关于足细胞中氨基酸代谢以及足细胞氨基酸代谢重编程的研究

ABSTRACT Podocytes, highly specialized glomerular epithelial cells, are essential for maintaining the filtration barrier integrity, yet they are particularly susceptible to metabolic stress. Recent advances have identified metabolic reprogramming as a central driver of podocyte injury in diverse glomerular diseases, including diabetic kidney disease and focal segmental glomerulosclerosis. Pathological stimuli, such as hyperglycemia, lipotoxicity, oxidative stress, and inflammatory cytokines, lead to profound alterations in podocyte metabolism, encompassing dysregulation of lipid, glucose, amino acid, and ion handling, as well as activation of immunometabolic pathways. These maladaptive changes result in mitochondrial dysfunction, cytoskeletal disorganization, and inflammatory forms of cell death including pyroptosis and ferroptosis. Mechanistic studies have elucidated the roles of nutrient-sensing pathways (AMPK, mTOR, SIRT1), innate immune sensors (NLRP3, cGAS-STING), and metabolic enzymes (CerS6, GLS2, ODC1) in orchestrating this reprogramming. Emerging evidence supports the therapeutic potential of modulating podocyte metabolism, as exemplified by the renoprotective effects of SGLT2 inhibitors, GLP-1 receptor agonists, PPAR agonists, and targeted inhibitors of inflammasome or lipid pathways. This Review synthesizes recent insights into the structural-metabolic coupling in podocytes, dissects the mechanisms of metabolic derangement in disease contexts, and discusses promising therapeutic strategies aimed at restoring metabolic homeostasis. Understanding the intersection between podocyte metabolism and injury response offers novel avenues for the prevention and treatment of chronic glomerular diseases.

Alteration of metabolic phenotype in podocytes directly contributes to the development of albuminuria and renal injury in conditions of diabetic kidney disease (DKD). This study aimed to identify and evaluate liver receptor homologue‐1 (LRH‐1) as a possible therapeutic target that alleviates glutamine (Gln) metabolism disorders and mitigates podocyte injury in DKD. Metabolomic and transcriptomic analyses were performed to characterize amino acid metabolism changes in the glomeruli of diabetic mice. Next, Western blotting, immunohistochemistry assays, and immunofluorescence staining were used to detect the expression of different genes in vitro and in vivo. Furthermore, Gln and glutamate (Glu) content as well as ATP generation were examined. A decrease in LRH‐1 and glutaminase 2 (GLS2) expression was detected in diabetic podocytes. Conversely, the administration of LRH‐1 agonist (DLPC) upregulated the expression of GLS2 and promoted glutaminolysis, with an improvement in mitochondrial dysfunction and less apoptosis in podocytes compared to those in vehicle‐treated db/db mice. Our study indicates the essential role of LRH‐1 in governing the Gln metabolism of podocytes, targeting LRH‐1 could restore podocytes from diabetes‐induced disturbed glutaminolysis in mitochondria.

Approximately 30-40% of patients with diabetes develop diabetic kidney disease (DKD). Identifying decisive factors for DKD initiation is crucial. Here, we observed that glomerular podocytes in male and female patients with DKD and db/db mice specifically displayed BCAA catabolic defects. Podocyte-specific PP2Cm (a key BCAA catabolism enzyme) knockout or exogenous BCAA supplementation induced DKD phenotypes including podocyte dysfunction/apoptosis, glomerular pathology, and proteinuria in high-fat (HF)-diet-fed male mice. Mechanistically, BCAAs promoted PKM2 depolymerization and inactivation in podocytes. Depolymerized PKM2 suppressed glucose oxidative phosphorylation (OXPHOS), diverting glucose metabolism towards serine biosynthesis and folate metabolism. Depolymerized PKM2 is also co-transported with DDIT3 into the nucleus, acting as a co-transcriptional factor to enhance DDIT3 transcriptional activity, which promotes Chac1 and Trib3 expression and directly inducing podocyte apoptosis. Thus, BCAA catabolic defects may be one of the missing factors that determine DKD initiation. Targeting BCAA catabolism or PKM2 activation is a promising DKD prevention strategy. Previous studies have suggested that branched chain amino acids (BCAA) may contribute to the development of diabetic kidney disease (DKD).Here the authors report that genetic disruption of BCAA catabolism in podocytes or exogenous BCAA supplementation can trigger DKD initiation via rewiring of glucose metabolism in mice.

No abstract available

Metabolic pathways play a critical role in driving differentiation but remain poorly understood in the development of kidney organoids. In this study, parallel metabolite and transcriptome profiling of differentiating human pluripotent stem cells (hPSCs) to multicellular renal organoids revealed key metabolic drivers of the differentiation process. In the early stage, transitioning from hPSCs to nephron progenitor cells (NPCs), both the glutamine and the alanine-aspartate-glutamate pathways changed significantly, as detected by enrichment and pathway impact analyses. Intriguingly, hPSCs maintained their ability to generate NPCs, even when deprived of both glutamine and glutamate. Surprisingly, single cell RNA-Seq analysis detected enhanced maturation and enrichment for podocytes under glutamine-deprived conditions. Together, these findings illustrate a novel role of glutamine metabolism in regulating podocyte development.

Fibroblast activation and proliferation is an essential phase in the progression of renal fibrosis. Despite the recognized significance of glutamine metabolism in cellular growth and proliferation, its precise pathophysiological relevance in renal fibrosis remains uncertain. Therefore, this study aims to investigate the involvement of glutamine metabolism in fibroblast activation and its possible mechanism. Our findings highlight the importance of glutamine metabolism in fibroblast activation and reveal that patients with severe fibrosis exhibit elevated serum glutamine levels and increased expression of kidney glutamine synthetase. Furthermore, the deprivation of glutamine metabolism in vitro and in vivo could inhibit fibroblast activation, thereby ameliorating renal fibrosis. It was also detected that glutamine metabolism is crucial for maintaining mitochondrial function and morphology. These effects may partially depend on the metabolic intermediate α-ketoglutaric acid. Moreover, glutamine deprivation led to upregulated mitochondrial fission in fibroblasts and the activation of the mammalian target of rapamycin / mitochondrial fission process 1 / dynamin-related protein 1 pathway. Thus, these results provide compelling evidence that the modulation of glutamine metabolism initiates the regulation of mitochondrial function, thereby facilitating the progression of renal fibrosis. Consequently, targeting glutamine metabolism emerges as a novel and promising avenue for therapeutic intervention and prevention of renal fibrosis.

Kidney metabolism may be greatly altered in chronic kidney disease. Here we report that arginine metabolism is the most altered in unilateral ureteral obstruction (UUO)-induced fibrosis of the kidneys in metabolomic analysis. Spermidine is the most increased metabolite of arginine. In human glomerulonephritis, the amount of spermidine shown by immunostaining is associated with the amount of fibrosis. In human proximal tubule cells, spermidine induces nuclear factor erythroid 2-related factor 2 (Nrf2). Subsequently, fibrotic signals, such as transforming growth factor β1 secretion, collagen 1 mRNA, and oxidative stress, represented by a decrease in the mitochondrial membrane potential is suppressed by spermidine. UUO kidneys of Arg2 knockout mice show less spermidine and significantly exacerbated fibrosis compared with wild-type mice. Nrf2 activation is reduced in Arg2 knockout UUO kidneys. Spermidine treatment prevents significant fibrotic progression in Arg2 knockout mice. Spermidine is increased in kidney fibrosis, but further increases in spermidine may reduce fibrosis. Arginine metabolism is activated and spermidine levels are increased in kidney fibrosis, resulting in activation of the Nrf2 pathway and subsequent protection against interstitial inflammation and fibrosis in renal tubular epithelial cells.

The Tricarboxylic Acid Cycle (TCA) cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), and combined metabolomics, transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in kidney epithelial cells. Our comparative approach shows that TCAi elicits a convergent rewiring of redox and amino acid metabolism dependent on the activation of ATF4 and the integrated stress response (ISR). Furthermore, we also uncover a divergent metabolic response, whereby acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis. Our work highlights an important interplay between the TCA cycle, redox biology and amino acid homeostasis. Highlights TCA cycle inhibition promotes GSH synthesis and impairs de novo aspartate and proline synthesis Disruption of mitochondrial thiol redox homeostasis phenocopies TCA cycle inhibition by promoting GSH synthesis and impairing proline and aspartate synthesis Acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis TCA cycle inhibition mimics an amino acid deprivation-type response and activates ATF4 via the integrated stress response to maintain redox and amino acid homeostasis

Metabolic reprogramming is emerging as a key pathological contributor to the progression of autosomal dominant polycystic kidney disease (ADPKD), but the molecular mechanisms underlying dysregulated cellular metabolism remain elusive. Here we report that amino acid biosynthesis is reprogrammed in Pkd2-knockout mouse kidneys via a defective PERK-eIF2ɑ-ATF4 pathway. Transcriptomic analysis revealed that the amino acid biosynthesis pathways such as serine, arginine and cysteine were impaired, and associated critical enzymes were downregulated in Pkd2-knockout mouse kidneys. ATF4 and CHOP, transcription factors downstream of the endoplasmic reticulum (ER) stress sensor PERK, were identified as master regulators of these enzymes' expression. PKD2 deficiency impaired the expression of ATF4 and amino acid synthesis enzymes in RCTEC cells under ER stress. Mechanistically, as an ER-resident protein, PKD2 interacts with TBL2, which functions as an adaptor bridging eIF2ɑ to PERK. PKD2 depletion impaired the recruitment of eIF2ɑ to TBL2, thus impeding activation of the PERK-eIF2ɑ-ATF4 pathway and downstream amino acid biosynthesis. These findings illuminate a molecular mechanism linking the PKD2-mediated PERK-eIF2ɑ-ATF4 pathway and amino acid metabolic reprogramming in ADPKD.

