, AGE-RAGE signalling pathway and MAPK signalling pathway might be the key pathways for the treatment of DN, and these pathways were involved in podocyte apoptosis, glomerular mesangial cell proliferation, inflammation and renal fibrosis. Based on the network pharmacology approach and molecular docking technology, we successfully predicted the active compounds and their respective targets. In addition, we illustrated the molecular mechanisms that mediate the therapeutic effects of rhubarb against DN. These findings provided an important scientific basis for further research of the mechanism of rhubarb in the treatment of DN.

In glomerular and tubulointerstitial disease, polymorphonuclear- and monocyte-derived reactive oxygen species may contribute to oxidative modification of proteins, lipids, and nucleic acids. In part, the processes instigated by reactive oxygen species parallel events that lead to the development of atherosclerosis. Myeloperoxidase (MPO), a heme protein and catalyst for (lipo)protein oxidation is present in these mononuclear cells. The ability of MPO to generate hypochlorous acid/hypochlorite (HOCl/OCl-) from hydrogen peroxide in the presence of chloride ions is a unique and defining activity for this enzyme. The MPO-hydrogen peroxide-chloride system leads to a variety of chlorinated protein and lipid adducts that in turn may cause dysfunction of cells in different compartments of the kidney. The aim of this article is to cover and interpret some experimental and clinical aspects in glomerular and tubulointerstitial diseases in which the MPO-hydrogen peroxide-chloride system has been considered an important pathophysiologic factor in the progression but also the attenuation of experimental renal disease. The colocalization of MPO and HOCl-modified proteins in glomerular peripheral basement membranes and podocytes in human membranous glomerulonephritis, the presence of HOCl-modified proteins in mononuclear cells of the interstitium and in damaged human tubular epithelia, the inflammation induced and exacerbated by MPO antibody complexes in necrotizing glomerulonephritis, and the presence of HOCl-modified epitopes in urine following hyperlipidemia-induced renal damage in rodents suggest that MPO is an important pathogenic factor in glomerular and tubulointerstitial diseases. Specifically, the interaction of MPO with nitric oxide metabolism adds to the complexity of actions of oxidants and may help to explain bimodal partly detrimental partly beneficial effects of the MPO-hydrogen peroxide-chloride system in redox-modulated renal diseases.

Alstonia scholaris was utilized as a medicinal herb for the management of diabetes traditionally, with diabetic nephropathy (DN) was one of its major complications. However, the effect of A. scholaris on DN have yet to be explored. To investigate the effect and mechanism of A. scholaris in treating DN. The high glucose (HG)-induced renal podocyte (MPC5) injury model was conducted in vitro, and DN mice induced by high fat diet and combined with streptozotocin (HFD + STZ) was employed to evaluate bioactivity in vivo. Transcriptome analysis was conducted to explore the potential targets of vallesamine, with findings further validated by RT-qPCR and WB analysis. Furthermore, the binding affinity of vallesamine to its potential target was investigated through molecular docking and dynamics simulation. Four major alkaloids of A. scholaris demonstrated significant efficacy in mitigating HG-induced MPC5 cell damage, and they also restored oxidation balance while reducing the release of nitric oxide and lactate dehydrogenase. Oral administration of the total alkaloids and the four compounds for 6 weeks, respectively, could ameliorate proteinuria, urinary protein-to-creatinine ratio, hyperglycemia and hyperlipidemia significantly, and as well elevate serum levels of total protein and albumin concurrently in HFD + STZ induced mice. Moreover, renal injury and matrix hyperplasia were also improved after the treatment. Notably, vallesamine (5 mg/kg) exerted a pronounced effect on DN through upregulating Ppar-δ, Fads2, Me1, Ehhadh, Lpl, Scd1, Acsl1, and downregulating Hmgcs5, Slc27a1, Dbil5 and Plin5 gene expressions of PPAR pathway. Meanwhile, proteins related to lipid metabolism (PPAR-δ and ACSL1, HMGCS2) as well as the associated with renal inflammation (PODOCIN, BCL-2, and IL-6) were regulated by vallesamine intervention. In addition, vallesamine-PPAR-δ complexes maintained structural integrity, with the binding free energy of -25.84 kJ/mol, indicating a particularly high affinity between the ligand and the receptor in molecular dynamics and docking. Total alkaloids from A. scholaris and its main components vallesamine alleviated kidney injury induced by HFD + STZ through modulation the PPAR-δ pathway, providing a potential strategy for the development of new botanical drug to treat DN.

Endothelial injury is an early event in chronic kidney disease (CKD) leading to renal hemodynamic disorders and even glomerulosclerosis. During this process, both oxidative stress and inflammation originating from injured endothelial cells can initiate pathogenic cell-to-cell interactions via a paracrine mechanism. Accumulating evidence underscores the pivotal role of mitochondrial dysfunction as a crucial mechanism underlying endothelial dysfunction. Lon protease 1 (LONP1) is a mitochondrial protease that plays a key role in maintaining mitochondrial homeostasis; however, its role in endothelial dysfunction-related renal disease is unknown. In CKD patients and mice subjected to 5/6 nephrectomy (5/6Nx), we observed decreased LONP1 expression in glomerular endothelial cells. Interestingly, endothelial cell-specific heterozygous knockout of LONP1 exacerbated glomerulosclerosis and aggravated renal function decline, proteinuria, hypertension and kidney inflammation in 5/6Nx mice. Mechanistically, our results suggest that the loss of LONP1 strikingly increased reactive oxygen species (ROS) levels by promoting the ubiquitination of mitochondrial superoxide dismutase 2 (SOD2); which in turn led to mitochondrial dysfunction and inflammation within endothelial cells. Additionally, the increase in mitochondrial ROS and subsequent production of inflammatory cytokines from damaged endothelial cells further trigger mesangial cell proliferation and podocyte injury, which together result in glomerulosclerosis and CKD progression. Taken together, our findings identify LONP1 as a therapeutic target for balancing glomerular redox, alleviating inflammation, and retarding glomerulosclerosis.

AMP-activated protein kinase (AMPK) has been postulated to be crucial in regulating various renal physiology and pathophysiology processes, including energy metabolism, ion and water transport, inflammation, and hypertrophy. However, the specific roles of AMPK in the podocyte, a cell critical for maintaining glomerular filtration, have not been fully explored using genetic model animals. In this study, we generated mice lacking both AMPK α1 and α2 catalytic subunits in glomerular podocytes (pmut). Our findings revealed that, surprisingly, AMPK is dispensable for normal podocyte function. These knockout mice could live as long as their wild-type littermates without showing any pathological alterations in their glomeruli or glomerular function at two years of age. However, under type 1 diabetic conditions, the diabetic pmut mice exhibited increased lipid and collagen accumulation and an elevated expression of mesenchymal proteins in their glomeruli. They also showed more significant albuminuria compared to control diabetic mice. Under high glucose culture conditions, glomeruli isolated from pmut mice demonstrated a reduced expression of mitochondrial genes (e.g., Ndufv2) and increased leakage of mitochondrial components. Additionally, there was heightened expression of genes associated with nucleotide sensing and pro-inflammatory pathways (including mb21d2, IL-1 beta, and NF-kB). These observations suggest that while AMPK is not necessary for podocyte function in healthy kidneys, it is crucial for preventing glomerular fibrosis resulting from lipotoxicity and inflammation under diabetic conditions.

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis during hyperhomocysteinemia (hHcys). However, it remains unclear whether the NLRP3 inflammasome can be a therapeutic target for treatment of hHcys-induced kidney injury. Given that DHA metabolites-resolvins have potent anti-inflammatory effects, the present study tested whether the prototype, resolvin D1 (RvD1), and 17S-hydroxy DHA (17S-HDHA), an intermediate product, abrogate hHcys-induced podocyte injury by targeting the NLRP3 inflammasome. In vitro, confocal microscopy demonstrated that 17S-HDHA (100 nM) and RvD1 (60 nM) prevented Hcys-induced formation of NLRP3 inflammasomes, as shown by reduced colocalization of NLRP3 with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) or caspase-1. Both DHA metabolites inhibited Hcys-induced caspase-1 activation and interleukin-1β production. However, DHA had no significant effect on these Hcys-induced changes in podocytes. In vivo, DHA lipoxygenase metabolites substantially inhibited podocyte NLRP3 inflammasome formation and activation and consequent glomerular sclerosis in mice with hHcys. Mechanistically, RvD1 and 17S-HDHA were shown to suppress Hcys-induced formation of lipid raft redox signaling platforms and subsequent O

Sirtuin 3 (SIRT3) is the primary mitochondrial deacetylase that controls the antioxidant pathway and energy metabolism. We previously found that renal

The urokinase-type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR -(s)uPAR- from circulation) is to regulate podocyte function through αvβ3 integrin/Rac-1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)-induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvβ3 integrin/Rac-1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ-induced up-regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvβ3 integrin/Rac-1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen-plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR-targeting approaches.

