Kidney organoids: accurate models or fortunate accidents.

There are now many reports of human kidney organoids generated via the directed differentiation of human pluripotent stem cells (PSCs) based on an existing understanding of mammalian kidney organogenesis. Such kidney organoids potentially represent tractable tools for the study of normal human development and disease with improvements in scale, structure, and functional maturation potentially providing future options for renal regeneration. The utility of such organotypic models, however, will ultimately be determined by their developmental accuracy. While initially inferred from mouse models, recent transcriptional analyses of human fetal kidney have provided greater insight into nephrogenesis. In this review, we discuss how well human kidney organoids model the human fetal kidney and how the remaining differences challenge their utility.

Podocyte-specific Nup160 knockout mice develop nephrotic syndrome and glomerulosclerosis.

More than 60 monogenic genes mutated in steroid-resistant nephrotic syndrome (SRNS) have been identified. Our previous study found that mutations in nucleoporin 160 kD (NUP160) are implicated in SRNS. The NUP160 gene encodes a component of the nuclear pore complex. Recently, two siblings with homozygous NUP160 mutations presented with SRNS and a nervous system disorder. However, replication of nephrotic syndrome (NS)-associated phenotypes in a mammalian model following loss of Nup160 is needed to prove that NUP160 mutations cause SRNS. Here, we generated a podocyte-specific Nup160 knockout (Nup160podKO) mouse model using CRISPR/Cas9 and Cre/loxP technologies. We investigated NS-associated phenotypes in these Nup160podKO mice. We verified efficient abrogation of Nup160 in Nup160podKO mice at both the DNA and protein levels. We showed that Nup160podKO mice develop typical signs of NS. Nup160podKO mice exhibited progression of proteinuria to average albumin/creatinine ratio (ACR) levels of 15.06 ± 2.71 mg/mg at 26 weeks, and had lower serum albumin levels of 13.13 ± 1.34 g/l at 30 weeks. Littermate control mice had urinary ACR mean values of 0.03 mg/mg and serum albumin values of 22.89 ± 0.34 g/l at the corresponding ages. Further, Nup160podKO mice exhibited glomerulosclerosis compared with littermate control mice. Podocyte-specific Nup160 knockout in mice led to NS and glomerulosclerosis. Thus, our findings strongly support that mutations in NUP160 cause SRNS. The newly generated Nup160podKO mice are a reliable mammalian model for future study of the pathogenesis of NUP160-associated SRNS.

Novel Antigens and Clinical Updates in Membranous Nephropathy.

Membranous nephropathy (MN), an autoimmune kidney disease and leading cause of nephrotic syndrome, leads to kidney failure in up to one-third of affected individuals. Most MN cases are due to an autoimmune reaction against the phospholipase A2 receptor (PLA2R) located on kidney podocytes. Serum PLA2R antibody quantification is now part of routine clinical practice because antibody titers correlate with disease activity and treatment response. Recent advances in target antigen detection have led to the discovery of more than 20 other podocyte antigens, yet the clinical impact of additional antigen detection remains unknown and is under active investigation. Here we review recent findings and hypothesize how current research will inform future care of patients with MN.

Ultrastructural analysis of whole glomeruli using array tomography.

The renal glomerulus produces primary urine from blood plasma by ultrafiltration. The ultrastructure of the glomerulus is closely related to filtration function and disease development. The ultrastructure of glomeruli has mainly been evaluated using transmission electron microscopy; however, the volume that can be observed using transmission electron microscopy is extremely limited relative to the total volume of the glomerulus. Consequently, observing structures that exist in only one location in each glomerulus, such as the vascular pole, and evaluating low-density or localized lesions are challenging tasks. Array tomography (AT) is a technique used to analyze the ultrastructure of tissues and cells via scanning electron microscopy of serial sections. In this study, we present an AT workflow that is optimized for observing complete serial sections of the whole glomerulus, and we share several analytical examples that use the optimized AT workflow, demonstrating the usefulness of this approach. Overall, this AT workflow can be a powerful tool for structural and pathological evaluation of the glomerulus. This workflow is also expected to provide new insights into the ultrastructure of the glomerulus and its constituent cells.