Autophagy is important for maintaining normal physiological functions and podocyte cell homeostasis. Amino acid signaling is an important upstream signaling pathway for autophagy regulation. However, the function and the associated mechanism of amino acid signaling in podocyte autophagy is unclear. The present study used normal culture medium and amino acid deprivation medium to culture podocytes in vitro. Multiple methods were utilized to detect autophagic activity including western blot analysis to measure the levels of microtubule-associated protein 1 light chain 3 (LC3) II and beclin1, reverse transcription-quantitative polymerase chain reaction was performed to evaluate the levels of LC3 mRNA and transmission electron microscopy was conducted to observe autophagosomes. In addition, tandem green fluorescent protein (GFP)-monomeric red fluorescent protein (mRFP)-LC3 adenoviruses were employed to transduce podocytes to observe autophagic flux. Furthermore, the present study examined the effects of amino acid signaling on mammalian target of rapamycin (mTOR) activity and the nuclear translocation of transcription factor EB (TFEB), a core regulator of autophagy, using western blotting and immunofluorescence. The results revealed that amino acid starvation promoted the expression of LC3II and beclin1, and increased the number of autophagosomes and autolysosomes. Amino acid starvation inhibited mTOR activity, and promoted nuclear translocation and TFEB activity. Inhibition of TFEB blocked amino acid starvation-induced autophagy. These results indicated that amino acid starvation stimulated podocyte autophagy, and thus suggested that mTOR suppression and TFEB activation may mediate amino acid starvation-induced autophagy in podocytes.

Background/Objectives: The pathogenesis of diabetic kidney disease (DKD) is complex and multifactorial. Because of its complications and reduced number of diagnostic biomarkers, it is important to explore new biomarkers with possible roles in the early diagnosis of DKD. Our study aims to investigate the pattern of previously identified metabolites and their association with biomarkers of endothelial dysfunction, proximal tubule (PT) dysfunction, and podocyte injury. Methods: A total of 110 participants, comprising 20 healthy individuals and 90 patients divided in three groups were enrolled in the study: normoalbuminuria, microalbuminuria, and macroalbuminuria. Untargeted and targeted metabolomic methods were employed to assess urinary and serum biomarkers, as well as indicators of endothelial dysfunction, podocyte damage, and PT dysfunction through ELISA techniques. Results: Our research uncovered specific metabolites that exhibit varying levels across different sub-groups. Notably, glycine serves as a distinguishing factor between group C and the normoalbuminuric group. Furthermore, glycine is correlated with endothelial markers, especially VCAM. We observed a gradual decrease in kynurenic acid levels from group C to group P3; this biomarker also demonstrates an inverse relationship with both p-selectin and VCAM. Additionally, tryptophan levels decline progressively from group C to group P3, accompanied by a negative correlation with p-selectin and VCAM. Urinary tiglylglycine also differentiates among the patient groups, with concentrations decreasing as the condition worsens. It shows a strong positive correlation with nephrin, podocalyxin, KIM1, and NAG. Conclusions: In conclusion, glycine, tiglylglycine, kynurenic acid and tryptophan may be considered putative biomarkers for early diagnosis of DKD and T2DM progression.

Background: Primary membranous nephropathy (pMN) is an autoimmune disease causing abnormalities in the glomerular basement membrane and podocytes, leading to proteinuria. These changes relate to autoantibodies and accumulated activated complement proteins on the glomerulus. Autoantibodies, primarily against phospholipase A2 receptor 1 (PLA2R) found in 80% of pMN patients, are thought to activate the complement system and contribute to pMN. Surprisingly, the predominant PLA2R autoantibodies in pMN patients are IgG4, which doesn’t activate the complement, while the complement-activating IgG1 and IgG3 are less common, questioning the role of PLA2R autoantibodies in pMN pathogenesis. Methods: We isolated monoclonal anti-PLA2R antibodies (mAbs) from pMN patients’ PBMC using single B cell sorting, then amplified the IgG gene for cloning into F293 cell vectors. We confirmed the mAbs’ characteristics via Western blot, ELISA, and a BLI system. These mAbs were found binding to the membrane-bound PLA2R on human podocyte cells through immunocytochemistry. By using a Lentivirus system, we created a PLA2R-overexpressing podocyte line for a disease model. We assessed the mAbs’ ability to trigger complement-dependent cytotoxicity (CDC) using a CellTiter-Glo assay to measure cell viability and complement-induced lysis. Results: We studied PLA2R-reactive IgGs’ role in complement activation in pMN, using a single-cell method to generate 16 anti-PLA2R mAbs from five patients. Our analysis revealed variable binding affinity across mAb groups, independent of binding sites. Using a CDC assay on PLA2R-overexpressing podocytes, we discovered that the combination of CTLD1 with another mAb significantly boosted CDC activation. After modifying the mAbs to IgG4, we found that a single IgG1 mAb was critical for CDC activation, but IgG4 could enhance it. Intriguingly, polyclonal IgGs from pMN patients, not from healthy volunteers, induced CDC. Furthermore, some patients with only CysR-IgG3 showed increased CDC activation with added CTLD1-IgG1 mAb. Conclusions: These results identify an essential role of complement-reactive IgGs, presumably IgG1 and IgG3 synergies between single-domain epitopes CysR and CTLD1, which underlies pMN. Funding

BackgroundSelenoproteins contain selenocysteine (Sec), commonly considered the 21st genetically encoded amino acid. Many selenoproteins, such as the glutathione peroxidases and thioredoxin reductases, protect cells against oxidative stress by functioning as antioxidants and/or through their roles in the maintenance of intracellular redox balance. Since oxidative stress has been implicated in the pathogenesis of diabetic nephropathy, we hypothesized that selenoproteins protect against this complication of diabetes.MethodsC57BL/6 mice that have a podocyte-specific inability to incorporate Sec into proteins (denoted in this paper as PodoTrsp-/-) and control mice were made diabetic by intraperitoneal injection of streptozotocin, or were injected with vehicle. Blood glucose, body weight, microalbuminuria, glomerular mesangial matrix expansion, and immunohistochemical markers of oxidative stress were assessed.ResultsAfter 3 and 6 months of diabetes, control and PodoTrsp-/- mice had similar levels of blood glucose. There were no differences in urinary albumin/creatinine ratios. Periodic acid-Schiff staining to examine mesangial matrix expansion also demonstrated no difference between control and PodoTrsp-/- mice after 6 months of diabetes, and there were no differences in immunohistochemical stainings for nitrotyrosine or NAD(P)H dehydrogenase, quinone 1.ConclusionLoss of podocyte selenoproteins in streptozotocin diabetic C57BL/6 mice does not lead to increased oxidative stress as assessed by nitrotyrosine and NAD(P)H dehydrogenase, quinone 1 immunostaining, nor does it lead to worsening nephropathy.

No abstract available

Diabetic nephropathy (DN) is a severe complication of diabetes, characterized by changes in kidney structure and function. The natural product rosmarinic acid (RA) has demonstrated therapeutic effects, including anti-inflammation and anti-oxidative-stress, in renal damage or dysfunction. In this study, we characterized the heterogeneity of the cellular response in kidneys to DN-induced injury and RA treatment at single cell levels. Our results demonstrated that RA significantly alleviated renal tubular epithelial injury, particularly in the proximal tubular S1 segment and on glomerular epithelial cells known as podocytes, while attenuating the inflammatory response of macrophages, oxidative stress, and cytotoxicity of natural killer cells. These findings provide a comprehensive understanding of the mechanisms by which RA alleviates kidney damage, oxidative stress, and inflammation, offering valuable guidance for the clinical application of RA in the treatment of DN.

Introduction Diabetic nephropathy (DN), a major complication of diabetes, presents with poor clinical outcomes and affects patients throughout their lifetime. α-Methyltryptophan (α-MT) is a blocker of the amino acid transporter. SLC6A14 and also an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1). Methods In this study, we employed a nuclear magnetic resonance-based metabolomic approach to investigate the therapeutic effects of α-MT in a db/db mouse model of DN and explore the underlying molecular mechanisms. Results The results of the study demonstrated that α-MT significantly reduced the urinary excretion of albumin and creatinine, improved kidney function, and decreased renal fibrosis in db/db mice. Metabolomic analyses of kidney tissues and urine samples indicated that db/db mice displayed increased activity of the enzyme IDO1, and alongside pronounced metabolic disturbances. These disturbances are chiefly characterized by alterations in amino acid metabolism, energy production pathways, membrane biochemical features, and nicotinamide metabolism, all of which have been implicated in mTOR signaling and apoptotic pathways. Discussion Administration of α-MT to db/db mice showed evidence of IDO1 inhibition and rectification of metabolic dysfunctions with concurrent suppression of mTOR signaling and apoptosis. These findings highlight the potential of α-MT as a promising therapeutic agent for diabetic nephropathy.

