Connective tissue growth factor modulates podocyte actin cytoskeleton and extracellular matrix synthesis and is induced in podocytes upon injury

Structural changes of podocytes and retraction of their foot processes are a critical factor in the pathogenesis of minimal change nephritis and glomerulosclerosis. Here we tested, if connective tissue growth factor (CTGF) is involved in podocyte injury during acute and chronic puromycin aminonucleoside nephrosis (PAN) as animal models of minimal change nephritis, and focal segmental glomerulosclerosis, respectively. Rats were treated once (acute PAN) or for 13 weeks (chronic PAN). In both experimental conditions, CTGF and its mRNA were found to be highly upregulated in podocytes. The upregulation correlated with onset and duration of proteinuria in acute PAN, and glomerulosclerosis and high expression of glomerular fibronectin, and collagens I, III, and IV in chronic PAN. In vitro, treatment of podocytes with recombinant CTGF increased amount and density of actin stress fibers, the expression of actin-associated molecules such as podocalyxin, synaptopodin, ezrin, and actinin-4, and activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Moreover, we observed increased podocyte expression of mRNA for transforming growth factor (TGF)-β2, TGF-β receptor II, fibronectin, and collagens I, III, and IV. Treatment of cultured podocytes with puromycin aminonucleoside resulted in loss of actin stress fibers and cell death, effects that were partially prevented when CTGF was added to the culture medium. Depletion of CTGF mRNA in cultured podocytes by RNA interference reduced both the number of actin stress fibers and the expression of actin-associated molecules. We propose that the expression of CTGF is acutely upregulated in podocytes as part of a cellular attempt to repair structural changes of the actin cytoskeleton. When the damaging effects on podocyte structure and function persist chronically, continuous CTGF expression in podocytes is a critical factor that promotes progressive accumulation of glomerular extracellular matrix and glomerulosclerosis.     

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.     

GADD45B mediates podocyte injury in zebrafish by activating the ROS-GADD45B-p38 pathway

GADD45 gene has been implicated in cell cycle arrest, cell survival or apoptosis in a cell type specific and context-dependent manner. Members of GADD45 gene family have been found differentially expressed in several podocyte injury models, but their roles in podocytes are unclear. Using an in vivo zebrafish model of inducible podocyte injury that we have previously established, we found that zebrafish orthologs of gadd45b were induced upon the induction of podocyte injury. Podocyte-specific overexpression of zebrafish gadd45b exacerbated edema, proteinuria and foot-process effacement, whereas knockdown of gadd45b by morpholino-oligos in zebrafish larvae ameliorated podocyte injury. We then explored the role of GADD45B induction in podocyte injury using in vitro podocyte culture. We confirmed that GADD45B was significantly upregulated during the early phase of podocyte injury in cultured human podocytes and that podocyte apoptosis induced by TGF-β and puromycin aminonucleoside (PAN) was aggravated by GADD45B overexpression but ameliorated by shRNA-mediated GADD45B knockdown. We also showed that ROS inhibitor NAC suppressed PAN-induced GADD45B expression and subsequent activation of p38 MAPK pathway in podocytes and that inhibition of GADD45B diminished PAN-induced p38 MAPK activation. Taken together, our findings demonstrated that GADD45B has an important role in podocyte injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases.Podocyte dysfunction, injury or loss is a common and decisive cause of various glomerular diseases and understanding the molecular mechanism underlying podocyte response to stress will be very helpful to undermine the pathogenesis of podocyte injury and the targeted therapy for glomerular diseases.The members of Gadd45 gene family, Gadd45a, Gadd45b and Gadd45r have been commonly implicated in stress signaling in response to physiological or environmental stressors, resulting in cell cycle arrest, DNA damage repair, cell survival, senescence and apoptosis.¹ Recently, this gene family has been found differentially expressed in several podocyte injury models. Zhang et al.² observed an induction of GADD45β mRNA expression by lipopolysaccharide in the lung, kidney and spleen, which had the highest GADD45β mRNA expression among all of the tissues examined. Jeffrey W Pippin reported that protein expression of GADD45 was increased in glomeruli from passive Heymann nephritis rats and cultured podocytes exposed in vitro to C5b-9. ³ More recently, Shi et al.⁴ reported that Gadd45b was upregulated in glomeruli of mice with podocyte-specific deletion of Dicer, suggesting the involvement of Gadd45b in podocyte injury. However, no functional characterization of Gadd45 genes in podocytes has been conducted to date and the role of GADD45B in the context of podocyte injury remains unclear.Zebrafish has emerged as a new vertebrate model system for renal glomerular research. The podocytes and renal glomeruli in zebrafish kidney are structurally, molecularly and functionally conserved, rendering zebrafish a valuable and relevant model for podocyte studies. To characterize the role of GADD45b in podocyte injury, we therefore employed zebrafish as an in vivo model system and human podocytes as an in vitro model. We observed the upregulation of GADD45B on podocyte injury in zebrafish renal glomeruli as well as in cultured human podocytes treated with TGF-β and PAN. We further showed that podocyte-specific overexpression of zebrafish orthologs of gadd45b predisposed podocytes to injury, whereas inhibition of gadd45b expression in zebrafish larvae ameliorated podocyte injury and reduced proteinuria. Furthermore, we found that the ROS-GADD45B-p38 pathway was involved in the regulation of GADD45B expression and deleterious role in podocyte injury. Collectively, we have identified GADD45B as an important player in podocyte injury.     