Effects of zinc in podocytes and cortical collecting duct in vitro and Dahl Salt-Sensitive rats in vivo.

Zinc is one of the essential divalent cations in the human body and a fundamental microelement involved in the regulation of many cellular and subcellular functions. Experimental studies reported that zinc deficiency is associated with renal damage and could increase blood pressure. It was proposed that zinc dietary supplementation plays a renoprotective role. Our study aimed to investigate the effects of zinc on intracellular signaling in renal cells and explore the correlation between dietary zinc and the progression of salt-induced hypertension. The impact of extracellular zinc concentrations on two different kidney epithelial cell types - podocytes and principal cells of the cortical collecting duct (CCD), was tested. In podocytes, a rise in extracellular zinc promotes TRPC6 channel-mediated calcium entry but not altered intracellular zinc levels. However, we observe the opposite effect in CCD cells with no alteration in calcium levels and steady-state elevation in intracellular zinc. Moreover, prolonged extracellular zinc exposure leads to cytotoxic insults in CCD cells but not in podocytes, characterized by increased cell death and disrupted cytoskeletal organization. Next, we tested if dietary zinc plays a role in the development of hypertension in Dahl salt-sensitive rats. Neither zinc-rich nor deficient diets impact the regular development of salt-sensitive hypertension. These results suggest specialized roles for zinc in renal function, implicating its involvement in proliferation and apoptosis in CCD cells and calcium signaling and cytoskeletal dynamics modulation in podocytes. Further research is required to elucidate the detailed mechanisms of zinc action and its implications in renal health and disease.

Ectodomain shedding of PLA2R1 is mediated by the metalloproteases ADAM10 and ADAM17.

Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer and is the major autoantigen in membranous nephropathy, a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts, and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1 and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by ß- or γ-secretase, suggesting that PLA2R1 may not be a substrate for regulated intramembrane proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C activator PMA, cytokines, and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer, and membranous nephropathy.

The role of the FSGS disease gene product and nuclear pore protein NUP205 in regulating nuclear localization and activity of transcriptional regulators YAP and TAZ.

Mutations in genes encoding nuclear pore proteins (NUPs) lead to the development of steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis (FSGS). However, the precise molecular mechanisms by which NUP dysfunction contributes to podocyte injury preceding FSGS remain unclear. The tightly regulated activity of Yes-associated protein (YAP) and WW-domain-containing transcription regulator 1 (TAZ), the transcriptional effectors of the Hippo pathway, is crucial for podocytes and the maintenance of the glomerular filter. In this study, we investigate the impact of NUPs on the regulation of YAP/TAZ nuclear import and activity in podocytes. In unbiased interactome studies using quantitative label-free mass spectrometry, we identify the FSGS disease gene products NUP107, NUP133, NUP205, and Exportin-5 (XPO5) as components of YAP and TAZ protein complexes in podocytes. Moreover, we demonstrate that NUP205 is essential for YAP/TAZ nuclear import. Consistently, both the nuclear interaction of YAP/TAZ with TEA domain transcription factor 1 and their transcriptional activity were dependent on NUP205 expression. Additionally, we elucidate a regulatory feedback mechanism whereby YAP activity is modulated in response to TAZ-mediated NUP205 expression. In conclusion, this study establishes a connection between the FSGS disease protein NUP205 and the activity of the transcriptional regulators and Hippo effectors YAP and TAZ and it proposes a potential pathological role of YAP/TAZ dysregulation in podocytes of patients with pathogenic NUP205 variants.

FTO-mediated m6A modification of serum amyloid A2 mRNA promotes podocyte injury and inflammation by activating the NF-κB signaling pathway.