BACKGROUND 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. METHODS 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. RESULTS 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 Prevotella and Bifidobacterium, which increased fecal and systemic SCFA concentrations. Fiber reduced expression of genes encoding inflammatory cytokines, chemokines, and fibrosis-promoting proteins in diabetic kidneys. SCFA-treated diabetic mice were protected from nephropathy, but not in the absence of GPR43 or GPR109A. In vitro, SCFAs modulated inflammation in renal tubular cells and podocytes under hyperglycemic conditions. CONCLUSIONS 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.

Abstract Aim Podocyte injury plays an important role in diabetic nephropathy (DN), yet the underlying molecular mechanisms of podocyte injury in DN is not clear. Here, we investigated the role of activating transcription factor 4 (ATF4) and HO-1 in DN-induced podocyte injury. Methods Protein expression was measured by western blotting (WB) and immunofluorescence. Cellular apoptosis was quantified by flow cytometry. ATF4 siRNA knockdown and HO-1 overexpression in podocyte were employed to evaluate the role of ER stress in DN-induced apoptosis and autophagy response. Urinary protein levels, nephrin expression, serum creatinine and BUN were evaluated and glomerulosclerosis was quantified by Periodic Acid-Schiff staining. Results Expression of ATF4 was increased in podocytes exposed to serum from DN mice. ATF4 knockdown enhanced DN-induced podocyte apoptosis. HO-1 overexpression reduced the decline of DN-induced podocyte autophagy and inhibited apoptosis and the beneficial effects of HO-1 overexpression in DN were blocked by ATF4 knockdown. The diabetic mice were significantly ameliorated by HO-1 agonist hemin treatment. Conclusions ATF4 induces autophagy by enhancing the expression of HO-1, and inhibits podocyte apoptosis in DN. Treatment with the HO-1 agonist reduced proteinuria, apoptosis, and enhanced autophagy response, and thus improved renal function in DN mice.

No abstract available

D-Amino acids, the enantiomers of L-amino acids, are increasingly recognized as novel biomarkers. Although the amounts of D-amino acids are usually very trace in human, some of them have sporadically been detected in blood from patients with kidney diseases. This study examined whether multiple chiral amino acids would be associated with kidney functions, comorbidities, and prognosis of chronic kidney disease (CKD) by enantioselective analyses of all chiral amino acids with a micro-two-dimensional high-performance liquid chromatograph (2D-HPLC)-based analytical platform. 16 out of 21 D-amino acids were detected in plasma from 108 CKD patients in a longitudinal cohort. The levels of D-Ser, D-Pro, and D-Asn were strongly associated with kidney function (estimated glomerular filtration ratio), the levels of D-Ala and D-Pro were associated with age, and the level of D-Asp and D-Pro were associated with the presence of diabetes mellitus. D-Ser and D-Asn were significantly associated with the progression of CKD in mutually-adjusted Cox regression analyses; the risk of composite end point (developing to ESKD or death before ESKD) was elevated from 2.7- to 3.8-fold in those with higher levels of plasma D-Ser and D-Asn. These findings identified chiral amino acids as potential biomarkers in kidney diseases.

No abstract available

Although gut dysbiosis had been demonstrated to be an important factor affecting hyperuricemia (HUA) and gout, little is known for its potential mechanistic connections. In this study, Uox-KO mice model that with spontaneously developed pronounced HUA and urate nephropathy was used to explore the pathophysiologic mechanism of microbiota alterations in HUA and gout with integrated multi-omics analysis. 16S rRNA gene sequencing was performed to characterize the characteristic bacteria, and untargeted LC/MS analysis was applied to reveal the featured metabolites. Our results showed there was a significant shift in gut microbiota composition and function in Uox-KO mice compared to WT mice and apparent metabolomics differences between the two groups. Among them, amino acids metabolism appears to play a critical role. Correlation analysis further revealed that the characteristic metabolites were strongly influenced by the discrepant bacterial genera. Furthermore, impairment of intestinal integrity and profound alterations in the profile of solute carrier family resulted in dysregulation of amino acids transportation, which subsequently impacted serum uric acid level and CD4+ Th17 driven inflammation. Together, these data indicate that gut dysbiosis promotes purine metabolism disorder and inflammation in Uox-KO mice. Remodeling the gut microbiota is a promising strategy to combat HUA and gout.

Background: Metabolomics is useful in elucidating the progression of diabetes; however, the follow-up changes in metabolomics among health, diabetes mellitus, and diabetic kidney disease (DKD) have not been reported. This study was aimed to reveal metabolomic signatures in diabetes development and progression. Methods: In this cross-sectional study, we compared healthy (n = 30), type 2 diabetes mellitus (T2DM) (n = 30), and DKD (n = 30) subjects with the goal of identifying gradual altering metabolites. Then, a prospective study was performed in T2DM patients to evaluate these altered metabolites in the onset of DKD. Logistic regression was conducted to predict rapid eGFR decline in T2DM subjects using altered metabolites. The prospective association of metabolites with the risk of developing DKD was examined using logistic regression and restricted cubic spline regression models. Results: In this cross-sectional study, impaired amino acid metabolism was the main metabolic signature in the onset and development of diabetes, which was characterized by increased N-acetylaspartic acid, L-valine, isoleucine, asparagine, betaine, and L-methionine levels in both the T2DM and DKD groups. These candidate metabolites could distinguish the DKD group from the T2DM group. In the follow-up study, higher baseline levels of L-valine and isoleucine were significantly associated with an increased risk of rapid eGFR decline in T2DM patients. Of these, L-valine and isoleucine were independent risk factors for the development of DKD. Notably, nonlinear associations were also observed for higher baseline levels of L-valine and isoleucine, with an increased risk of DKD among patients with T2DM. Conclusion: Amino acid metabolism was disturbed in diabetes, and N-acetylaspartic acid, L-valine, isoleucine, asparagine, betaine, and L-methionine could be biomarkers for the onset and progression of diabetes. Furthermore, high levels of L-valine and isoleucine may be risk factors for DKD development.

No abstract available

Amino acids have crucial roles in central metabolism, both anabolic and catabolic. To elucidate these roles, steady-state concentrations of amino acids alone are insufficient, as each amino acid participates in multiple pathways and functions in a complex network, which can also be compartmentalized. Stable Isotope-Resolved Metabolomics (SIRM) is an approach that uses atom-resolved tracking of metabolites through biochemical transformations in cells, tissues, or whole organisms. Using different elemental stable isotopes to label multiple metabolite precursors makes it possible to resolve simultaneously the utilization of these precursors in a single experiment. Conversely, a single precursor labeled with two (or more) different elemental isotopes can trace the allocation of e.g. C and N atoms through the network. Such dual-label experiments however challenge the resolution of conventional mass spectrometers, which must distinguish the neutron mass differences among different elemental isotopes. This requires ultrahigh resolution Fourier transform mass spectrometry (UHR-FTMS). When combined with direct infusion nano-electrospray ion source (nano-ESI), UHR-FTMS can provide rapid, global, and quantitative analysis of all possible mass isotopologues of metabolites. Unfortunately, very low mass polar metabolites such as amino acids can be difficult to analyze by current models of UHR-FTMS, plus the high salt content present in typical cell or tissue polar extracts may cause unacceptable ion suppression for sources such as nano-ESI. Here we describe a modified method of ethyl chloroformate (ECF) derivatization of amino acids to enable rapid quantitative analysis of stable isotope labeled amino acids using nano-ESI UHR-FTMS. This method showed excellent linearity with quantifiable limits in the low nanomolar range represented in microgram quantities of biological specimens, which results in extracts with total analyte abundances in the low to sub-femtomole range. We have applied this method to profile amino acids and their labeling patterns in 13C and 2H doubly labeled PC9 cell extracts, cancerous and non-cancerous tissue extracts from a lung cancer patient and their protein hydrolysates as well as plasma extracts from mice fed with a liquid diet containing 13C6-glucose (Glc). The multi-element isotopologue distributions provided key insights into amino acid metabolism and intracellular pools in human lung cancer tissues in high detail. The 13C labeling of Asp and Glu revealed de novo synthesis of these amino acids from 13C6-Glc via the Krebs cycle, specifically the elevated level of 13C3-labeled Asp and Glu in cancerous versus non-cancerous lung tissues was consistent with enhanced pyruvate carboxylation. In addition, tracking the fate of double tracers, (13C6-Glc + 2H2-Gly or 13C6-Glc + 2H3-Ser) in PC9 cells clearly resolved pools of Ser and Gly synthesized de novo from 13C6-Glc (13C3-Ser and 13C2-Gly) versus Ser and Gly derived from external sources (2H3-Ser, 2H2-Gly). Moreover the complex 2H labeling patterns of the latter were results of Ser and Gly exchange through active Ser-Gly one-carbon metabolic pathway in PC9 cells.