Systemic administration of naked plasmid encoding HGF attenuates puromycin aminonucleoside-induced damage of murine glomerular podocytes

Podocyte injury is considered to play important roles in the pathogenesis of human glomerular disease. There is accumulating evidence suggesting that hepatocyte growth factor (HGF) elicits preventive activity for glomerular cells in animal models of chronic renal diseases. In this study, we demonstrated that delivery of a naked plasmid vector encoding the human HGF gene into mice by a hydrodynamic-based in vivo gene transfection approach markedly reduced proteinuria and attenuated podocyte injury in a mouse model induced by puromycin aminonucleoside (PAN) injection. Systemic administration by rapid injection via the tail vein of a naked plasmid containing HGF cDNA driven under a cytomegalovirus promoter (pCMV-HGF) produced a remarkable level of human HGF protein in the circulation. Tissue distribution studies suggested that the kidney expressed a high level of the HGF transgene. Meanwhile, compared with tubules and interstitium, a higher level of exogenous HGF protein was detected in the glomeruli. Administration of pCMV-HGF dramatically abated the urine albumin excretion and podocyte injury in PAN nephropathy in mice. Exogenous expression of HGF produced evidently beneficial effects, leading to restoration of Wilms' tumor-1 (WT1) and α-actinin-4 expression and attenuation of ultrastructural damage of the podocytes. In vitro, HGF not only restored WT1 and α-actinin-4 expression but also inhibited albumin leakage of podocytes incubated with PAN in a Transwell culture chamber. These results suggest that HGF might provide a novel strategy for amelioration of podocyte injury.     

GAP-43 ameliorates Podocyte injury by decreasing nuclear NFATc1 expression

Podocyte injury is sufficient to cause glomerulosclerosis and proteinuria, eventually leading to kidney failure. Previous studies found that podocytes and neurons had similar biological characteristics. Growth-associated protein-43 (GAP-43) is a growth cone protein in neurons, and a marker of axonal and synaptic growth. However, it is not known whether GAP-43 is expressed in podocytes. Compared with normal glomerular podocytes, GAP-43 was significantly reduced in patients with glomerular diseases. GAP-43 also significantly reduced in lipopolysaccharide (LPS)-treated podocytes. We found that the decreased expression of nephrin, the cell marker of the podocyte, was significantly recovered with GAP-43 overexpression. In contrast, the migration ability in LPS-treated podocyte was reduction after GAP-43 overexpressing. Moreover, overexpression of GAP-43 attenuated podocyte apoptosis by up-regulating the ratio of Bcl-2/Bax with LPS treatment. Finally, Plaue and Rcan1 which are downstream target gene of NFATc1 decreased with overexpression of GAP-43 podocytes. We concluded that GAP-43 attenuated podocyte injury by inhibiting calcineurin/NFATc1 signaling. The findings may provide a promising treatment for podocyte injury-related diseases.     