Diabetic kidney disease (DKD) is one of the severe complications of diabetes mellitus, yet there is no effective treatment. Exploring the development of DKD is essential to treatment. Podocyte injury and inflammation are closely related to the development of DKD. However, the mechanism of podocyte injury and progression in DKD remains largely unclear. Here, we observed that FTO expression was significantly upregulated in high glucose-induced podocytes and that overexpression of FTO promoted podocyte injury and inflammation. By performing RNA-seq and MeRIP-seq with control podocytes and high glucose-induced podocytes with or without FTO knockdown, we revealed that serum amyloid A2 (SAA2) is a target of FTO-mediated m6A modification. Knockdown of FTO markedly increased SAA2 mRNA m6A modification and decreased SAA2 mRNA expression. Mechanistically, we demonstrated that SAA2 might participate in podocyte injury and inflammation through activation of the NF-κB signaling pathway. Furthermore, by generating podocyte-specific adeno-associated virus 9 (AAV9) to knockdown SAA2 in mice, we discovered that the depletion of SAA2 significantly restored podocyte injury and inflammation. Together, our results suggested that upregulation of SAA2 promoted podocyte injury through m6A-dependent regulation, thus suggesting that SAA2 may be a therapeutic target for diabetic kidney disease.

miR-4645-3p attenuates podocyte injury and mitochondrial dysfunction in diabetic kidney disease by targeting Cdk5.

Podocyte injury plays a critical role in the progression of diabetic kidney disease (DKD), but the underlying cellular and molecular mechanisms remain poorly understanding. MicroRNAs (miRNAs) can disrupt gene expression by inducing translation inhibition and mRNA degradation, and recent evidence has shown that miRNAs may play a key role in many kidney diseases. In this study, we identified miR-4645-3p by global transcriptome expression profiling as one of the major downregulated miRNAs in high glucose-cultured podocytes. Moreover, whether DKD patients or STZ-induced diabetic mice, expression of miR-4645-3p was also significantly decreased in kidney. In the podocytes cultured by normal glucose, inhibition of miR-4645-3p expression promoted mitochondrial damage and podocyte apoptosis. In the podocytes cultured by high glucose (30 mM glucose), overexpression of miR-4645-3p significantly attenuated mitochondrial dysfunction and podocyte apoptosis induced by high glucose. Furthermore, we found that miR-4645-3p exerted protective roles by targeting Cdk5 inhibition. In vitro, miR-4645-3p obviously antagonized podocyte injury by inhibiting overexpression of Cdk5. In vivo of diabetic mice, podocyte injury, proteinuria, and impaired renal function were all effectively ameliorated by treatment with exogenous miR-4645-3p. Collectively, these findings demonstrate that miR-4645-3p can attenuate podocyte injury and mitochondrial dysfunction in DKD by targeting Cdk5. Sustaining the expression of miR-4645-3p in podocytes may be a novel strategy to treat DKD.

A molecular mechanism for angiotensin II receptor blocker-mediated slit membrane protection: Angiotensin II increases nephrin endocytosis via AT1-receptor-dependent ERK 1/2 activation.

Albuminuria is characterized by a disruption of the glomerular filtration barrier, which is composed of the fenestrated endothelium, the glomerular basement membrane, and the slit diaphragm. Nephrin is a major component of the slit diaphragm. Apart from hemodynamic effects, Ang II enhances albuminuria by ß-Arrestin2-mediated nephrin endocytosis. Blocking the AT1 receptor with candesartan and irbesartan reduces the Ang II-mediated nephrin-ß-Arrestin2 interaction. The inhibition of MAPK ERK 1/2 blocks Ang II-enhanced nephrin-ß-Arrestin2 binding. ERK 1/2 signaling, which follows AT1 receptor activation, is mediated by G-protein signaling, EGFR transactivation, and ß-Arrestin2 recruitment. A mutant AT1 receptor defective in EGFR transactivation and ß-Arrestin2 recruitment reduces the Ang II-mediated increase in nephrin ß-Arrestin2 binding. The mutation of ß-Arrestin2K11,K12, critical for AT1 receptor binding, completely abrogates the interaction with nephrin, independent of Ang II stimulation. ß-Arrestin2K11R,K12R does not influence nephrin cell surface expression. The data presented here deepen our molecular understanding of a blood-pressure-independent molecular mechanism of AT-1 receptor blockers (ARBs) in reducing albuminuria.