No abstract available

Modern mass spectrometry-based metabolomics is a key technology for biomedicine, enabling discovery and quantification of a wide array of biomolecules critical for human physiology. Yet, only a fraction of human metabolites have been structurally determined, and the majority of features in typical metabolomics data remain unknown. To date, metabolite identification relies largely on comparing MS2 fragmentation patterns against known standards, related compounds or predicted spectra. Here, we propose an orthogonal approach to identification of endogenous metabolites, based on mass isotopomer distributions (MIDs) measured in an isotope-labeled reference material. We introduce a computational measure of pairwise distance between metabolite MIDs that allows identifying novel metabolites by their similarity to previously known peaks. Using cell material labeled with 20 individual 13C tracers, this method identified 62% of all unknown peaks, including previously never seen metabolites. Importantly, MID-based identification is highly complementary to MS2-based methods in that MIDs reflect the biochemical origin of metabolites, and therefore also yields insight into their synthesis pathways, while MS2 spectra mainly reflect structural features. Accordingly, our method performed best for small molecules, while MS2-based identification was stronger on lipids and complex natural products. Among the metabolites discovered was trimethylglycyl-lysine, a novel amino acid derivative that is altered in human muscle tissue after intensive lifestyle treatment. MID-based annotation using isotope-labeled reference materials enables identification of novel endogenous metabolites, extending the reach of mass spectrometry-based metabolomics.

We report the method of LC/ESI-MS analysis of underivatized amino acids with corresponding mass spectrum and fragmentation patterns. Diagnostic product ions determined by mass spectrometry can support the qualitative identification and quantitative estimation of individual amino acids. Therefore, the optimization of chromatographic separation using the ion-pairing reagent (i.e., Nonafluoropentanoic acid, NFPA) is useful for the evaluation of target amino acid and for further compound-specific nitrogen isotope studies of amino acids.

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.

The aim of this study is to analyze the effect of serum metabolites on diabetic nephropathy (DN) and predict the prevalence of DN through a machine learning approach. The dataset consists of 548 patients from April 2018 to April 2019 in Second Affiliated Hospital of Dalian Medical University (SAHDMU). We select the optimal 38 features through a Least absolute shrinkage and selection operator (LASSO) regression model and a 10-fold cross-validation. We compare four machine learning algorithms, including eXtreme Gradient Boosting (XGB), random forest, decision tree and logistic regression, by AUC-ROC curves, decision curves, calibration curves. We quantify feature importance and interaction effects in the optimal predictive model by Shapley Additive exPlanations (SHAP) method. The XGB model has the best performance to screen for DN with the highest AUC value of 0.966. The XGB model also gains more clinical net benefits than others and the fitting degree is better. In addition, there are significant interactions between serum metabolites and duration of diabetes. We develop a predictive model by XGB algorithm to screen for DN. C2, C5DC, Tyr, Ser, Met, C24, C4DC, and Cys have great contribution in the model, and can possibly be biomarkers for DN.

Isotope labeling is a fundamental staple for the study of cellular metabolism and protein function. The conventional techniques that allow resolution and identification of isotopically-labeled biomarkers, such as mass spectrometry and infrared spectroscopy, are macroscopic in nature and have the disadvantage of requiring relatively large quantities of material and lacking spatial resolution. Here, we record the vibrational spectra of an α-amino acid, L-alanine, using spatially-resolved monochromated electron energy loss spectroscopy (EELS) to directly resolve carbon-site-specific isotopic labels in a scanning transmission electron microscope. The EELS is acquired in aloof mode, meaning the probe is positioned away from the sample (~20 nm) sparing the sensitive biomolecule from the high-energy excitations, while the vibrational modes are investigated. An isotopic red-shift of 5.3 meV was obtained for the C=O stretching mode in the carboxylic acid group for 13C-enriched L-alanine when compared with naturally occurring 12C L-alanine, which is confirmed by macroscopic infrared spectroscopy measurements and theoretical calculations. The EELS experiments presented here are the first demonstration of non-destructive resolution and identification of isotopically-labeled amino acids in the electron microscope, opening a new door for the study of biological matter at the nanoscale.

Chronic kidney disease (CKD) is a major health issue, with podocyte injury with senescence playing a central role in glomerulosclerosis. This study investigates the link between glycolysis-derived serine metabolism and podocyte injury with senescence, focusing on the role of phosphoglycerate kinase 1 (PGK1) in the regulation of L-serine synthesis and podocyte homeostasis. Using in vivo and in vitro models, we examined the effects of angiotensin II (Ang II)-induced metabolic dysregulation on serine metabolism and its impact on podocyte function. The results demonstrate that Ang II downregulates PGK1 expression through the transcription factor FOXA1, leading to reduced L-serine biosynthesis, mitochondrial dysfunction, and increased cellular senescence in podocytes. Supplementing with L-serine or enhancing PGK1 expression in podocytes alleviated these pathological changes, restored mitochondrial function, and reduced senescence-associated phenotypes in CKD mouse models. Moreover, PGK1 was found to interact with keratin, type II cytoskeletal 1 (KRT1), stabilizing the cytoskeletal integrity of podocytes. These findings identify a novel metabolic pathway linking glycolysis, serine metabolism, and podocyte injury with senescence, suggesting that targeting the PGK1-serine axis may offer therapeutic potential for slowing podocyte senescence and CKD progression.

Inflammatory kidney disease is a major clinical problem that can result in end-stage renal failure. In this article, we show that Ab-mediated inflammatory kidney injury and renal disease in a mouse nephrotoxic serum nephritis model was inhibited by amino acid metabolism and a protective autophagic response. The metabolic signal was driven by IFN-γ-mediated induction of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme activity with subsequent activation of a stress response dependent on the eIF2α kinase general control nonderepressible 2 (GCN2). Activation of GCN2 suppressed proinflammatory cytokine production in glomeruli and reduced macrophage recruitment to the kidney during the incipient stage of Ab-induced glomerular inflammation. Further, inhibition of autophagy or genetic ablation of Ido1 or Gcn2 converted Ab-induced, self-limiting nephritis to fatal end-stage renal disease. Conversely, increasing kidney IDO1 activity or treating mice with a GCN2 agonist induced autophagy and protected mice from nephritic kidney damage. Finally, kidney tissue from patients with Ab-driven nephropathy showed increased IDO1 abundance and stress gene expression. Thus, these findings support the hypothesis that the IDO-GCN2 pathway in glomerular stromal cells is a critical negative feedback mechanism that limits inflammatory renal pathologic changes by inducing autophagy.

LAT3 is a Na+-independent neutral l-amino acid transporter recently isolated from a human hepatocellular carcinoma cell line. Although liver, skeletal muscle, and pancreas are known to express LAT3, the tissue distribution and physiologic function of this transporter are not completely understood. Here, we observed that glomeruli express LAT3. Immunofluorescence, confocal microscopy, and immunoelectron microscopy revealed that LAT3 localizes to the apical plasma membrane of podocyte foot processes. In mice, starvation upregulated glomerular LAT3, phosphorylated AKT1, reconstituted the actin network, and elongated foot processes. In the fetal kidney, we observed intense LAT3 expression at the capillary loops stage of renal development. Finally, zebrafish morphants lacking lat3 function showed collapsed glomeruli with thickened glomerular basement membranes. Permeability studies of the glomerular filtration barrier in these zebrafish morphants demonstrated a disruption of selective glomerular permeability. Our data suggest that LAT3 may play a crucial role in the development and maintenance of podocyte structure and function by regulating protein synthesis and the actin cytoskeleton.

It has recently been shown that formation of podocyte foot processes is dependent on a constant source of lipids and proteins (Simons M, Saffrich R, Reiser J, and Mundel P. J Am Soc Nephrol 10: 1633-1639, 1999). Here we characterize amino acid transport mechanisms in differentiated cultured podocytes and investigate whether it may be disturbed during podocyte injury. RT-PCR studies detected mRNA for transporters of neutral amino acids (ASCT1, ASCT2, and B(0/+)), cationic AA (CAT1 and CAT3), and anionic AA (EAAT2 and EAAT3). Alanine (Ala), asparagine, cysteine (Cys), glutamine (Gln), glycine (Gly), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), glutamic acid (Glu), arginine (Arg), and histidine (His) depolarized podocytes and increased their whole cell conductances. Depletion of extracellular Na(+) completely inhibited the depolarization induced by Ala, Gln, Glu, Gly, Leu, and Pro and decreased the depolarization induced by Arg and His, indicating the presence of Na(+)-dependent amino acid transport. Incubation of podocytes with 100 microg/ml puromycin aminonucleoside for 24 h significantly attenuated the effects induced by the various amino acids by approximately 70%. The data indicate the existence of different amino acid transporter systems in podocytes. Alteration of amino acid transport may participate in podocyte injury and disturbed foot process formation.