Receptor activator of NF-kappaB and podocytes: towards a function of a novel receptor-ligand pair in the survival response of podocyte injury

Background

Glomerulosclerosis correlates with reduction in podocyte number that occurs through mechanisms which include apoptosis. Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of glomerulosclerosis. However, the mechanism by which podocytes respond to injury is poorly understood. TNF and TNF receptor superfamilies are important in the pathogenesis of podocyte injury and apoptosis. The ligand of receptor activator of NF-kappaB (RANKL) and receptor activator of NF-kappaB (RANK) are members of the TNF and receptor superfamilies. We investigated whether RANK - RANKL is a receptor - ligand complex for podocytes responding to injury.

Methodology/Principal Findings

In this study, RANKL and RANK were examined in human podocyte diseases and a rat model of puromycin aminonucleoside nephrosis (PAN). Compared with controls, RANK and RANKL were increased in both human podocyte diseases and the rat PAN model; double immunofluorescence staining revealed that RANK protein expression was mainly attributed to podocytes. Immunoelectron microscopy showed that RANK was localized predominantly at the top of the foot process membrane and the cytoplasm of rat podocyte. In addition, RANK was upregulated in mouse podocytes in vitro after injury induced by puromycin aminonucleoside (PA). Knockdown of RANK expression by small interference RNA (siRNA) exacerbated podocyte apoptosis induced by PA. However, RANKL inhibited significantly the apoptosis of podocytes induced by PA.

Conclusions/Significance

These findings suggest the increase in RANK–RANKL expression is a response to podocyte injury, and RANK–RANKL may be a novel receptor–ligand complex for the survival response during podocyte injury.     

The Calcineurin Inhibitor Tacrolimus Reduces Proteinuria in Membranous Nephropathy Accompanied by a Decrease in Angiopoietin-Like-4

Tacrolimus is an anticalcineurinic agent with potent immunosuppressive activity that has recently been shown to have the added benefit of reducing proteinuria in membranous nephropathy (MN) patients. However, its potential mechanisms remain unknown. To reveal the mechanism, rat cohorts were administered tacrolimus or vehicle from days 7 to 28 after the induction of passive Heymann nephritis (PHN). PHN induction resulted in heavy proteinuria and increased expression of desmin, a marker of injured podocytes. We also showed that the glomerular expression of angiopoietin-like-4 (Angptl4) was markedly upregulated in PHN rats and human MN followed by an increase in urine Angptl4 excretion. In addition, increased Angptl4 expression may be related to podocyte injury and proteinuria. Furthermore, upregulated Angptl4 expression primarily colocalized with podocytes rather than endothelial or mesangial cells, indicating that podocytes may be the source of Angptl4, which then gradually migrated to the glomerular basement membrane over time. However, tacrolimus treatment markedly reduced glomerular and urinary Angptl4, accompanied by a reduction in the established proteinuria and the promotion of podocyte repair. Additionally, glomerular immune deposits and circulating IgG levels induced by PHN clearly decreased following tacrolimus treatment. In conclusion, this is the first demonstration that the calcineurin inhibitor tacrolimus can reduce Angptl4 in podocytes accompanied by a decrease in established proteinuria and promotion of podocyte repair in MN.     