Adiponectin is known to take part in the regulation of energy metabolism. AdipoRon, an orally-active synthetic adiponectin agonist, binds to both adiponectin receptors (AdipoR)1/R2 and ameliorates diabetic complications. Among the lipid metabolites, the ceramide subspecies of sphingolipids have been linked to features of lipotoxicity, including inflammation, cell death, and insulin resistance. We investigated the role of AdipoRon in the prevention and development of type 2 diabetic nephropathy. AdipoRon (30 mg/kg) was mixed into the standard chow diet and provided to db/db mice (db + AdipoRon, n = 8) and age-matched male db/m mice (dm + AdipoRon, n = 8) from 17 weeks of age for 4 weeks. Control db/db (db cont, n = 8) and db/m mice (dm cont, n = 8) were fed a normal diet of mouse chow. AdipoRon-fed db/db mice showed a decreased amount of albuminuria and lipid accumulation in the kidney with no significant changes in serum adiponectin, glucose, and body weight. Restoring expression of adiponectin receptor-1 and -2 in the renal cortex was observed in db/db mice with AdipoRon administration. Consistent up-regulation of phospho-Thr AdipoRon may prevent lipotoxicity in the kidney particularly in both GECs and podocytes through an improvement in lipid metabolism, as shown by the ratio of ceramide to sphingosines, and further contribute to prevent deterioration of renal function, independent of the systemic effects of adiponectin. The reduction in oxidative stress and apoptosis by AdipoRon provides protection against renal damage, thereby ameliorating endothelial dysfunction in type 2 diabetic nephropathy.

The current treatment for diabetic nephropathy (DN) is still limited. NaoXinTong Capsule (NXT) is a Chinese Medicine prescribed to patients with cardiovascular disease. It can also ameliorate metabolic syndromes in patients indicating its anti-diabetic properties. Herein we report the therapeutic effects of NXT on the developed DN. The db/db diabetic mice at ˜12 weeks old, the age with DN at middle/advanced stages, were treated with NXT for 12 weeks. We found NXT treatment reduced diabetes-induced hyperglycemia and dyslipidemia, thereby substantially reduced DN progress. In the kidney, NXT reduced mesangial matrix expansion and glomerulosclerosis by inhibiting extracellular matrix accumulation through activation of matrix metalloproteinase 2/9 and inactivating transforming growth factor β1 expression. NXT reduced podocyte injury by reducing renal inflammation and expression of adhesion molecules. Mechanically, NXT potently activated AMPKα in multiple tissues thereby enhancing energy metabolism. In the liver, NXT increased glucokinase expression and insulin sensitivity by increasing insulin receptor substrate 1/2 and protein kinase B (AKT) 1/2 expression/phosphorylation. In skeletal muscle, NXT activated expression of glucose transporter type 4, AKT, glycogen synthase and peroxisome proliferator activated receptor α/γ. In adipose tissue, NXT reduced fatty acid synthase while activating hormone-sensitive lipase expression. Taken together, our study demonstrates that NXT reduced progress of the developed DN by ameliorating glucose, lipid and energy metabolism, maintaining renal structural and functional integrity. Our study also indicates the potential application of NXT for DN treatment in clinics.

Increased intercellular reactive oxygen species (ROS) levels are the major cause of podocyte injury with proteinuria. Caveolin‑1 (CAV‑1) is an essential protein component of caveolae. CAV‑1 participates in signal transduction and endocytic trafficking. Recent research has indicated that CAV‑1 regulates oxidative stress‑induced pathways. The present study used hydrogen peroxide (H2O2) at nontoxic concentrations to elevate the level of ROS in E11 podocytes. Treatment with 500 and 1,000 µM H2O2 for 1 h significantly reduced CAV‑1 expression levels. Simultaneously, the treatment significantly reduced the expression of the antioxidant enzymes glutamine‑cysteine ligase catalytic subunit, superoxide dismutase 2 and catalase. To determine the role of CAV‑1 in mediating oxidative stress, E11 podocytes were administered antenapedia‑CAV‑1 (AP‑CAV‑1) peptide for 48 h. The AP‑CAV‑1 treatment enhanced CAV‑1 expression and inhibited cyclophilin A expression, thus reducing ROS‑induced inflammation. Moreover, CAV‑1 protected against H2O2‑induced oxidative stress responses by enhancing the expression of antioxidant enzymes. Furthermore, CAV‑1 attenuated H2O2‑induced changes oxidative phosphorylation, and the expression of optic atrophy 1 and translocase of the inner membrane 23, as well as preserving mitochondrial function. CAV‑1 treatment significantly suppressed apoptosis, as indicated by a higher B‑cell lymphoma 2/BCL2‑associated X protein ratio. Therefore, enhancing the expression of CAV‑1 may be an important therapeutic consideration in treating podocyte injury.

Diabetic kidney disease (DKD) is a multifactorial complication of diabetes involving mitochondrial dysfunction and immune cell infiltration. However, the causal relationships remain unclear. We applied Mendelian randomization (MR) and single-cell RNA sequencing (scRNA-seq) to investigate the roles of mitochondrial gene expression and immune cells in DKD. Additionally, peripheral blood mononuclear cells (PBMCs) from DKD patients were analyzed for differential gene expression. Higher expression of mitochondrial genes PCCB, ACADM, ADHFE1, OCIAD1, and FIS1 increased DKD risk, while genes like NT5DC2, ATP5MC3, and GLYCTK decreased risk. Immune traits, including human leukocyte antigen (HLA)-DR + plasmacytoid dendritic cells (pDCs), mediated the effects of mitochondrial dysfunction on DKD. scRNA-seq revealed significant downregulation of ATP5MC3, GLYCTK, and NT5DC2 in podocytes (PODOs) and tubular cells in DKD kidneys, alongside increased infiltration of helper T cells, B cells, dendritic cells (DCs), and plasma cells. PBMC analysis highlighted the upregulation of proinflammatory genes (CXCL2, CXCL3, and others) in DKD patients. This study highlights the complex interplay between mitochondrial dysfunction and immune cell infiltration in DKD pathogenesis. Key mitochondrial genes and immune traits identified here offer novel therapeutic targets such as ATP5MC3, GLYCTK, and DC pathways.

Ectopic lipid deposition, mitochondrial injury, and inflammatory responses contribute to the development of diabetic kidney disease (DKD); however, the mechanistic link between these processes remains unclear. In this study, we demonstrate that the ceramide synthase 6 (CerS6) is primarily localized in podocytes of the glomeruli and is upregulated in two different models of diabetic mice. Podocyte-specific CerS6 knockout ameliorates glomerular injury and inflammatory responses in male diabetic mice and in male mice with adriamycin-induced nephropathy. In contrast, podocyte-specific overexpression of CerS6 sufficiently induces proteinuria. Mechanistically, CerS6-derived ceramide (d18:1/16:0) can bind to the mitochondrial channel protein VDAC1 at Glu59 residue, initiating mitochondrial DNA (mtDNA) leakage, activating the cGAS-STING signaling pathway, and ultimately promoting an immune-inflammatory response in the kidney. Importantly, CERS6 expression is increased in podocytes from kidney biopsies of patients with DKD and focal segmental glomerulosclerosis (FSGS), and the expression level of CERS6 is correlated negatively with glomerular filtration rate and positively with proteinuria. Thus, our findings suggest that targeting CerS6 may be a potential therapeutic strategy for proteinuric kidney diseases.

Heteroplasmic pathogenic mitochondrial DNA (mtDNA) mutations are key drivers of mitochondrial diseases, yet their tissue-specific and cell-specific accumulation patterns during aging and the mechanistic links to pathology remain poorly understood. In this study, we employed DddA-derived cytosine base editor technology to generate three mouse models harboring distinct pathogenic mitochondrial tRNA mutations. These mutations exhibited age-dependent accumulation in the kidneys, leading to severe kidney defects that well recapitulate human mitochondrial kidney disease. Mitochondrial single-cell assay for transposase-accessible chromatin with sequencing (mtscATAC-seq) revealed unique heteroplasmy dynamics across different kidney cell types: podocytes exhibited a positive selection for mutant mtDNA, whereas tubular epithelial cells displayed neutral drift of mutations during aging. Integrative analyses combining mtscATAC-seq, single-cell RNA sequencing and spatially enhanced resolution omics sequencing further identified molecular changes in high-mutant defective cells, including increased AP-1 family transcription factor activity, tubular epithelial cell proliferation and immune activation, which contribute to disease progression. Our study underscores the importance of kidney function monitoring in patients with mitochondrial disease, particularly in older adults, and establishes robust preclinical models to facilitate the development of therapeutic strategies.