Cystinosis is a rare, incurable lysosomal storage disease caused by mutations in the CTNS gene encoding the cystine transporter cystinosin, which leads to lysosomal cystine accumulation in all cells of the body. Patients with cystinosis display signs of podocyte damage characterized by extensive loss of podocytes into the urine at early disease stages, glomerular proteinuria, and the development of focal segmental glomerulosclerosis (FSGS) lesions. Although standard treatment with cysteamine decreases cellular cystine levels, it neither reverses glomerular injury nor prevents the loss of podocytes. Thus, pathogenic mechanisms other than cystine accumulation are involved in podocyte dysfunction in cystinosis. We used immortalized patient-derived cystinosis, healthy, and CTNS knockdown podocytes to investigate podocyte dysfunction in cystinosis. The results were validated in our newly in-house developed fluorescent ctns In the current study, we discovered that cystinosis podocytes demonstrate increased ferroptotic cell death caused by mitochondrial reactive oxygen species (ROS)-driven membrane lipid peroxidation. Moreover, cystinosis cells present a fragmented mitochondrial network with impaired tricarboxylic acid cycle (TCA) cycle and energy metabolism. Targeting mitochondrial ROS and lipid peroxidation improved podocyte function in vitro and rescued proteinuria in vivo in cystinosis zebrafish larvae. Mitochondrial ROS contribute to podocyte injury in cystinosis by driving lipid peroxidation and ferroptosis, which in turn lead to podocyte detachment. This finding adds cystinosis to the list of podocytopathies associated with mitochondrial dysfunction. The identified mechanisms reveal new therapeutic targets and highlight lipid peroxidation as an exploitable vulnerability of cystinosis podocytes.

Nutrients such as glucose, amino acids and lipids are fundamental sources for the maintenance of essential cellular processes and homeostasis in all organisms. The nutrient-sensing kinases mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are expressed in many cell types and have key roles in the control of cell growth, proliferation, differentiation, metabolism and survival, ultimately contributing to the physiological development and functions of various organs, including the kidney. Dysregulation of these kinases leads to many human health problems, including cancer, neurodegenerative diseases, metabolic disorders and kidney diseases. In the kidney, physiological levels of mTOR and AMPK activity are required to support kidney cell growth and differentiation and to maintain kidney cell integrity and normal nephron function, including transport of electrolytes, water and glucose. mTOR forms two functional multi-protein kinase complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Hyperactivation of mTORC1 leads to podocyte and tubular cell dysfunction and vulnerability to injury, thereby contributing to the development of chronic kidney diseases, including diabetic kidney disease, obesity-related kidney disease and polycystic kidney disease. Emerging evidence suggests that targeting mTOR and/or AMPK could be an effective therapeutic approach to controlling or preventing these diseases.

Compromised glycolysis in podocytes contributes to the initiation of diabetic kidney disease (DKD). Podocyte injury is characterized by cytoskeletal remodeling and foot process fusion. Compromised glycolysis in diabetes likely leads to switch of energy supply in podocyte. However, the underlying mechanism by which disturbed energy supply in podocytes affects the cytoskeletal structure of podocytes remains unclear. Metabolomic and transcriptomic analyses were performed on the glomeruli of db/db mice to examine the catabolism of glucose, fatty, and amino acids. Ornithine catabolism was targeted in db/db and podocyte-specific pyruvate kinase M2 knockout (PKM2-podoKO) mice. In vitro, expression of ornithine decarboxylase (ODC1) was modulated to investigate the effect of ornithine catabolism on mammalian target of rapamycin (mTOR) signaling and cytoskeletal remodeling in cultured podocytes. Multi-omic analyses of the glomeruli revealed that ornithine metabolism was enhanced in db/db mice compared with that in db/m mice under compromised glycolytic conditions. Additionally, ornithine catabolism was exaggerated in podocytes of diabetic PKM2-podoKO mice compared with that in diabetic PKM2 These findings demonstrate that compromised glycolysis in podocytes under diabetic conditions enhances ornithine catabolism. The metabolites of ornithine catabolism contribute to mTOR signaling activation via Rheb and cytoskeletal remodeling in podocytes in DKD.

Diabetic kidney disease (DKD) is a main cause of end-stage renal disorder, yet its pathogenesis is still incompletely understood. Ferroptosis has been implicated in DKD progression; however, its regulatory mechanisms remain unclear. Phosphoglycerate dehydrogenase (PHGDH), a key enzyme in serine biosynthesis, has been minimally studied in DKD development. To elucidate the roles of PHGDH in ferroptosis and its underlying mechanism in podocytes and DKD, we conducted this study. Our findings demonstrate that PHGDH deficiency exacerbates podocyte injury, characterized by cytoskeletal disorganization, and promotes ferroptosis in both podocytes and DKD renal tissues. Conversely, PHGDH overexpression alleviates podocyte injury, reduces ferroptosis, and improves renal function in DKD mice. Mechanistically, we identified that PHGDH mediates ferroptosis by regulating SLC7A11 expression, a key ferroptosis-related protein. Specifically, PHGDH stabilizes Y-box binding protein 1 (YB1) by inhibiting its K48-linked ubiquitination and degradation, thereby enhancing SLC7A11 mRNA stability and expression. In conclusion, our study reveals a novel PHGDH-YB1-SLC7A11 regulatory axis that is responsible for suppressing ferroptosis and protecting against podocyte and renal injury in DKD. Our findings shed new light into the molecular mechanism underlying ferroptosis in DKD and highlight PHGDH as a therapeutic target for mitigating ferroptosis-mediated renal damage.

l-Glutamate (l-Glu) plays an essential role in the central nervous system (CNS) as an excitatory neurotransmitter, and exerts its effects by acting on a large number of ionotropic and metabotropic receptors. These receptors are also expressed in several peripheral tissues, including the kidney. This review summarizes the general properties of ionotropic and metabotropic l-Glu receptors, focusing on N-methyl-d-aspartate (NMDA) and Group 1 metabotropic glutamate receptors (mGluRs). NMDA receptors are expressed in the renal cortex and medulla, and appear to play a role in the regulation of renal blood flow, glomerular filtration, proximal tubule reabsorption and urine concentration within medullary collecting ducts. Sustained activation of NMDA receptors induces Ca(2+) influx and oxidative stress, which can lead to glomerulosclerosis, for example in hyperhomocysteinemia. Group 1 mGluRs are expressed in podocytes and probably in other cell types. Mice in which these receptors are knocked out gradually develop albuminuria and glomerulosclerosis. Several endogenous agonists of l-Glu receptors, which include sulfur-containing amino acids derived from l-homocysteine, and quinolinic acid (QA), as well as the co-agonists glycine and d-serine, are present in the circulation at concentrations capable of robustly activating ionotropic and metabotropic l-Glu receptors. These endogenous agonists may also be secreted from renal parenchymal cells, or from cells that have migrated into the kidney, by exocytosis or by transporters such as system x(-)(c), or by transporters involved in ammonia secretion. l-Glu receptors may be useful targets for drug therapy, and many selective orally-active compounds exist for investigation of these receptors as potential drug targets for various kidney diseases.

Cordycepin (CRD) has been identified to alleviate diabetes-induced injuries and complications including diabetic nephropathy (DN). Here, this work focused on probing the specific effects and potential mechanisms of CRD on DN progression. High glucose (HG)-induced mouse podocyte cell line (MPC5) was used for in vitro functional analyses. Cell proliferation and apoptosis were determined using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry, respectively. ELISA analysis detected inflammatory factors. Cell ferroptosis was assessed by measuring the levels of Fe2+, glutathione, reactive oxygen species, and malonaldehyde. CRD treatment suppressed HG-induced apoptosis, inflammation, and ferroptosis in podocytes. CRD treatment elevated SLC7A11 and GPX4 expression in HG-treated podocytes. The overexpression of SLC7A11 or GPX4 suppressed HG-evoked apoptosis, inflammation, and ferroptosis in podocytes. Moreover, the silencing of SLC7A11 or GPX4 abolished the protective effects of CRD on HG-treated podocytes. Moreover, CRD ameliorated renal structure injury and inflammation in STZ-induced diabetic mice by modulating SLC7A11 or GPX4 expression. Cordycepin suppressed HG-induced apoptosis, inflammation, and ferroptosis in podocytes in vitro, and ameliorated renal injury and inflammation in STZ-induced diabetic mice by activating the SLC7A11/GPX4 pathway.