The activation of extracellular signal-regulated kinase is responsible for podocyte injury

Podocyte and its slit diaphragm play an important role in maintaining normal glomerular filtration barrier function and structure. Podocyte apoptosis and slit diaphragm injury leads to proteinuria and glomerulosclerosis. However, the molecular mechanism of podocyte injury remains poorly understood. The family of mitogen-activated protein kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase, and p38 signal pathways, are implicated in the progression of various glomerulopathies. However, the role of the activated signal pathway(s) in podocyte injury is elusive. This study examined phosphorylation of ERK in rat puromycin aminonucleoside (PAN) nephropathy as well as conditionally immortalized mouse podocyte treated with PAN in vitro. The effect of treatment with U0126, an inhibitor of ERK, was also investigated. In PAN nephropathy, the phosphorylation of ERK was marked. In podocyte injury, the marked and sustained activation of ERK pathway was also observed before the appearance of significant podocyte apoptosis. Pretreatment with U0126 to podocyte completely inhibited ERK activation, with complete suppression podocyte apoptosis and ameliorated nephrin protein expression along with the phosphorylation of nephrin in podocyte injury. In cultured podocyte, PAN induced actin recorganition, and U0126 inhibited such change. However, U0126 did not recovery the phosphorylation change of neph1 in podocyte injury. We concluded that the sustained activation of ERK along with the phosphorylation of neph1 might be necessary for podocyte injury. The study here suggested that ERK might become a potential target for therapeutic intervention to prevent podocytes from injury which will result in proteinuria.     

The cytoprotective role of autophagy in puromycin aminonucleoside treated human podocytes

Autophagy is a ubiquitous catabolic process involving degradation of damaged organelles and protein aggregates. It shows cytoprotective effects in many cell types and helps to maintain cell homeostasis. In many glomerular diseases, podocyte damage leads to the disruption of the renal filtration barrier and subsequent proteinuria. Puromycin aminonucleoside (PAN) which induces podocyte apoptosis in vitro and in vivo is widely used for studying the pathophysiology of glomerular diseases. It has been shown that PAN induces autophagy in podocytes. However, the relationship between autophagy and apoptosis in PAN treated human podocytes is not known and the role of PAN-induced autophagy in podocyte survival remains unclear. Here we demonstrate that PAN induced autophagy in human podocytes prior to apoptosis which was featured with the activation of mTOR complex 1 (mTORC1). When the PAN-induced autophagy was inhibited by 3-methyladenine (3-MA) or chloroquine (CQ), podocyte apoptosis increased significantly along with the elevation of active caspase-3. Under such circumstance, the podocyte cytoskeleton was also disrupted. Collectively, our results suggested that the induced autophagy may be an early adaptive cytoprotective mechanism for podocyte survival after PAN treatment.     

A vital role for myosin-9 in puromycin aminonucleoside-induced podocyte injury by affecting actin cytoskeleton

Podocyte injury is an early pathological change of many kidney diseases. In particular, the actin cytoskeleton plays an important role in maintaining the normal function of podocytes. Disruption of the actin cytoskeleton is a feature of podocyte injury in proteinuric nephropathies. Recent studies showed that myosin-9 was localized in the podocyte foot processes and was necessary in maintaining podocyte structural homeostasis. However, it is unclear whether myosin-9 maintains podocyte structure by affecting actin cytoskleton. Here, the role of myosin-9 in puromycin aminonucleoside (PAN)-induced podocyte injury was explored both in vitro and in vivo. In cultured mouse podocytes (MPC5), it was determined that PAN downregulated myosin-9 expression, disrupted the actin cytoskeleton and reduced the adhesion ability. Reduced myosin-9 expression by siRNA precipitated podocyte cytoskeletal damage and accelerated PAN-induced podocyte detachment. Overexpression of myosin-9 protected against PAN-induced podocyte detachment. Furthermore, administration of an antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) inhibited PAN-induced podocyte cytoskeletal damage and podocyte detachment by restoring the expression of myosin-9. In the rat PAN nephropathy model, MnTBAP could also attenuate PAN-induced reduction of myosin-9 and podocyte loss. Taken together, these findings pinpointed that oxidative stress contributed to PAN-induced podocyte injury through the repression of a cytoskeletal protein myosin-9, which provided novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies.     