'HSP90AB1 protein macromolecule plays an important role in various cellular stress responses, but its specific mechanism in podiatocyte injury and mitochondrial dysfunction remains unclear. The aim of this study was to investigate the mechanism of how 'HSP90AB1 mediates mitochondrial dysfunction and leads to podiocyte injury through regulation of ATP5A1 and PARK2. Clinical podocyte samples were collected and the MPC5 mouse podocyte cell line was used for experiments. The interaction of 'HSP90AB1 with ATP5A1 and PARK2 was analyzed by transcriptome sequencing, cell culture, 'HSP90AB1 overexpression and knockdown construction, combined immunoprecipitation (CoIP) and immunofluorescence detection. CCK8 was used to measure cell viability, Westernblot and qPCR were used to assess protein and mRNA expression levels, and statistical methods were used to analyze the data. Bioinformatics analysis revealed physical or functional interactions between 'HSP90AB1, ATP5A1, and PARK2 proteins. The interaction between 'HSP90AB1 and these two proteins was verified by cell experiments, and 'HSP90AB1 played an important role in podiatocyte injury. In ADR-induced podocyte injury model, mRNA and protein expressions of 'HSP90AB1, ATP5A1 and PARK2 were significantly changed, and the expression of mitochondrial autophagy related proteins was also changed. Further analysis showed that the interaction between 'HSP90AB1, ATP5A1 and PARK2 played a key role in the process of podiocyte injury. This study revealed that 'HSP90AB1 mediates mitochondrial dysfunction by regulating the interaction of ATP5A1 and PARK2, thereby promoting podiocyte injury. This discovery provides new potential targets for the treatment of podocyte injury and contributes to the understanding of the pathological mechanisms of related diseases.

Toll-like receptor 9 (TLR9) senses bacterial DNA characteristic of unmethylated CpG motifs to induce innate immune response. TLR9 is de novo expressed in podocytes of some patients with glomerular diseases, but its role in podocyte injury remains undetermined. Since TLR9 activates p38 MAPK and NFkB that are known to mediate podocyte apoptosis, we hypothesized that TLR9 induces podocyte apoptosis in glomerular diseases. We treated immortalized podocytes with puromycin aminonucleosides (PAN) and observed podocyte apoptosis, accompanied by TLR9 upregulation. Prevention of TLR9 upregulation by siRNA significantly attenuated NFκB p65 or p38 activity and apoptosis, demonstrating that TLR9 mediates podocyte apoptosis. We next showed that endogenous mitochondrial DNA (mtDNA), whose CpG motifs are also unmethylated, is the ligand for TLR9, because PAN induced mtDNA accumulation in endolysosomes where TLR9 is localized, overexpression of endolysosomal DNase 2 attenuated PAN-induced p38 or p65 activity and podocyte apoptosis, and DNase 2 silencing was sufficient to activate p38 or p65 and induce apoptosis. In PAN-treated rats, TLR9 was upregulated in the podocytes, accompanied by increase of apoptosis markers. Thus, de novo expressed TLR9 may utilize endogenous mtDNA as the ligand to facilitate podocyte apoptosis, a novel mechanism underlying podocyte injury in glomerular diseases.

Mineralocorticoid receptor (MR) overactivation plays a crucial role in the pathogenesis of chronic kidney disease, as well as several cardiovascular and arterial diseases. Current studies determined the mechanisms of the beneficial kidney effects of the nonsteroidal MR antagonist finerenone (FN) in a mouse model of Western diet-induced obesity and insulin resistance. Ten-week-old male C57BL/6J mice were fed a low-fat (LF) or a Western diet (WD) for 12 weeks followed by treatment with either vehicle or FN for another 14 weeks (intervention studies) until they were 36 weeks old. Finerenone treatment prevented

The pathophysiology of glomerular lesions of membranous nephropathy (MN), including seldom-reported IgG4-related disease, is still elusive. Unlike in idiopathic MN where IgG4 prevails, in this patient IgG3 was predominant in glomerular deposits in the absence of circulating anti-phospholipase A2 receptor antibodies, suggesting a distinct pathologic process. Here we documented that IgG4 retrieved from the serum of our propositus reacted against carbonic anhydrase II (CAII) at the podocyte surface. In patient's biopsy, glomerular CAII staining increased and co-localized with subepithelial IgG4 deposits along the capillary walls. Patient's IgG4 caused a drop in cell pH followed by mitochondrial dysfunction, excessive ROS production and cytoskeletal reorganization in cultured podocytes. These events promoted mitochondrial superoxide-dismutase-2 (SOD2) externalization on the plasma membrane, becoming recognizable by complement-binding IgG3 anti-SOD2. Among patients with IgG4-related disease only sera of those with IgG4 anti-CAII antibodies caused low intracellular pH and mitochondrial alterations underlying SOD2 externalization. Circulating IgG4 anti-CAII can cause podocyte injury through processes of intracellular acidification, mitochondrial oxidative stress and neoantigen induction in patients with IgG4 related disease. The onset of MN in a subset of patients could be due to IgG4 antibodies recognizing CAII with consequent exposure of mitochondrial neoantigen in the context of multifactorial pathogenesis of disease.

BACKGROUND Diabetic kidney disease (DKD) is characterized by progressive injury to glomerular podocytes due to sustained mechanical stress within the glomerulus. Piezo proteins, acting as cellular mechanosensors, play a pivotal role in mechanotransduction by sensing mechanical forces and regulating intracellular ion flux. This study investigates the role of Piezo1 in the progression of DKD and its mechanistic involvement in podocyte injury. METHODS Podocyte-specific Piezo1 knockout mice were generated using the streptozotocin plus high-fat diet model of DKD. In vitro studies included the use of Piezo1 inhibitors to assess calcium influx, podocyte cytoskeletal rearrangement, and apoptosis under stiff matrix conditions. Additionally, nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and transient receptor potential cation channel 6 (TRPC6) signaling pathways were explored to establish their role in Piezo1-mediated podocyte injury. Adeno-associated virus -TRPC6 was utilized to overexpress TRPC6 in podocyte-specific Piezo1 knockout mice to assess the in vivo interaction between Piezo1 and TRPC6. RESULTS Podocyte-specific deletion of Piezo1 significantly ameliorated the progression of DKD in diabetic mice. Inhibition of Piezo1 reduced calcium influx, cytoskeletal rearrangement, and podocyte apoptosis in vitro. Mechanistically, Piezo1 activation triggered a signaling loop involving NFATc1 and TRPC6, leading to increased calcium influx, perpetuating podocyte injury. TRPC6 overexpression in vivo counteracted the protective effects of Piezo1 deletion, confirming the critical role of the Piezo1/NFATc1/TRPC6 axis in DKD progression. CONCLUSIONS Piezo1 plays a key mechanosensory role in podocyte injury during DKD progression by mediating calcium influx and activating the NFATc1/TRPC6 signaling pathway.

Recent studies have shown the crucial role of podocyte injury in the development of diabetic kidney disease (DKD). Deubiquitinating modification of proteins is widely involved in the occurrence and development of diseases. Here, we explore the role and regulating mechanism of a deubiquitinating enzyme, OTUD5, in podocyte injury and DKD. RNA-seq analysis indicates a significantly decreased expression of OTUD5 in HG/PA-stimulated podocytes. Podocyte-specific Otud5 knockout exacerbates podocyte injury and DKD in both type 1 and type 2 diabetic mice. Furthermore, AVV9-mediated OTUD5 overexpression in podocytes shows a therapeutic effect against DKD. Mass spectrometry and co-immunoprecipitation experiments reveal an inflammation-regulating protein, TAK1, as the substrate of OTUD5 in podocytes. Mechanistically, OTUD5 deubiquitinates K63-linked TAK1 at the K158 site through its active site C224, which subsequently prevents the phosphorylation of TAK1 and reduces downstream inflammatory responses in podocytes. Our findings show an OTUD5-TAK1 axis in podocyte inflammation and injury and highlight the potential of OTUD5 as a promising therapeutic target for DKD. Deubiquitinating of key proteins may be involved in podocyte injury and diabetic kidney disease (DKD). Here, the authors show that OTUD5 in podocytes alleviates DKD through deubiquitinating TAK1 at the K158 site, preventing TAK1 phosphorylation and inflammatory responses in podocytes.