Hypertension is a persistent epidemic across the developed world that is closely associated with kidney disease. Here, we applied a metabolomic, phosphoproteomic, and proteomic strategy to analyze the effect of hypertensive insults on kidneys. Our data revealed the metabolic aspects of hypertension-induced glomerular sclerosis, including lipid breakdown at early disease stages and activation of anaplerotic pathways to regenerate energy equivalents to counter stress. For example, branched-chain amino acids and proline, required for collagen synthesis, were depleted in glomeruli at early time points. Furthermore, indicators of metabolic stress were reflected by low amounts of ATP and NADH and an increased abundance of oxidized lipids derived from lipid breakdown. These processes were specific to kidney glomeruli where metabolic signaling occurred through mTOR and AMPK signaling. Quantitative phosphoproteomics combined with computational modeling suggested that these processes controlled key molecules in glomeruli and specifically podocytes, including cytoskeletal components and GTP-binding proteins, which would be expected to compete for decreasing amounts of GTP at early time points. As a result, glomeruli showed increased expression of metabolic enzymes of central carbon metabolism, amino acid degradation, and lipid oxidation, findings observed in previously published studies from other disease models and patients with glomerular damage. Overall, multilayered omics provides an overview of hypertensive kidney damage and suggests that metabolic or dietary interventions could prevent and treat glomerular disease and hypertension-induced nephropathy.

The SLC43 family is composed of only three genes coding for the plasma membrane facilitator system l amino acid transporters LAT3 (SLC43A1; TC 2.A.1.44.1) and LAT4 (SLC43A2; TC 2.A.1.44.2), and the orphan protein EEG1 (SLC43A3; TC 2.A.1.44.3). Besides the known mechanism of transport of LAT3 and LAT4, their physiological roles still remain quite obscure. Morphants suggested a role of LAT3 in renal podocyte development in zebrafish. Expression in liver and skeletal muscle, and up-regulation by starvation suggest a role of LAT3 in the flux of branched-chain amino acids (BCAAs) from liver and skeletal muscle to the bloodstream. Finally, LAT3 is up-regulated in androgen-dependent cancers, suggesting a role in mTORC1 signaling in this type of tumors. In addition, LAT4 might contribute to the transfer of BCAAs from mother to fetus. Unfortunately, the EEG1 mouse model (EEG1(Y221∗)) described here has not yet offered a clue to the physiological role of this orphan protein.

Glomerular podocytes act as a part of the filtration barrier in the kidney. The activity of this filter is regulated by ionotropic and metabotropic glutamate receptors. Adjacent podocytes can potentially release glutamate into the intercellular space; however, little is known about how podocytes release glutamate. Here, we demonstrated vesicular glutamate transporter 3 (VGLUT3)-dependent glutamate release from podocytes. Immunofluorescence analysis revealed that rat glomerular podocytes and an immortal mouse podocyte cell line (MPC) express VGLUT1 and VGLUT3. Consistent with this finding, quantitative RT-PCR revealed the expression of VGLUT1 and VGLUT3 mRNA in undifferentiated and differentiated MPCs. In addition, the exocytotic proteins vesicle-associated membrane protein 2, synapsin 1, and synaptophysin 1 were present in punctate patterns and colocalized with VGLUT3 in MPCs. Interestingly, approximately 30% of VGLUT3 colocalized with VGLUT1. By immunoelectron microscopy, VGLUT3 was often observed around clear vesicle-like structures in differentiated MPCs. Differentiated MPCs released glutamate following depolarization with high potassium levels and after stimulation with the muscarinic agonist pilocarpine. The depletion of VGLUT3 in MPCs by RNA interference reduced depolarization-dependent glutamate release. These results strongly suggest that VGLUT3 is involved in glutamatergic signalling in podocytes and may be a new drug target for various kidney diseases.

Oxidative metabolism in mitochondria regulates cellular differentiation and gene expression through intermediary metabolites and reactive oxygen species. Its role in kidney development and pathogenesis is not completely understood. Here we inactivated ubiquinone-binding protein QPC, a subunit of mitochondrial complex III, in two types of kidney progenitor cells to investigate the role of mitochondrial electron transport in kidney homeostasis. Inactivation of QPC in sine oculis-related homeobox 2 (SIX2)-expressing cap mesenchyme progenitors, which give rise to podocytes and all nephron segments except collecting ducts, resulted in perinatal death from severe kidney dysplasia. This was characterized by decreased proliferation of SIX2 progenitors and their failure to differentiate into kidney epithelium. QPC inactivation in cap mesenchyme progenitors induced activating transcription factor 4-mediated nutritional stress responses and was associated with a reduction in kidney tricarboxylic acid cycle metabolites and amino acid levels, which negatively impacted purine and pyrimidine synthesis. In contrast, QPC inactivation in ureteric tree epithelial cells, which give rise to the kidney collecting system, did not inhibit ureteric differentiation, and resulted in the development of functional kidneys that were smaller in size. Thus, our data demonstrate that mitochondrial oxidative metabolism is critical for the formation of cap mesenchyme-derived nephron segments but dispensable for formation of the kidney collecting system. Hence, our studies reveal compartment-specific needs for metabolic reprogramming during kidney development.

Diabetic kidney disease (DKD) progression involves NIMA-related kinase 7 (NEK7)-driven podocyte pyroptosis, with hyperglycemia-induced O-GlcNAcylation as a key posttranslational regulator. This study elucidates how O-GlcNAc modification governs NEK7 stability and its pathological role. We used clinical DKD specimens, high-glucose-stimulated podocytes, and streptozotocin-induced diabetic mice to first examine NEK7, O-GlcNAc, O-GlcNAc transferase (OGT), and glutamine fructose-6-phosphate amidotransferase 1 (GFPT1) expression, confirming the pyroptosis role of NEK7 via siRNA knockdown. Bioinformatic analysis predicted O-GlcNAcylation motifs, validated by T302A mutagenesis and coimmunoprecipitation. Protein stability was assessed using cycloheximide chase and ubiquitination assays. Therapeutic efficacy of the GFPT1 inhibitor (6-diazo-5-oxo-l-norleucine) DON was evaluated in vitro and in vivo through biochemical parameters, histopathology, and pyroptosis markers. Chronic hyperglycemia activated the hexosamine biosynthetic pathway (HBP), elevating pathology-associated O-GlcNAc modifications that promoted NEK7 accumulation via posttranslational stabilization. This was accompanied by upregulated O-GlcNAc, OGT, and GFPT1 in DKD glomeruli and high-glucose podocytes. Crucially, threonine 302 was identified as the primary O-GlcNAcylation site of NEK7. This modification reduced proteasomal degradation, extended NEK7 half-life, and enhanced NLRP3 inflammasome activation and interleukin release. Pharmacological HBP inhibition using DON normalized O-GlcNAcylation, suppressed pyroptosis, and mitigated renal injury. We report the discovery of the glucose/O-GlcNAc/NEK7/NLRP3 axis driving podocyte pyroptosis in DKD, proposing threonine 302 as a potential therapeutic target. These findings establish a novel posttranslational modification mechanism and suggest a dual-target therapeutic strategy for DKD management. We identify threonine 302 as the critical O-GlcNAcylation site on NIMA-related kinase 7 (NEK7), which stabilizes NEK7 by inhibiting its proteasomal degradation, thereby enhancing NLRP3 inflammasome activation and podocyte pyroptosis in diabetic kidney disease (DKD). Chronic hyperglycemia activates the hexosamine biosynthetic pathway (HBP), driving pathological O-GlcNAcylation and significant upregulation of NEK7, O-GlcNAc transferase, and glutamine fructose-6-phosphate amidotransferase 1 in glomeruli from patients with DKD and experimental models. This study establishes the discovery of the pathogenic glucose/O-GlcNAc/NEK7/NLRP3 signaling axis, identifying a novel posttranslational mechanism driving podocyte loss in DKD progression. Pharmacological inhibition of the HBP with 6-diazo-5-oxo-l-norleucine normalizes O-GlcNAcylation, suppresses NEK7-driven pyroptosis, and mitigates renal injury, demonstrating the therapeutic potential of targeting threonine 302, NEK7, or the HBP for DKD management.

Mitochondrial dysfunction is central to the pathogenesis of podocytopathies, yet the determinants of metabolic resilience versus failure remain elusive. We investigated how distinct disruptions of mitochondrial architecture, specifically hyperfusion via OMA1 deletion versus compromised inner mitochondrial membrane (IMM) integrity via PHB2 knockdown, influence the metabolic fate and insulin responsiveness of podocytes. To this end, we analyzed conditionally immortalized mouse podocytes with genetic OMA1 deletion or inducible PHB2 knockdown and employed an integrated approach combining bioenergetic studies, quantitative proteomics, phosphoproteomics, metabolomics, and stable isotope tracing studies with

Nephrotic syndrome (NS) is a clinical syndrome resulting from abnormal glomerular permeability, mainly manifesting as edema and proteinuria. Qingrekasen granule (QRKSG), a Chinese Uyghur folk medicine, is a single-flavor preparation made from chicory (Cichorium intybus L.), widely used in treating dysuria and edema. Chicory, the main component in QRKSG, effectively treats edema and protects kidneys. However, the active components in QRKSG and its underlying mechanism for treating NS remain unclear. This study explored the specific mechanism and composition of QRKSG on an NS rat model using integrated metabolomics and network pharmacology. First, metabolomics explored the relevant metabolic pathways impacted by QRKSG in the treatment of NS. Secondly, network pharmacology further explored the possible metabolite targets. Afterward, a comprehensive network was constructed using the results from the network pharmacology and metabolomics analysis. Finally, the interactions between the active components and targets were predicted by molecular docking, and the differential expression levels of the target protein were verified by Western blotting. The metabolomics results showed "D-Glutamine and D-glutamate metabolism" and "Alanine, aspartate, and glutamate metabolism" as the main targeted metabolic pathways for treating NS in rats. AKT1, BCL2L1, CASP3, and MTOR were the core QRKSG targets in the treatment of NS. Molecular docking revealed that these core targets have a strong affinity for flavonoids, terpenoids, and phenolic acids. Moreover, the expression levels of p-PI3K, p-AKT1, p-mTOR, and CASP3 in the QRKSG group significantly decreased, while BCL2L1 increased compared to the model group. These findings established the underlying mechanism of QRKSG, such as promoting autophagy and anti-apoptosis through the expression of AKT1, CASP3, BCL2L1, and mTOR to protect podocytes and maintain renal tubular function.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) and N-acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O-glycosylation, one of key post-translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT-PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3 +/- 0.2 nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1 +/- 0.2 nmol/min/mg protein) in a significant part, involved a sodium-dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50 microM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6 mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment.