FilGAP controls cell-extracellular matrix adhesion and process formation of kidney podocytes.

The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin-driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase-activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin-based protein complex closely associated with stress fibers, that mediates cell-extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process-like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP-depleted podocyte cells was rescued by inhibition of Rac1 or P21-activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell-ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.

Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Single-cell transcriptomics identifies aberrant glomerular angiogenic signalling in the early stages of WT1 kidney disease.

WT1 encodes a podocyte transcription factor whose variants can cause an untreatable glomerular disease in early childhood. Although WT1 regulates many podocyte genes, it is poorly understood which of them are initiators in disease and how they subsequently influence other cell-types in the glomerulus. We hypothesised that this could be resolved using single-cell RNA sequencing (scRNA-seq) and ligand-receptor analysis to profile glomerular cell-cell communication during the early stages of disease in mice harbouring an orthologous human mutation in WT1 (Wt1R394W/+). Podocytes were the most dysregulated cell-type in the early stages of Wt1R394W/+ disease, with disrupted angiogenic signalling between podocytes and the endothelium, including the significant downregulation of transcripts for the vascular factors Vegfa and Nrp1. These signalling changes preceded glomerular endothelial cell loss in advancing disease, a feature also observed in biopsy samples from human WT1 glomerulopathies. Addition of conditioned medium from murine Wt1R394W/+ primary podocytes to wild-type glomerular endothelial cells resulted in impaired endothelial looping and reduced vascular complexity. Despite the loss of key angiogenic molecules in Wt1R394W/+ podocytes, the pro-vascular molecule adrenomedullin was upregulated in Wt1R394W/+ podocytes and plasma and its further administration was able to rescue the impaired looping observed when glomerular endothelium was exposed to Wt1R394W/+ podocyte medium. In comparative analyses, adrenomedullin upregulation was part of a common injury signature across multiple murine and human glomerular disease datasets, whilst other gene changes were unique to WT1 disease. Collectively, our study describes a novel role for altered angiogenic signalling in the initiation of WT1 glomerulopathy. We also identify adrenomedullin as a proangiogenic factor, which despite being upregulated in early injury, offers an insufficient protective response due to the wider milieu of dampened vascular signalling that results in endothelial cell loss in later disease. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

miRNA-193a-mediated WT1 suppression triggers podocyte injury through activation of the EZH2/ß-catenin/NLRP3 pathway in children with diabetic nephropathy.

Diabetic nephropathy (DN), an eminent etiology of renal disease in patients with diabetes, involves intricate molecular mechanisms. Recent investigations have elucidated microRNA-193a (miR-193a) as a pivotal modulator in DN, although its precise function in podocyte impairment remains obscure. The present study investigated the role of miR-193a in podocyte injury via the WT1/EZH2/ß-catenin/NLRP3 pathway. This study employed a comprehensive experimental approach involving both in vitro and in vivo analyses. We utilized human podocyte cell lines and renal biopsy samples from pediatric patients with DN. The miR-193a expression levels in podocytes and glomeruli were quantified via qRT‒PCR. Western blotting and immunofluorescence were used to assess the expression of WT1, EZH2, ß-catenin, and NLRP3 inflammasome components. Additionally, the study used luciferase reporter assays to confirm the interaction between miR-193a and WT1. The impact of miR-193a manipulation was observed by overexpressing WT1 and inhibiting miR-193a in podocytes, followed by analysis of downstream pathway activation and inflammatory markers. We found upregulated miR-193a in podocytes and glomeruli, which directly targeted and suppressed WT1, a crucial podocyte transcription factor. WT1 suppression, in turn, activated the EZH2/ß-catenin/NLRP3 pathway, leading to inflammasome assembly and proinflammatory cytokine production. Overexpression of WT1 or inhibition of miR-193a attenuated these effects, protecting podocytes from injury. This study identified a novel mechanism by which miR-193a-mediated WT1 suppression triggers podocyte injury in DN via the EZH2/ß-catenin/NLRP3 pathway. Targeting this pathway or inhibiting miR-193a may be potential therapeutic strategies for DN.