Emerging evidence suggests that GSK3β, a redox-sensitive transducer downstream of insulin signaling, acts as a convergent point for myriad pathways implicated in kidney injury, repair, and regeneration. However, its role in diabetic kidney disease remains controversial. In cultured glomerular podocytes, exposure to a milieu of type 2 diabetes elicited prominent signs of podocyte injury and degeneration, marked by loss of homeostatic marker proteins like synaptopodin, actin cytoskeleton disruption, oxidative stress, apoptosis, and stress-induced premature senescence, as shown by increased staining for senescence-associated β-galactosidase activity, amplified formation of γH2AX foci, and elevated expression of mediators of senescence signaling, like p21 and p16INK4A. These degenerative changes coincided with GSK3β hyperactivity, as evidenced by GSK3β overexpression and reduced inhibitory phosphorylation of GSK3β, and were averted by tideglusib, a highly-selective small molecule inhibitor of GSK3β. In agreement, post-hoc analysis of a publicly-available glomerular transcriptomics dataset from patients with type 2 diabetic nephropathy revealed that the curated diabetic nephropathy-related gene set was enriched in high GSK3β expression group. Mechanistically, GSK3β-modulated nuclear factor Nrf2 signaling is involved in diabetic podocytopathy, because GSK3β knockdown reinforced Nrf2 antioxidant response and suppressed oxidative stress, resulting in an improvement in podocyte injury and senescence. Conversely, ectopic expression of the constitutively active mutant of GSK3β impaired Nrf2 antioxidant response and augmented oxidative stress, culminating in an exacerbated diabetic podocyte injury and senescence. Moreover, IRS-1 was found to be a cognate substrate of GSK3β for phosphorylation at IRS-1S332, which negatively regulates IRS-1 activity. GSK3β hyperactivity promoted IRS-1 phosphorylation, denoting a desensitized insulin signaling. Consistently, in vivo in db/db mice with diabetic nephropathy, GSK3β was hyperactive in glomerular podocytes, associated with IRS-1 hyperphosphorylation, impaired Nrf2 response and premature senescence. Our finding suggests that GSK3β is likely a novel therapeutic target for treating type 2 diabetic glomerular injury.

Podocytes are essential to maintaining the integrity of the glomerular filtration barrier, yet they are frequently affected in lupus nephritis (LN). Here, we show that the significant upregulation of Drp1S616 phosphorylation in podocytes promotes mitochondrial fission, leading to mitochondrial dysfunction and podocyte injury in LN. Inhibition or knockdown of Drp1 promotes mitochondrial fusion and protects podocytes from injury induced by LN serum. In vivo, pharmacological inhibition of Drp1 reduces the phosphorylation of Drp1S616 in podocytes in lupus-prone mice. Podocyte injury is reversed when Drp1 is inhibited, resulting in the alleviation of proteinuria. Mechanistically, complement component C5a (C5a) upregulates the phosphorylation of Drp1S616 and promotes mitochondrial fission in podocytes. Moreover, the expression of C5a receptor 1 (C5aR1) is notably upregulated in podocytes in LN. C5a-C5aR1 axis-controlled phosphorylation of Drp1S616 and mitochondrial fission are substantially suppressed when C5aR1 is knocked down by siRNA. Moreover, lupus-prone mice treated with C5aR inhibitor show reduced phosphorylation of Drp1S616 in podocytes, resulting in significantly less podocyte damage. Together, this study uncovers a novel mechanism by which the C5a-C5aR1 axis promotes podocyte injury by enhancing Drp1-mediated mitochondrial fission, which could have significant implications for the treatment of LN.

Retinoic acid receptor responder protein-1 (RARRES1) is a podocyte-enriched transmembrane protein whose increased expression correlates with human glomerular disease progression. RARRES1 promotes podocytopenia and glomerulosclerosis via p53-mediated podocyte apoptosis. Importantly, the cytopathic actions of RARRES1 are entirely dependent on its proteolytic cleavage into a soluble protein (sRARRES1) and subsequent podocyte uptake by endocytosis, as a cleavage mutant RARRES1 exerted no effects in vitro or in vivo. As RARRES1 expression is upregulated in human glomerular diseases, here we investigated the functional consequence of podocyte-specific overexpression of RARRES1 in mice in the experimental focal segmental glomerulosclerosis and diabetic kidney disease. We also examined the effects of long-term RARRES1 overexpression on slowly developing aging-induced kidney injury. As anticipated, the induction of podocyte overexpression of RARRES1 (Pod-RARRES1WT) significantly worsened glomerular injuries and worsened kidney function in all three models, while overexpression of RARRES1 cleavage mutant (Pod-RARRES1MT) did not. Remarkably, direct uptake of sRARRES1 was also seen in proximal tubules of injured Pod-RARRES1WT mice and associated with exacerbated tubular injuries, vacuolation, and lipid accumulation. Single cell RNA sequence analysis of mouse kidneys demonstrated RARRES1 led to a marked deregulation of lipid metabolism in proximal tubule subsets. We further identified matrix metalloproteinase 23 (MMP23) as a highly podocyte-specific metalloproteinase and responsible for RARRES1 cleavage in disease settings, as adeno-associated virus 9-mediated knockdown of MMP23 abrogated sRARRES1 uptake in tubular cells in vivo. Thus, our study delineates a previously unrecognized mechanism by which a podocyte-derived protein directly facilitates podocyte and tubular injury in glomerular diseases and suggests that podocyte-specific functions of RARRES1 and MMP23 may be targeted to ameliorate glomerular disease progression in vivo.

Visual Abstract Significance Statement Autophagy protects podocytes from injury in diabetic kidney disease (DKD). Restoring glomerular autophagy is a promising approach to limit DKD. This study demonstrates a novel regulatory mechanism of autophagy that blocks this critical protection of the glomerular filtration barrier. We demonstrated that TRPC6 induced in podocytes in mouse models of diabetes mediates calpain activation, thereby impairing podocyte autophagy, causing injury and accelerating DKD. Furthermore, this study provides proof of principle for druggable targets for DKD because restoration of podocyte autophagy by calpain inhibitors effectively limits glomerular destruction. Background Diabetic kidney disease is associated with impaired podocyte autophagy and subsequent podocyte injury. The regulation of podocyte autophagy is unique because it minimally uses the mTOR and AMPK pathways. Thus, the molecular mechanisms underlying the impaired autophagy in podocytes in diabetic kidney disease remain largely elusive. Methods This study investigated how the calcium channel TRPC6 and the cysteine protease calpains deleteriously affect podocyte autophagy in diabetic kidney disease in mice. We demonstrated that TRPC6 knockdown in podocytes increased the autophagic flux because of decreased cysteine protease calpain activity. Diabetic kidney disease was induced in vivo using streptozotocin with unilateral nephrectomy and the BTBRob/ob mouse models. Results Diabetes increased TRPC6 expression in podocytes in vivo with decreased podocyte autophagic flux. Transgenic overexpression of the endogenous calpain inhibitor calpastatin, as well as pharmacologic inhibition of calpain activity, normalized podocyte autophagic flux, reduced nephrin loss, and prevented the development of albuminuria in diabetic mice. In kidney biopsies from patients with diabetes, we further confirmed that TRPC6 overexpression in podocytes correlates with decreased calpastatin expression, autophagy blockade, and podocyte injury. Conclusions Overall, we discovered a new mechanism that connects TRPC6 and calpain activity to impaired podocyte autophagy, increased podocyte injury, and development of proteinuria in the context of diabetic kidney disease. Therefore, targeting TRPC6 and/or calpain to restore podocyte autophagy might be a promising therapeutic strategy for diabetic kidney disease.

ABSTRACT Circular RNAs (circRNAs) are special non-coding RNA (ncRNA) molecules that play a significant role in many diseases. However, the biogenesis and regulation of circRNAs in diabetic nephropathy (DN) are largely unknown. Here, we investigated the expression profile of circRNAs in kidney of DN mice through circular RNA sequencing (circRNA-seq). The renal biopsy samples of patients with DN had low circ -0,000,953 expression, which was significantly associated with renal function. Furthermore, loss-of-function and gain-of-function experiments were carried out to prove the role of circ -0,000,953 in DN. Podocyte conditional knockin (cKI) or systemic overexpression of circ -0,000,953 alleviated albuminuria and restored macroautophagy/autophagy in kidney of diabetic mice. However, circ -0,000,953 knockdown exacerbated albuminuria and podocyte injury. Mechanistically, we found circ -0,000,953 directly binds to Mir665-3p-Atg4b to perform its function. Silencing of Mir665-3p or overexpression of Atg4b recovered podocyte autophagy both in vitro and in vivo. To examine the cause of circ -0,000,953 downregulation in DN, bioinformatics prediction found that circ -0,000,953 sequence has a high possibility of containing an m6A methylation site. Additionally, METTL3 was proved to regulate the expression and methylation level of circ -0,000,953 through YTHDF2 (YTH N6-methyladenosine RNA binding protein 2). In conclusion, this study revealed that circ -0,000,953 regulates podocyte autophagy by targeting Mir665-3p-Atg4b in DN. Therefore, circ -0,000,953 is a potential biomarker for prevention and cure of DN. Abbreviation: CCL2/MCP-1: C-C motif chemokine ligand 2; ceRNA: competing endogenous RNA; circRNA: circular RNA; cKI: conditional knockin; cKO: conditional knockout; CRE: creatinine; DM: diabetes mellitus; DN: diabetic nephropathy; ESRD: end-stage renal disease; HG: high glucose; IF: immunofluorescence; MAP1LC3/LC3B: microtubule-associated protein 1 light chain 3 beta; MPC5: mouse podocyte clone 5; MTECs: mouse tubular epithelial cells; MTOR: mechanistic target of rapamycin kinase; NC: normal control; ncRNA: non-coding RNA; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PAS: periodic acid-Schiff; RELA/p65: v-rel reticuloendotheliosis viral oncogene homolog A (avian); SDs: slit diaphragm proteins; Seq: sequencing; STZ: streptozotocin; SV40: SV40-MES13-cells, mouse mesangial cell line; T1D: type 1 diabetes mellitus; T2D: type 2 diabetes mellitus; TEM: transmission electron microscopy; TNF/TNF-α: tumor necrosis factor; VECs: vascular endothelial cells; WT1: WT1 transcription factor; YTHDF2: YTH N6-methyladenosine RNA binding protein 2.