We aimed to determine the metabolomic profile of kidney cells under high glucose conditions and following sodium-glucose cotransporter 2 (SGLT2) inhibitor treatment. Targeted metabolomics using the Absolute IDQ-p180 kit was applied to quantify metabolites in kidney cells stimulated with high glucose (25 and 50 mM) and treated with SGLT2 inhibitor, dapagliflozin (2 µM). Primary cultured human tubular epithelial cells and podocytes were used to identify the metabolomic profile in high glucose conditions following dapagliflozin treatment. The levels of asparagine, PC ae C34:1, and PC ae C36:2 were elevated in tubular epithelial cells stimulated with 50 mM glucose and were significantly decreased after 2 µM dapagliflozin treatment. The level of PC aa C32:0 was significantly decreased after 50 mM glucose treatment compared with the control, and its level was significantly increased after dapagliflozin treatment in podocytes. The metabolism of glutathione, asparagine and proline was significantly changed in tubular epithelial cells under high-glucose stimulation. And the pathway analysis showed that aminoacyl-tRNA biosynthesis, arginine and proline metabolism, glutathione metabolism, valine, leucine and isoleucine biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, beta-alanine metabolism, phenylalanine metabolism, arginine biosynthesis, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism were altered in tubular epithelial cells after dapagliflozin treatment following 50 mM glucose compared to those treated with 50 mM glucose.

Podocyte apoptosis induced by hyperglycemia is considered a critical factor in the development of diabetic nephropathy. Recent studies have implicated Notch signaling in podocyte apoptosis; however, its regulatory mechanisms are not fully understood. In this study, we found that high-glucose treatment increased Notch1 and Jagged-1 expression, the transcriptional activity of Hes, and podocyte apoptosis, and decreased the expression of coactivator-associated arginine methyltransferase 1 (CARM1) in rat podocytes. Transient transfection of CARM1 reversed high-glucose-induced Notch1 expression, the transcriptional activity of Hes, and podocyte apoptosis. Moreover, the silencing of CARM1 using siRNA increased Notch1 expression, the transcriptional activity of Hes, and podocyte apoptosis. However, the Glu(266)-mediated enzymatic activity of CARM1 was not necessary for Notch signaling activation and podocyte apoptosis. Here, we demonstrate that AMP-activated protein kinase alpha (AMPKα) and cannabinoid receptor 1 (CB1R) are regulated by CARM1 and that high-glucose-induced podocyte apoptosis is mediated by a CARM1-AMPKα-Notch1-CB1R signaling axis. We also show that high-glucose-induced CARM1 downregulation is due to ubiquitination-dependent CARM1 degradation. Finally, we demonstrate that CARM1 expression in podocytes was diminished in rats with streptozotocin-induced diabetes compared to vehicle-treated rats. Together, our data provide evidence that ubiquitination-dependent CARM1 degradation in podocytes in diabetes promotes podocyte apoptosis via Notch1 activation. Strategies to preserve CARM1 expression or reduce the enzymatic activity of a ubiquitin ligase specific for CARM1 could be used to prevent podocyte loss in diabetic nephropathy.

Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that

Nephrotic syndrome is recognized by the presence of proteinuria in excess of 3.5 g/24 h along with hypoalbuminemia, edema, hyperlipidemia (hypertriglyceridemia and hypercholesterolemia), and lipiduria. Each component has been investigated individually over the past four decades with some success. Studies published recently have started unraveling the molecular basis of proteinuria and its relationship with other components. We now have improved understanding of the threshold for nephrotic-range proteinuria and the pathogenesis of hypertriglyceridemia. These studies reveal that modifying sialylation of the soluble glycoprotein angiopoietin-like 4 or changing key amino acids in its sequence can be used successfully to treat proteinuria. Treatment strategies on the basis of fundamental relationships among different components of nephrotic syndrome use naturally occurring pathways and have great potential for future development into clinically relevant therapeutic agents.

Although diabetic nephropathy (DN) is the most common cause for end-stage renal disease in western societies, its pathogenesis still remains largely unclear. A different gene pattern of diabetic and healthy kidney cells is one of the probable explanations. Numerous signalling pathways have emerged as important pathophysiological mechanisms for diabetes-induced renal injury. Glomerular cells, as podocytes or mesangial cells, are predominantly involved in the development of diabetic renal lesions. While many gene assays concerning DN are performed with whole kidney or renal cortex tissue, we isolated glomeruli from black and tan, brachyuric (BTBR) obese/obese (ob/ob) and wildtype mice at four different timepoints (4, 8, 16 and 24 weeks) and performed an mRNA microarray to identify differentially expressed genes (DEGs). In contrast to many other diabetic mouse models, these homozygous ob/ob leptin-deficient mice develop not only a severe type 2 diabetes, but also diabetic kidney injury with all the clinical and especially histologic features defining human DN. By functional enrichment analysis we were able to investigate biological processes and pathways enriched by the DEGs at different disease stages. Altered expression of nine randomly selected genes was confirmed by quantitative polymerase chain reaction from glomerular RNA. Ob/ob type 2 diabetic mice showed up- and downregulation of genes primarily involved in metabolic processes and pathways, including glucose, lipid, fatty acid, retinol and amino acid metabolism. Members of the CYP4A and ApoB family were found among the top abundant genes. But more interestingly, altered gene loci showed enrichment for processes and pathways linked to angioneogenesis, complement cascades, semaphorin pathways, oxidation and reduction processes and renin secretion. The gene profile of BTBR ob/ob type 2 diabetic mice we conducted in this study can help to identify new key players in molecular pathogenesis of diabetic kidney injury.

Diabetic kidney disease is a major complication in diabetes mellitus, and the most common reason for end-stage renal disease. Patients suffering from diabetes mellitus encounter glomerular damage by basement membrane thickening, and develop albuminuria. Subsequently, albuminuria can deteriorate the tubular function and impair the renal outcome. The impact of diabetic stress conditions on the metabolome was investigated by untargeted gas chromatography-mass spectrometry (GC-MS) analyses. The results were validated by qPCR analyses. In total, four cell lines were tested, representing the glomerulus, proximal nephron tubule, and collecting duct. Both murine and human cell lines were used. In podocytes, proximal tubular and collecting duct cells, high glucose concentrations led to global metabolic alterations in amino acid metabolism and the polyol pathway. Albumin overload led to the further activation of the latter pathway in human proximal tubular cells. In the proximal tubular cells, aldo-keto reductase was concordantly increased by glucose, and partially increased by albumin overload. Here, the combinatorial impact of two stressful agents in diabetes on the metabolome of kidney cells was investigated, revealing effects of glucose and albumin on polyol metabolism in human proximal tubular cells. This study shows the importance of including highly concentrated albumin in in vitro studies for mimicking diabetic kidney disease.

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.

As common pathogenic agents in the world and widely distributed globally, T-2 toxin and selenium deficiency might exacerbate toxic effects by combined exposure, posing a dramatic health hazard to humans and animals. In this study, we aim to elucidate the underlying mechanisms of renal fibrosis triggered by T-2 toxin and selenium deficiency exposure. A total of thirty-two rats are randomly divided into the normal control, T-2 toxin, selenium deficiency, and combined intervention groups. T-2 toxin (100 ng/g) is intragastric gavaged to the rats in compliance with the body weight. Both the standard (containing selenium 0.20 mg/Kg) and selenium-deficient (containing selenium 0.02 mg/Kg) diets were manufactured adhering to the AIN-93 formula. After 12 weeks of intervention, renal tissue ultrastructural and pathological changes, inflammatory infiltration, epithelial mesenchymal transition (EMT), and extracellular matrix (ECM) deposition are evaluated, respectively. Metabolomics analysis is conducted to explore the underlying pathology of renal fibrosis, followed by the validation of potential mechanisms at gene and protein levels. T-2 toxin and selenium deficiency exposure results in podocyte foot process elongation or fusion, tubular vacuolization and dilatation, and collagen deposition in the kidneys. Additionally, it also increases inflammatory infiltration, EMT conversion, and ECM deposition. Metabolomics analysis suggests that T-2 toxin and selenium deficiency influence amino acid and cholesterol metabolism, respectively, and the estrogen signaling pathway is probably engaged in renal fibrosis progression. Moreover, T-2 toxin and selenium deficiency are found to regulate the expressions of the ERα/PI3K/Akt signaling pathway. In conclusion, T-2 toxin and selenium deficiency synergistically exacerbate renal fibrosis through regulating the ERα/PI3K/Akt signaling pathway, and inflammatory infiltration, EMT and ECM deposition are involved in this process.