C5a-C5aR1 axis controls mitochondrial fission to promote podocyte injury in lupus nephritis.

Podocytes are essential to maintaining the integrity of the glomerular filtration barrier, but 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.

LRG1 loss effectively restrains glomerular TGF-ß signaling to attenuate diabetic kidney disease.

Transforming growth factor (TGF)-ß signaling is a well-established pathogenic mediator of diabetic kidney disease (DKD). However, owing to its pleiotropic actions, its systemic blockade is not therapeutically optimal. The expression of TGF-ß signaling regulators can substantially influence TGF-ß's effects in a cell- or context-specific manner. Among these, leucine-rich α2-glycoprotein 1 (LRG1) is significantly increased in glomerular endothelial cells (GECs) in DKD. As LRG1 is a secreted molecule that can exert autocrine and paracrine effects, we examined the effects of LRG1 loss in kidney cells in diabetic OVE26 mice by single-cell transcriptomic analysis. Gene expression analysis confirmed a predominant expression of Lrg1 in GECs, which further increased in diabetic kidneys. Loss of Lrg1 led to the reversal of angiogenic and TGF-ß-induced gene expression in GECs, which were associated with DKD attenuation. Notably, Lrg1 loss also mitigated the increased TGF-ß-mediated gene expression in both podocytes and mesangial cells in diabetic mice, indicating that GEC-derived LRG1 potentiates TGF-ß signaling in glomerular cells in an autocrine and paracrine manner. Indeed, a significant reduction in phospho-Smad proteins was observed in the glomerular cells of OVE26 mice with LRG1 loss. These results indicate that specific antagonisms of LRG1 may be an effective approach to curb the hyperactive glomerular TGF-ß signaling to attenuate DKD.

Effectiveness of a novel rat model of off-target PLA2R1 knockout to renal impairment.

Phospholipase A2 receptor 1 (PLA2R1) plays a crucial role in various diseases, including membranous nephropathy. However, the precise implications of PLA2R1 deficiency remain poorly understood. In this study, we created PLA2R1 knockout rats to explore potential consequences resulting from the loss of the PLA2R1 gene. Unexpectedly, our PLA2R1 knockout rats exhibited symptoms resembling those of chronic kidney disease after an 8-week observation period. Notably, several rats developed persistent proteinuria, a hallmark of renal dysfunction. Immunohistochemical and immunofluorescence analyses revealed insignificant glomerular fibrosis, reduced podocyte count, and augmented glomerular expression of complement C3 (C3) compared to immunoglobin A (IgA) and immunoglobin G(IgG) in the rat model. These findings suggest that the loss of PLA2R1 may contribute to the pathogenesis of membranous nephropathy and related conditions. Our knockout rat model provides a valuable tool for investigating the underlying pathology of PLA2R1-associated diseases, and may facilitate the development of targeted therapies for membranous nephropathy and other related disorders.

Epithelial-mesenchymal plasticity in kidney fibrosis.

Epithelial-mesenchymal transition (EMT) is an important biological process contributing to kidney fibrosis and chronic kidney disease. This process is characterized by decreased epithelial phenotypes/markers and increased mesenchymal phenotypes/markers. Tubular epithelial cells (TECs) are commonly susceptible to EMT by various stimuli, for example, transforming growth factor-ß (TGF-ß), cellular communication network factor 2, angiotensin-II, fibroblast growth factor-2, oncostatin M, matrix metalloproteinase-2, tissue plasminogen activator (t-PA), plasmin, interleukin-1ß, and reactive oxygen species. Similarly, glomerular podocytes can undergo EMT via these stimuli and by high glucose condition in diabetic kidney disease. EMT of TECs and podocytes leads to tubulointerstitial fibrosis and glomerulosclerosis, respectively. Signaling pathways involved in EMT-mediated kidney fibrosis are diverse and complex. TGF-ß1/Smad and Wnt/ß-catenin pathways are the major venues triggering EMT in TECs and podocytes. These two pathways thus serve as the major therapeutic targets against EMT-mediated kidney fibrosis. To date, a number of EMT inhibitors have been identified and characterized. As expected, the majority of these EMT inhibitors affect TGF-ß1/Smad and Wnt/ß-catenin pathways. In addition to kidney fibrosis, these EMT-targeted antifibrotic inhibitors are expected to be effective for treatment against fibrosis in other organs/tissues.