No abstract available

Abstract Objectives Diabetic nephropathy (DN) is the most common microvascular complication of diabetes mellitus. This study investigated the mechanism of triptolide (TP) in podocyte injury in DN. Methods DN mouse models were established by feeding with a high-fat diet and injecting with streptozocin and MPC5 podocyte injury models were induced by high-glucose (HG), followed by TP treatment. Fasting blood glucose and renal function indicators, such as 24 h urine albumin (UAlb), serum creatinine (SCr), blood urea nitrogen (BUN), and kidney/body weight ratio of mice were examined. H&E and TUNEL staining were performed for evaluating pathological changes and apoptosis in renal tissue. The podocyte markers, reactive oxygen species (ROS), oxidative stress (OS), serum inflammatory cytokines, nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway-related proteins, and pyroptosis were detected by Western blotting and corresponding kits. MPC5 cell viability and pyroptosis were evaluated by MTT and Hoechst 33342/PI double-fluorescence staining. Nrf2 inhibitor ML385 was used to verify the regulation of TP on Nrf2. Results TP improved renal function and histopathological injury of DN mice, alleviated podocytes injury, reduced OS and ROS by activating the Nrf2/heme oxygenase-1 (HO-1) pathway, and weakened pyroptosis by inhibiting the nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome pathway. In vitro experiments further verified the inhibition of TP on OS and pyroptosis by mediating the Nrf2/HO-1 and NLRP3 inflammasome pathways. Inhibition of Nrf2 reversed the protective effect of TP on MPC5 cells. Conclusions Overall, TP alleviated podocyte injury in DN by inhibiting OS and pyroptosis via Nrf2/ROS/NLRP3 axis.

Activation of the renin-angiotensin system is associated with podocyte injury and has been well demonstrated as a pivotal factor in the progression of chronic kidney disease. Podocyte energy metabolism is crucial for maintaining their physiological functions. However, whether renin-angiotensin system activation promotes chronic kidney disease progression by disturbing the energy metabolism of podocytes has not been elucidated. Angiotensin II, the main active molecule of the renin-angiotensin system, plays a crucial role in chronic kidney disease initiation and progression, but its impact on podocyte metabolism remains unclear. Here, we demonstrate a rapid decrease in the expression of pyruvate kinase M2, a key glycolytic enzyme, and reduced glycolytic flux in podocytes exposed to angiotensin II in vivo and in vitro. Podocyte-specific deletion of pyruvate kinase M2 in mice aggravated angiotensin II-induced glomerular and podocyte injury with foot process effacement and proteinuria. The inhibition of glycolysis was accompanied by adenosine triphosphate deficiency, cytoskeletal remodeling and podocyte apoptosis. Mechanistically, we found that angiotensin II-induced glycolysis impairment contributed to an insufficient energy supply to the foot process, leading to podocyte injury. Additionally, pyruvate kinase M2 expression was found to be reduced in podocytes from kidney biopsies of patients with hypertensive nephropathy and diabetic kidney disease. Thus, our findings suggest that glycolysis activation is a potential therapeutic strategy for podocyte injury.

The use of mesenchymal stem cells (MSCs) has become a new strategy for treating diabetic kidney disease (DKD). However, the role of placenta derived mesenchymal stem cells (P-MSCs) in DKD remains unclear. This study aims to investigate the therapeutic application and molecular mechanism of P-MSCs on DKD from the perspective of podocyte injury and PINK1/Parkin-mediated mitophagy at the animal, cellular, and molecular levels. Western blotting, reverse transcription polymerase chain reaction, immunofluorescence, and immunohistochemistry were used to detect the expression of podocyte injury-related markers and mitophagy-related markers, SIRT1, PGC-1α, and TFAM. Knockdown, overexpression, and rescue experiments were performed to verify the underlying mechanism of P-MSCs in DKD. Mitochondrial function was detected by flow cytometry. The structure of autophagosomes and mitochondria were observed by electron microscopy. Furthermore, we constructed a streptozotocin-induced DKD rat model and injected P-MSCs into DKD rats. Results showed that as compared with the control group, exposing podocytes to high-glucose conditions aggravated podocyte injury, represented by a decreased expression of Podocin along with increased expression of Desmin, and inhibited PINK1/Parkin-mediated mitophagy, manifested as a decreased expression of Beclin1, the LC3II/LC3I ratio, Parkin, and PINK1 associated with an increased expression of P62. Importantly, these indicators were reversed by P-MSCs. In addition, P-MSCs protected the structure and function of autophagosomes and mitochondria. P-MSCs increased mitochondrial membrane potential and ATP content and decreased the accumulation of reactive oxygen species. Mechanistically, P-MSCs alleviated podocyte injury and mitophagy inhibition by enhancing the expression of the SIRT1-PGC-1α-TFAM pathway. Finally, we injected P-MSCs into streptozotocin-induced DKD rats. The results revealed that the application of P-MSCs largely reversed the markers related to podocyte injury and mitophagy and significantly increased the expression of SIRT1, PGC-1α, and TFAM compared with the DKD group. In conclusion, P-MSCs ameliorated podocyte injury and PINK1/Parkin-mediated mitophagy inhibition in DKD by activating the SIRT1-PGC-1α-TFAM pathway.

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9–mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

Dysfunction of podocytes has been regarded as an important early pathologic characteristic of diabetic kidney disease (DKD), but the regulatory role of long noncoding RNAs (lncRNAs) in this process remains largely unknown. Here, we performed RNA sequencing in kidney tissues isolated from DKD patients and nondiabetic renal cancer patients undergoing surgical resection and discovered that the novel lncRNA ENST00000436340 was upregulated in DKD patients and high glucose-induced podocytes, and we showed a significant correlation between ENST00000436340 and kidney injury. Gain- and loss-of-function experiments showed that silencing ENST00000436340 alleviated high glucose-induced podocyte injury and cytoskeleton rearrangement. Mechanistically, we showed that fat mass and obesity- associate gene (FTO)-mediated m6A induced the upregulation of ENST00000436340. ENST00000436340 interacted with polypyrimidine tract binding protein 1 (PTBP1) and augmented PTBP1 binding to RAB3B mRNA, promoted RAB3B mRNA degradation, and thereby caused cytoskeleton rearrangement and inhibition of GLUT4 translocation to the plasma membrane, leading to podocyte injury and DKD progression. Together, our results suggested that upregulation of ENST00000436340 could promote podocyte injury through PTBP1-dependent RAB3B regulation, thus suggesting a novel form of lncRNA-mediated epigenetic regulation of podocytes that contributes to the pathogenesis of DKD.

No abstract available

Podocyte injury is a hallmark of glomerular disease and one of the leading causes of chronic kidney disease (CKD). Peroxisome proliferator-activated receptor α (PPARα) plays a key role in podocyte fatty acid oxidation (FAO). However, the underlying regulatory mechanisms remain unresolved. Trim63 is an E3 ubiquitin ligase that has been shown to inhibit PPARα activity; however, its role in fatty acid metabolism in the kidney has not been elucidated to date. In this study, we investigated the effects of overexpression and knockdown of Trim63 in Adriamycin (ADR)-induced nephropathy and diabetic nephropathy models and a podocyte cell line. In both rodents and human patients with proteinuric CKD, Trim63 was upregulated, particularly in the podocytes of injured glomeruli. In the ADR-induced nephropathy model, ectopic Trim63 application aggravated FAO deficiency and mitochondrial dysfunction and triggered intense lipid deposition, podocyte injury, and proteinuria. Notably, Trim63 inhibition alleviated FAO deficiency and mitochondrial dysfunction, and markedly restored podocyte injury and renal fibrosis in ADR-induced and diabetic nephropathy (DN) models. Additionally, Trim63 was observed to mediate PPARα ubiquitination and degradation, leading to podocyte injury. We demonstrate the pathological role of Trim63, which was previously unrecognized in kidney tissue, in FAO deficiency and podocyte injury. Targeting Trim63 may represent a viable therapeutic strategy for podocyte injury and proteinuria.