Membranous glomerulonephritis (MGN) is one of the most frequent causes of nephrotic syndrome in adults. It is characterized by the thickening of the glomerular basement membrane in the renal tissue. The current diagnosis of MGN is based on renal biopsy and the detection of antibodies to the few podocyte antigens. Due to the limitations of the current diagnostic methods, including invasiveness and the lack of sensitivity of the current biomarkers, there is a requirement to identify more applicable biomarkers. The present study aimed to identify diagnostic metabolites that are involved in the development of the disease using topological features in the component‑reaction‑enzyme‑gene (CREG) network for MGN. Significant differential metabolites in MGN compared with healthy controls were identified using proton nuclear magnetic resonance and gas chromatography‑mass spectrometry techniques, and multivariate analysis. The CREG network for MGN was constructed, and metabolites with a high centrality and a striking fold‑change in patients, compared with healthy controls, were introduced as putative diagnostic biomarkers. In addition, a protein‑protein interaction (PPI) network, which was based on proteins associated with MGN, was built and analyzed using PPI analysis methods, including molecular complex detection and ClueGene Ontology. A total of 26 metabolites were identified as hub nodes in the CREG network, 13 of which had salient centrality and fold‑changes: Dopamine, carnosine, fumarate, nicotinamide D‑ribonucleotide, adenosine monophosphate, pyridoxal, deoxyguanosine triphosphate, L‑citrulline, nicotinamide, phenylalanine, deoxyuridine, tryptamine and succinate. A total of 13 subnetworks were identified using PPI analysis. In total, two of the clusters contained seed proteins (phenylalanine‑4‑hydroxlylase and cystathionine γ‑lyase) that were associated with MGN based on the CREG network. The following biological processes associated with MGN were identified using gene ontology analysis: 'Pyrimidine‑containing compound biosynthetic process', 'purine ribonucleoside metabolic process', 'nucleoside catabolic process', 'ribonucleoside metabolic process' and 'aromatic amino acid family metabolic process'. The results of the present study may be helpful in the diagnostic and therapeutic procedures of MGN. However, validation is required in the future.

Meprin metalloproteases are abundantly expressed in the brush border membranes of kidney proximal tubules and small intestines. Meprins are also expressed in podocytes and leukocytes (monocytes and macrophages). Meprins are implicated in the pathophysiology of diabetic nephropathy (DN) but underlying mechanisms are not fully understood. Single nucleotide polymophisms (SNPs) in the meprin β gene were associated with DKD in human subjects. Furthermore, meprin α and β double deficiency resulted in more severe kidney injury and higher mortality rates in mice with Streptozotocin (STZ)-induced type 1 diabetes. Identification of meprin substrates has provided insights on how meprins could modulate kidney injury. Meprin targets in the kidney include extracellular matrix (ECM) proteins, modulators of inflammation, and proteins involved in the protein kinase A (PKA) and PKC signaling pathways. The current study used a global metabolomics approach to determine how meprin β expression impacts the metabolite milieu in diabetes and DKD. Low dose STZ was used to induce type 1 diabetes in 8-week old wild-type (WT) and meprin β knockout (βKO) mice. Blood and urine samples were obtained at 4 and 8 weeks post-STZ injection. Assays for albumin, creatinine, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule - 1 (KIM-1), and cystatin C were used for biochemical assessment of kidney injury. Data for biomarkers of kidney injury utilized two-way ANOVA. Metabolomics data analysis utilized UPLC-QTOF MS and multivariate statistics. The number of metabolites with diabetes-associated changes in levels were significantly higher in the WT mice when compared to meprin βKO counterparts. Annotated meprin β expression-associated metabolites with strong variable importance in projection (VIP) scores play roles in lipid metabolism (LysoPC(16:1(9Z)), taurocholic acid), amino acid metabolism (indoxyl sulfate, hippuric acid), and neurotransmitter/stress hormone synthesis (cortisol, 3-methoxy-4-hydroxyphenylethylene glycolsulfate, homovanillic acid sulfate). Metabolites that associated with meprin β deficiency include; 3,5-dihydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavone 3-glucuronide, pantothenic acid, and indoxyl glucuronide (all decreased in plasma). Taken together, the annotated metabolites suggest that meprin β impacts complications of diabetes such as DKD by altering distinct metabolite profiles.

Focal segmental glomerulosclerosis (FSGS) is a common glomerulonephritis, and its rates of occurrence are increasing worldwide. Proteinuria is a clinical defining feature of FSGS which correlates with the severity of podocyte injury in patients with nephrotic-range protein excretion. Metabolite biomarkers corresponding with the level of proteinuria could be considered as non-invasive complementary prognostic factors to proteinuria. The urine samples of 15 patients (n = 6 women and n = 9 men) with biopsy-proven FSGS were collected and subjected to nuclear magnetic resonance (NMR) analysis for metabolite profiling. Multivariate statistical analyses, including principal component analysis and orthogonal projection to latent structure discriminant analysis, were applied to construct a predictive model based on patients with proteinuria >3000 mg/day and <3000 mg/day. In addition, random forest was performed to predict differential metabolites, and pathway analysis was performed to find the defective pathways responsible for proteinuria. Ten metabolites, significant in both statistical methods (orthogonal projection to latent structure discriminant analysis and random forest), were considered as prognostic biomarkers for FSGS: citrulline, dimethylamine, proline, acetoacetate, alpha-ketoisovaleric acid, valine, isobutyrate, D-Palmitylcarnitine, histidine, and N-methylnicotinamide. Pathway analysis revealed impairment of the branched-chain amino acid degradation pathways in patients with massive proteinuria. This study shows that metabolomics can reveal the molecular changes corresponding with disease progression in patients with FSGS and provide a new insight for pathogenic pathways. Copyright © 2016 John Wiley & Sons, Ltd.

Membranous nephropathy is an important glomerular disease characterized by podocyte injury and proteinuria, but no metabolomics research was reported as yet. Here, we performed a parallel metabolomics study, based on human urine and serum, to comprehensively profile systematic metabolic variations, identify differential metabolites, and understand the pathogenic mechanism of membranous nephropathy. There were obvious metabolic distinctions between the membranous nephropathy patients with urine protein lower than 3.5 g/24 h (LUPM) and those higher than 3.5 g/24 h (HUPM) by Partial Least Squares Discriminant Analysis (PLS-DA) model analysis. In total, 26 urine metabolites and 9 serum metabolites were identified to account for such differences, and the majority of metabolites were significantly increased in HUPM patients for both urines and serums. Combining the results of urine with serum, all differential metabolites were classified to 5 classes. This classification helps globally probe the systematic metabolic alterations before and after blood flowing through kidney. Citric acid and 4 amino acids were markedly increased only in the serum samples of HUPM patients, implying more impaired filtration function of kidneys of HUPM patients than LUPM patients. The dicarboxylic acids, phenolic acids, and cholesterol were significantly elevated only in urines of HUPM patients, suggesting more severe oxidative attacks than LUPM patients. Parallel metabolomics of urine and serum revealed the systematic metabolic variations associated with LUPM and HUPM patients, where HUPM patients suffered more severe injury of kidney function and oxidative stresses than LUPM patients. This research exhibited a promising application of parallel metabolomics in renal diseases.

Managing tryptophan (TRP) availability is important for cell homeostasis, and a dynamic balance between dietary intake and its catabolism is crucial. The enzymes of the kynurenine pathway (KP) mediate the main catabolic route for TRP. Its intermediary products, collectively known as kynurenines, are considered metabolically active and highly pleiotropic. Some progress has been made in the description of the biological function of the kynurenines, and despite the growing number of studies that show an association between TRP metabolism and kidney function, not much is known about the cellular mechanisms involved. To assess if the kynurenines play a role in glomerular dysfunction, we carried out a series of experiments aimed at describing the effect of changes in the relative abundance of the kynurenines on cells of the glomerulus, both in vivo and in vitro. We used a transgenic zebrafish line as a model to show that systemic changes in the KP either by morpholino knockdown, enzymatic inhibition, or kynurenine supplementation, lead to pericardial effusion, yolk sac edema, and excretion of high molecular weight proteins, all signs of impaired glomerular filtration. Cultured podocytes incubated with a KP inhibitor show changes in cell size, morphology and focal adhesions, leading to a higher detachment rate. Additionally, there is a change in the polarization status of the mitochondria, showing a loss of membrane potential and an alteration of bioenergetics parameters. Taken together, our results highlight the importance of kynurenine metabolite levels in the maintenance of a functioning filtration barrier.