Cuproptosis associated cytoskeletal destruction contributes to podocyte injury in chronic kidney disease.

The podocyte cytoskeleton determines the stability of podocyte structure and function, and their imbalance plays a pathogenic role in podocyte diseases. However, the underlying mechanism of podocyte cytoskeleton damage is not fully understood. Here, we investigate the specific role of cuproptosis in inducing podocyte cytoskeleton injury. In in vitro and in vivo studies, exposure to high levels of copper and adriamycin (ADR) caused significant increases in copper concentration in intracellular and renal tissue. Moreover, excessive accumulation of copper induced cuproptosis, resulting in the destruction of the podocyte cytoskeleton. However, inhibition of copper accumulation to reduce cuproptosis also significantly alleviated the damage of podocyte cytoskeleton. In addition, inhibition of cuproptosis mitigated ADR-induced mitochondrial damage as well as the production of reactive oxygen species and depolarization of mitochondrial membrane potential, and restored adenosine triphosphate (ATP) synthesis. Among the transcriptome sequencing data, the difference of CXCL5 (C-X-C motif chemokine ligand 5) was the most significant. Both high copper and ADR exposure can cause upregulation of CXCL5, and CXCL5 deletion inhibits the occurrence of cuproptosis, thereby alleviating the podocyte cytoskeleton damage. This suggests that CXCL5 may act upstream of cuproptosis that mediates podocyte cytoskeleton damage. In conclusion, cuproptosis induced by excessive copper accumulation may induce podocyte cytoskeleton damage by promoting mitochondrial dysfunction, thereby causing podocyte injury. This indicates that cuproptosis plays an important role in the pathogenesis of podocyte injury and provides a basis for seeking potential targets for the treatment of chronic kidney disease.NEW & NOTEWORTHY Cuproptosis induced by excessive copper accumulation leads to podocyte cytoskeleton damage by promoting mitochondrial dysfunction, and CXCL5 acts as an upstream signal mediating the occurrence of cuproptosis.

Dihydroxyacetone phosphate accumulation leads to podocyte pyroptosis in diabetic kidney disease.

Diabetic kidney disease (DKD) can lead to accumulation of glucose upstream metabolites due to dysfunctional glycolysis. But the effects of accumulated glycolysis metabolites on podocytes in DKD remain unknown. The present study examined the effect of dihydroxyacetone phosphate (DHAP) on high glucose induced podocyte pyroptosis. By metabolomics, levels of DHAP, GAP, glucose-6-phosphate and fructose 1, 6-bisphosphate were significantly increased in glomeruli of db/db mice. Furthermore, the expression of LDHA and PKM2 were decreased. mRNA sequencing showed upregulation of pyroptosis-related genes (Nlrp3, Casp1, etc.). Targeted metabolomics demonstrated higher level of DHAP in HG-treated podocytes. In vitro, ALDOB expression in HG-treated podocytes was significantly increased. siALDOB-transfected podocytes showed less DHAP level, mTORC1 activation, reactive oxygen species (ROS) production, and pyroptosis, while overexpression of ALDOB had opposite effects. Furthermore, GAP had no effect on mTORC1 activation, and mTORC1 inhibitor rapamycin alleviated ROS production and pyroptosis in HG-stimulated podocytes. Our findings demonstrate that DHAP represents a critical metabolic product for pyroptosis in HG-stimulated podocytes through regulation of mTORC1 pathway. In addition, the results provide evidence that podocyte injury in DKD may be treated by reducing DHAP.