Epigenetic changes are present in many physiological and pathological processes. The N6-methyladenosine (m6A) modification is the most common modification in eukaryotic mRNA. However, the role of m6A modification in diabetic nephropathy (DN) remains elusive. Here, we found that m6A modification was significantly upregulated in the kidney of type 1 and type 2 diabetic mice, which was caused by elevated levels of METTL3. Moreover, METTL3 is increased in podocyte of renal biopsy from patients with DN, which is related to renal damage. METTL3 knockout significantly reduced the inflammation and apoptosis in high glucose (HG)-stimulated podocytes, while its overexpression significantly aggravated these responses in vitro. Podocyte-conditional knockout METTL3 significantly alleviated podocyte injury and albuminuria in streptozotocin (STZ)-induced diabetic mice. Therapeutically, silencing METTL3 with adeno-associated virus serotype-9 (AAV9)-shMETTL3 in vivo mitigated albuminuria and histopathological injury in STZ-induced diabetic mice and db/db mice. Mechanistically, METTL3 modulated Notch signaling via the m6A modification of TIMP2 in an insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2)-dependent manner and exerted pro-inflammatory and pro-apoptotic effects. In summary, this study suggested that METTL3-mediated m6A modification is an important mechanism of podocyte injury in DN. Targeting m6A through the writer enzyme METTL3 is a potential approach for the treatment of DN.

Summary Diabetic kidney disease (DKD) is the leading cause of end-stage renal diseases. DKD does not have efficacious treatment. The cGAS-STING pathway is activated in podocytes at the early stage of kidney dysfunction, which is associated with the activation of STING downstream effectors TBK1 and NF-κB but not IRF3. Lipotoxicity induces mitochondrial damage and mtDNA leakage to the cytosol through Bcl-2 associated X protein (BAX) in podocytes. BAX-mediated mtDNA cytosolic leakage can activate the cGAS-STING pathway in the absence of lipotoxicity and is sufficient to cause podocyte injury. Depletion of cytosolic mtDNA, genetic STING knockdown, or pharmacological inhibition of STING or TBK1 alleviates podocyte injury and improves renal functions in cultured podocytes or mouse models of diabetes and obesity. These results suggest that the mtDNA-cGAS-STING pathway promotes podocyte injury and is a potential therapeutic target for DKD or other obesity-related kidney diseases.

A major cause of proteinuria in lupus nephritis (LN) is podocyte injury, and determining potential therapeutic targets to prevent podocyte injury is important from a clinical perspective in the treatment of LN. CD36 is involved in podocyte injury in several glomerulopathies and was reported to be a vital candidate gene in LN. Here, we determined the role of CD36 in the podocyte injury of LN and the underlying mechanisms. We observed that CD36 and NLRP3 (NLR family pyrin domain containing 3) were upregulated in the podocytes of lupus nephritis patients and MRL/lpr mice with renal impairment. In vitro, CD36, NLRP3 inflammasome, and autophagy were elevated accompanied with increased podocyte injury stimulated by IgG extracted from lupus nephritis patients compared that from healthy donors. Knocking out CD36 with the CRISPR/cas9 system decreased the NLRP3 inflammasome levels, increased the autophagy levels and alleviated podocyte injury. By enhancing autophagy, NLRP3 inflammasome was decreased and podocyte injury was alleviated. These results demonstrated that, in lupus nephritis, CD36 promoted podocyte injury by activating NLRP3 inflammasome and inhibiting autophagy by enhancing which could decrease NLRP3 inflammasome and alleviate podocyte injury.

Although tissue-resident memory T (TRM) cells, a recently identified non-circulating memory T cell population, play a crucial role in mediating local immune responses and protect against pathogens upon local reinfection, the composition, effector function, and specificity of TRM cells in the kidney and their relevance for chronic kidney disease remain unknown. In this study, we found that renal tissue displayed high abundance of tissue-resident lymphocytes and the proportion of CD8+ TRM cells was significantly increased in the kidney from patients and mice with focal segmental glomerulosclerosis (FSGS), diabetic kidney disease (DKD) and lupus nephritis (LN). Mechanistically, IL-15 significantly promoted CD8+ TRM cell formation and activation, thereby promoting podocyte injury and glomerulosclerosis. Interestingly, Sparsentan, the dual angiotensin II (Ang II) receptor and endothelin Type A receptor antagonist, can also reduce TRM cell responses by intervening IL-15 signaling, exploring its new pharmacological functions. Mechanistically, Sparsentan inhibited Ang II or endothelin-1 (ET-1)-mediated IL-15 signaling, thereby further regulating renal CD8+ TRM cell fates. Collectively, our studies provide direct evidence for the pivotal role of renal CD8+ TRM cells in podocyte injury, and further strengthen that targeting TRM cells represents a novel therapeutic strategy for patients with glomerular diseases.

Podocyte pyroptosis is an inflammatory form of cell death associated with Diabetic nephropathy (DN). It is reported that hyposialylated Angiopoietin-like-4 (Angptl4) secreted by glomerular podocytes plays an important role in the formation of proteinuria. Previous study indicated that supplementation of sialic acid precursor N-acetylmannosamine (ManNAc) could inhibit podocyte apoptosis and actin cytoskeleton rearrangement. Nevertheless, whether ManNAc could improve diabetic kidney damage by inhibiting podocyte pyroptosis remains unclear. This study aimed to explore the effect of ManNAc therapy on alleviating diabetic renal injury and podocyte pyroptosis, and its possible mechanism was also figured out. The male 8-week-old C57BL/6 mice were divided into three groups: control group, Streptozocin (STZ)-induced DN group, and ManNAc treated diabetic group. Then, the changes in renal function, renal histopathology, podocyte pyroptosis, reactive oxygen species (ROS), and mitochondrial dysfunction were measured. Herein, we observed that the upregulated expression of Angptl4 was involved in podocyte injury. ManNAc treatment ameliorated podocyte ultrastructure, renal function, and renal histopathology in STZ-induced DN mice. In addition, ManNAc administration attenuated podocyte cell death and suppressed the activation of Nucleotide leukin-rich polypeptide 3 (NLRP3), caspase-1, and interleukin-1β (IL-1β), and the cleavage of gasdermin-D (GSDMD). Moreover, ManNAc inhibited ROS production and restored mitochondrial morphology in vivo and vitro. Further, ManNAc administration significantly alleviated podocyte pyroptosis through inhibiting ROS/NLRP3 signaling pathway. Therefore, these results elucidated that the upregulated expression of Angptl4 was involved in podocyte injury and ManNAc treatment protected against podocyte pyroptosis via inhibiting mitochondrial injury and ROS/NLRP3 signaling pathway in DN mice.

Sestrin2 is identified as a stress-induced protein and could functionate in many aspects. In our study, we investigated the latent impact of Sestrin2 on podocyte injury and its molecular mechanism in vivo and in vitro in diabetic kidney disease (DKD). Sestrin2 was low-expressed in renal biopsies from individuals with DKD, the glomeruli from diabetic mice, and mouse podocytes exposed to high glucose (HG). Sestrin2 overexpression ameliorated HG-induced phenotypic alterations, apoptosis, and oxidative stress in conditionally immortalized mouse podocytes and modulated the activity of Thrombospondin-1 (TSP-1)/transforming growth factor (TGF-β1)/Smad3 pathway in podocytes. Moreover, TSP-1 inhibitor LSKL or TGF-β blocker Pirfenidone arrested podocyte injury induced by HG. Streptozotocin (STZ) was employed to render equivalent diabetes in B6-TgN (CMV-Sestrin2) (TgN) and wild-type (WT) control mice. Sestrin2 alleviated increased levels of 24‐h urinary protein, blood urea nitrogen, serum creatinine and triglyceride, and urine 8-OHdG in diabetic mice. Podocyte phenotypic alterations, increased expression of apoptosis-associated proteins and podocyte loss were observed in WT but not in diabetic TgN mice, as well as oxidative stress. Additionally, TSP-1/TGF-β1/Smad3 signaling pathway was also suppressed in glomeruli of diabetic TgN mice. Thus, Sestrin2 mitigates podocyte injury in DKD via orchestrating TSP-1/TGF-β1/Smad3 pathway, underlining Sestrin2 as a promising therapeutic target for DKD.

Diabetic nephropathy (DN) is one of the primary complications of diabetes. Fisetin is a flavonoid polyphenol that is present in several vegetables and fruits. The present study investigated the mechanisms of fisetin in DN-induced podocyte injury both in vitro and in vivo. The results revealed that fisetin ameliorated high glucose (HG)-induced podocyte injury and streptozotocin (STZ)-induced DN in mice. CDKN1B mRNA expression in the glomeruli of patients with DN decreased based on the Nephroseq dataset, and fisetin reversed CDKN1B expression at mRNA and protein levels in a dose-dependent manner in podocytes and mice kidney tissues. Furthermore, fisetin suppressed the phosphorylation of P70S6K, a downstream target of CDKN1B, activated autophagosome formation, and inhibited Nod-like receptor protein 3 (NLRP3) inflammasomes. Interfering CDKN1B reduced the protective effects of fisetin against high glucose-induced podocyte injury. Molecular docking results revealed a potential interaction between fisetin and CDKN1B. In summary, the present study revealed that fisetin alleviated high glucose-induced podocyte injury and STZ-induced DN in mice by restoring autophagy-mediated CDKN1B/P70S6K pathway and inhibiting NLRP3 inflammasome.

Glomerular podocyte damage is considered to be one of the main mechanisms leading to Diabetic nephropathy (DN). However, the relevant mechanism of podocyte injury is not yet clear. This study aimed to investigate the effect of peroxiredoxin 6 (Prdx6) on the pathogenesis of podocyte injury induced by high glucose (HG). The mouse glomerular podocyte MPC5 was stimulated with 30 nM glucose, and the Prdx6 overexpression vector or specificity protein 1 (Sp1) overexpression vector was transfected into MPC5 cells before the high glucose stimulation. As results, HG treatment significantly reduced the expression of Prdx6 and Sp1 in MPC5 cells. Prdx6 overexpression increased cell viability, while inhibited podocyte death, inflammation and podocyte destruction in HG-induced MPC5 cells. Prdx6 overexpression inhibited HG-induced ROS and MDA production, while restored SOD and GSH activity in MPC5 cells. Prdx6 overexpression also eliminated ferroptosis caused by HG, which was reflected in the suppression of iron accumulation and the increase in SLC7A11 and GPX4 expression. The improvement effect of Prdx6 on HG-induced podocyte damage could be eliminated by erastin. Moreover, Sp1 could bind to the three Sp1 response elements in the Prdx6 promoter, thereby directly regulating the transcriptional activation of Prdx6 in podocytes. Silencing Sp1 could eliminate the effect of Prdx6 on HG-induced podocyte damage. Further, Prdx6 overexpression attenuated renal injuries in streptozotocin-induced DN mice. Sp1-mediated upregulation of Prdx6 expression prevents podocyte injury in diabetic nephropathy via mitigation of oxidative stress and ferroptosis, which may provide new insights for the study of the mechanism of DN.

ABSTRACT Diabetic nephropathy (DN) is a complication of diabetes. This study sought to explore the mechanism of triptolide (TP) in podocyte injury in DN. DN mice were induced by high-fat diet&streptozocin and treated with TP. Fasting blood glucose, 24 h urine microalbumin (UMA), the pathological changes of renal tissues, and ultrastructure of renal podocytes were observed. Podocytes (MPC5) were induced by high-glucose (HG) in vitro and treated with TP or microRNA (miR)-155-5p mimics, with Irbesartan as positive control. Reactive oxygen species (ROS) and levels of oxidative stress (OS) and inflammatory factors in MPC5 were detected. The levels of miR-155-5p, podocyte marker protein Nephrin, and inflammatory factors in mice and MPC5 were detected. The targeting relationship between miR-155-5p and brain-derived neurotrophic factor (BDNF) was verified. The expression levels of BDNF were detected. miR-155-5p mimics and overexpressed (oe)-BDNF plasmids were co-transfected into mouse podocytes treated with HG and TP. TP reduced fasting glucose and 24 h UMA of DN mice, alleviated the pathological damage and podocyte injury, up-regulated Nephrin level, and down-regulated miR-155-5p. TP down-regulated the high expression of miR-155-5p in HG-induced MPC5 cells and inhibited HG-induced OS and inflammatory injury, and the improvement effect of TP was better than Irbesartan. Overexpression of miR-155-5p reversed the protective effect of TP on injured mouse podocytes. miR-155-5p targeted BDNF. oe-BDNF reversed the inhibitory effect of oe-miR-155-5p on TP protection on podocyte injury in mice. Overall, TP up-regulated BDNF by inhibiting miR-155-5p, thus inhibiting OS and inflammatory damage and alleviating podocyte injury in DN mice. Graphical abstract

Background: C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in mediating podocyte dysfunction, proteinuria and glomerulosclerosis. However, the underlying mechanism remains poorly understood. Here we studied the role of β-catenin in mediating CXCR4-triggered podocyte injury. Methods: Mouse models of proteinuric kidney diseases were used to assess CXCR4 and β-catenin expression. We utilized cultured podocytes and glomeruli to delineate the signal pathways involved. Conditional knockout mice with podocyte-specific deletion of CXCR4 were generated and used to corroborate a role of CXCR4/β-catenin in podocyte injury and proteinuria. Results: Both CXCR4 and β-catenin were induced and colocalized in the glomerular podocytes in several models of proteinuric kidney diseases. Activation of CXCR4 by its ligand SDF-1α stimulated β-catenin activation but did not affect the expression of Wnt ligands in vitro. Blockade of β-catenin signaling by ICG-001 preserved podocyte signature proteins and inhibited Snail1 and MMP-7 expression in vitro and ex vivo. Mechanistically, activation of CXCR4 by SDF-1α caused the formation of CXCR4/β-arrestin-1/Src signalosome in podocytes, which led to sequential phosphorylation of Src, EGFR, ERK1/2 and GSK-3β and ultimately β-catenin stabilization and activation. Silencing β-arrestin-1 abolished this cascade of events and inhibited β-catenin in response to CXCR4 stimulation. Podocyte-specific knockout of CXCR4 in mice abolished β-catenin activation, preserved podocyte integrity, reduced proteinuria and ameliorated glomerulosclerosis after Adriamycin injury. Conclusion: These results suggest that CXCR4 promotes podocyte dysfunction and proteinuria by assembling CXCR4/β-arrestin-1/Src signalosome, which triggers a cascade of signal events leading to β-catenin activation.

Podocytes are known to play a determining role in the progression of proteinuric kidney disease. N6-methyladenosine (m6A), as the most abundant chemical modification in eukaryotic mRNA, has been reported to participate in various pathological processes. However, its role in podocyte injury remains unclear. In this study, we observed the elevated m6A RNA levels and the most upregulated METTL14 expression in kidneys of mice with adriamycin (ADR) and diabetic nephropathy. METTL14 was also evidently increased in renal biopsy samples from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy and in cultured human podocytes with ADR or advanced glycation end product (AGE) treatment in vitro. Functionally, we generated mice with podocyte-specific METTL14 deletion, and identified METTL14 knockout in podocytes improved glomerular function and alleviated podocyte injury, characterized by activation of autophagy and inhibition of apoptosis and inflammation, in mice with ADR nephropathy. Similar to the results in vivo, knockdown of METTL14 facilitated autophagy and alleviated apoptosis and inflammation in podocytes under ADR or AGE condition in vitro. Mechanically, we identified METTL14 knockdown upregulated the level of Sirt1, a well-known protective deacetylase in proteinuric kidney diseases, in podocytes with ADR or AGE treatment. The results of MeRIP-qPCR and dual-luciferase reporter assay indicated METTL14 promoted Sirt1 mRNA m6A modification and degradation in injured podocytes. Our findings suggest METTL14-dependent RNA m6A modification contributes to podocyte injury through posttranscriptional regulation of Sirt1 mRNA, which provide a potential approach for the diagnosis and treatment of podocytopathies.

Background: The total flavones of Abelmoschus manihot (TFA), a compound that is extracted from Abelmoschus manihot, has been widely used in China to reduce podocyte injury in diabetic kidney disease (DKD). However, the mechanisms underlying the therapeutic action of this compound have yet to be elucidated. Podocyte pyroptosis is characterized by activation of the NLRP3 inflammasome and plays an important role in inflammation-mediated diabetic kidneys. Regulation of the PTEN/PI3K/Akt pathway is an effective strategy for improving podocyte damage in DKD. Previous research has also shown that N6-methyladenosine (m6A) modification is involved in DKD and that m6A-modified PTEN regulates the PI3K/Akt pathway. In this study, we investigated whether TFA alleviates podocyte pyroptosis and injury by targeting m6A modification-mediated NLRP3-inflammasome activation and PTEN/PI3K/Akt signaling. Methods: We used MPC-5 cells under high glucose (HG) conditions to investigate the key molecules that are involved in podocyte pyroptosis and injury, including activation of the NLRP3 inflammasome and the PTEN/PI3K/Akt pathway. We detected alterations in the levels of three methyltransferases that are involved in m6A modification. We also investigated changes in the levels of these key molecules in podocytes with the overexpression or knockdown of methyltransferase-like (METTL)3. Results: Analysis showed that TFA and MCC950 protected podocytes against HG-induced pyroptosis and injury by reducing the protein expression levels of gasdermin D, interleukin-1β, and interleukin-18, and by increasing the protein expression levels of nephrin, ZO-1, WT1 and podocalyxin. TFA and 740Y-P inhibited activation of the NLRP3 inflammasome via the PI3K/Akt pathway by inhibiting the protein levels of NIMA-related kinase7, NLRP3, ASC, and caspase-1, and by increasing the protein expression levels of p-PI3K and p-Akt. TFA improved pyroptosis and injury in HG-stimulated podocytes by regulating METTL3-dependent m6A modification. Conclusion: Collectively, our data indicated that TFA could ameliorate pyroptosis and injury in podocytes under HG conditions by adjusting METTL3-dependent m6A modification and regulating NLRP3-inflammasome activation and PTEN/PI3K/Akt signaling. This study provides a better understanding of how TFA can protect podocytes in DKD.

No abstract available

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca2+ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca2+-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)–induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca2+ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca2+ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.

Podocyte damage is strongly associated with the progression of diabetic nephropathy. Mitotic catastrophe plays an essential role in accelerating podocyte loss and detachment from the glomerular basement membrane. In the current study, we observed that the long non-coding RNA (lncRNA) MIAT was noticeably upregulated in the plasma and kidney tissues of patients with diabetic nephropathy, and this upregulation was accompanied by higher albumin/creatinine ratios and serum creatinine levels. By generating CRISPR-Cas9 Miat-knockout (KO) mice in vivo and employing vectors in vitro, we found that the depletion of Miat expression significantly restored slit-diaphragm integrity, attenuated foot process effacement, prevented dedifferentiation, and suppressed mitotic catastrophe in podocytes during hyperglycemia. The mechanistic investigation revealed that Miat increased Sox4 expression and subsequently regulated p53 ubiquitination and acetylation, thereby inhibiting the downstream factors CyclinB/cdc2 by enhancing p21cip1/waf1 activity, and that Miat interacted with Sox4 by sponging miR-130b-3p. Additionally, the inhibition of miR-130b-3p with an antagomir in vivo effectively enhanced glomerular podocyte injury and mitotic dysfunction, eventually exacerbating proteinuria. Based on these findings, MIAT may represent a therapeutic target for diabetic nephropathy.

Background and purpose: Mitophagy plays a significant role in the progression of diabetic nephropathy (DN), although the regulatory mechanisms remain unclear. Recently, accumulating evidence demonstrated that impaired mitochondrial function and mitophagy are involved in DN. Here, we are aimed to explore the role of c-Src (Src) and FUNDC1-related mitophagy in the development of DN. Methods: The db/db mice were used to establish a DN mice model. The mice accepted PP2 (Src inhibitor) treatment to study the role of Src in DN. Kidney function was measured via biochemical testing. Renal histopathology and morphometric analysis were conducted via hematoxylin-eosin (HE), periodic acid-Schiff (PAS), Masson’s staining, and transmission electron microscopy (TEM). We measured degree of apoptosis in kidney by TUNEL assay. Indices of mitophagy (LC3 and p62) were evaluated by Western blotting and immunofluorescence. Complementary in vitro assays were conducted using human podocytes subjected to high glucose in combination with PP2 treatment or FUNDC1 small interfering RNAs (siRNAs). Flow cytometry was used to detect the apoptotic cells. Mitochondrial function was evaluated by JC-1 staining. Double immunofluorescence labeling of LC3 and TOMM20 used to assess the degree of mitophagy. Results: Increased Src activation was detected in the kidneys of db/db mice, and its expression was positively correlated with mitochondrial damage, podocyte apoptosis, and renal dysfunction. Inhibition of Src activation with PP2 protected against mitochondrial damage and podocyte apoptosis. In vitro experiments in podocytes established that high glucose increased Src activation, promoting FUNDC1 phosphorylation and inhibiting mitophagy. Consistent with the mouse model, inhibiting Src activity protected podocytes against mitochondrial damage. FUNDC1 silencing negated the actions of PP2, indicating that FUNDC1-mediated mitophagy is downstream pathway of Src. Conclusion: In summary, our data indicated that Src is a culprit factor in diabetic renal damage via suppression of FUNDC1-mediated mitophagy, promoting the development of DN.

Background LncRNA AK044604 (regulator of insulin sensitivity and autophagy, Risa ) and autophagy-related factors Sirt1 and GSK3β play important roles in diabetic nephropathy (DN). In this study, we sought to explore the effect of Risa on Sirt1/GSK3β-induced podocyte injury. Methods Diabetic db/db mice received Risa -inhibition adeno-associated virus (AAV) via tail vein injection, and intraperitoneal injection of lithium chloride (LiCl). Blood, urine, and kidney tissue samples were collected and analyzed at different time points. Immortalized mouse podocyte cells (MPCs) were cultured and treated with Risa -inhibition lentivirus (LV), EX-527, and LiCl. MPCs were collected under different stimulations as noted. The effects of Risa on podocyte autophagy were examined by qRT-PCR, Western blotting analysis, transmission electron microscopy, Periodic Acid-Schiff staining, and immunofluorescence staining. Results Risa and activated GSK3β were overexpressed, but Sirt1 was downregulated in DN mice and high glucose-treated MPCs ( P  < 0.001, db/m vs. db/db, NG or HM vs. HG), which was correlated with poor prognosis. Risa overexpression attenuated Sirt1-mediated downstream autophagy levels and aggravated podocyte injury by inhibiting the expression of Sirt1 ( P  < 0.001, db/m vs. db/db, NG or HM vs. HG). In contrast, Risa suppression enhanced Sirt1-induced autophagy and attenuated podocyte injury, which could be abrogated by EX-527 ( P  < 0.001, db/db +  Risa -AAV vs. db/db, HG +  Risa -LV vs. HG). Furthermore, LiCl treatment could restore GSK3β-mediated autophagy of podocytes ( P  < 0.001, db/db + LiCl vs. db/db, HG + LiCl vs. HG), suggesting that Risa overexpression aggravated podocyte injury by decreasing autophagy. Conclusion Risa could inhibit autophagy by regulating the Sirt1/GSK3β axis, thereby aggravating podocyte injury in DN. Risa may serve as a therapeutic target for the treatment of DN.

Astragaloside II (AS II), a novel saponin purified from Astragalus membranes, has been reported to modulate the immune response, repair tissue injury, and prevent inflammatory response. However, the protective effects of AS II on podocyte injury in diabetic nephropathy (DN) have not been investigated yet. In this study, we aimed to investigate the beneficial effects of AS II on podocyte injury and mitochondrial dysfunction in DN. Diabetes was induced with streptozotocin (STZ) by intraperitoneal injection at 55 mg/kg in rats. Diabetic rats were randomly divided into four groups, namely, diabetic rats and diabetic rats treated with losartan (10 mg·kg−1·d−1) or AS II (3.2 and 6.4 mg·kg−1·d−1) for 9 weeks. Normal Sprague-Dawley rats were chosen as nondiabetic control group. Urinary albumin/creatinine ratio (ACR), biochemical parameters, renal histopathology and podocyte apoptosis, and morphological changes were evaluated. Expressions of mitochondrial dynamics-related and autophagy-related proteins, such as Mfn2, Fis1, P62, and LC3, as well as Nrf2, Keap1, PINK1, and Parkin, were examined by immunohistochemistry, western blot, and real-time PCR, respectively. Our results indicated that AS II ameliorated albuminuria, renal histopathology, and podocyte foot process effacement and podocyte apoptosis in diabetic rats. AS II also partially restored the renal expression of mitochondrial dynamics-related and autophagy-related proteins, including Mfn2, Fis1, P62, and LC3. AS II also increased the expression of PINK1 and Parkin associated with mitophagy in diabetic rats. Moreover, AS II facilitated antioxidative stress ability via increasing Nrf2 expression and decreasing Keap1 protein level. These results suggested that AS II ameliorated podocyte injury and mitochondrial dysfunction in diabetic rats partly through regulation of Nrf2 and PINK1 pathway. These important findings might provide an innovative therapeutic strategy for the treatment of DN.