Angiopoietin-Like-4, a Potential Target of Tacrolimus,Predicts Earlier Podocyte Injury in Minimal Change Disease

Podocyte injury plays central roles in proteinuria and kidney dysfunction, therefore, identifying specific biomarker to evaluate earlier podocyte injury is highly desirable. Podocyte-secreted angiopoietin-like-4 (Angptl4) mediates proteinuria in different types of podocytopathy. In the present study, we established an experimental minimal change disease (MCD) rat model, induced by adriamycin (ADR) and resulted in definite podocyte injury, to identify the dynamic changes in Angptl4 expression. We also investigated the direct effects of tacrolimus on Angptl4 and podocyte repair. We determined that the glomerular Angptl4 expression was rapidly upregulated and reached a peak earlier than desmin, an injured podocyte marker, in the ADR rats. Furthermore, this upregulation occurred prior to heavy proteinuria and was accompanied by increased urinary Angptl4. We observed that the Angptl4 upregulation occurred only when podocyte was mainly damaged since we didn’t observe little Angptl4 upregulation in MsPGN patients. In addition, we observed the glomerular Angptl4 mainly located in injured podocytes rather than normal podocytes. Moreover, we found that tacrolimus treatment significantly promoted podocyte repair and reduced glomerular and urinary Angptl4 expression at an earlier stage with a significant serum Angptl4 upregulation. And similar results were confirmed in MCD patients. In conclusion, this study represents the first investigation to demonstrate that Angptl4 can predict podocyte injury at earlier stages in MCD and the identification of earlier podocyte injury biomarkers could facilitate the prompt diagnosis and treatment of patients with podocytopathy, as well as determination of the prognosis and treatment efficacy in these diseases.     

Dual Involvement of Growth Arrest-Specific Gene 6 in the Early Phase of Human IgA Nephropathy

Background

Gas6 is a growth factor that causes proliferation of mesangial cells in the development of glomerulonephritis. Gas6 can bind to three kinds of receptors; Axl, Dtk, and Mer. However, their expression and functions are not entirely clear in the different glomerular cell types. Meanwhile, representative cell cycle regulatory protein p27 has been reported to be expressed in podocytes in normal glomeruli with decreased expression in proliferating glomeruli, which inversely correlated with mesangial proliferation in human IgA nephropathy (IgAN).

Methods

The aim of this study is to clarify Gas6 involvement in the progression of IgAN. Expression of Gas6/Axl/Dtk was examined in 31 biopsy proven IgAN cases. We compared the expression levels with histological severity or clinical data. Moreover, we investigated the expression of Gas6 and its receptors in cultured podocytes.

Results

In 28 of 31 cases, Gas6 was upregulated mainly in podocytes. In the other 3 cases, Gas6 expression was induced in endothelial and mesangial cells, which was similar to animal nephritis models. Among 28 podocyte type cases, the expression level of Gas6 correlated with the mesangial hypercellularity score of IgAN Oxford classification and urine protein excretion. It also inversely correlated with p27 expression in glomeruli. As for the receptors, Axl was mainly expressed in endothelial and mesangial cells, while Dtk was expressed in podocytes. In vitro, Dtk was expressed in cultured murine podocytes, and the expression of p27 was decreased by Gas6 stimulation.

Conclusions

Gas6 was uniquely upregulated in either endothelial/mesangial cells or podocytes in IgAN. The expression pattern can be used as a marker to classify IgAN. Gas6 has a possibility to be involved in not only mesangial proliferation via Axl, but also podocyte injury via Dtk in IgAN.     

Trehalose,an mTOR Independent Autophagy Inducer,Alleviates Human Podocyte Injury after Puromycin Aminonucleoside Treatment

Glomerular diseases are commonly characterized by podocyte injury including apoptosis, actin cytoskeleton rearrangement and detachment. However, the strategies for preventing podocyte damage remain insufficient. Recently autophagy has been regarded as a vital cytoprotective mechanism for keeping podocyte homeostasis. Thus, it is reasonable to utilize this mechanism to attenuate podocyte injury. Trehalose, a natural disaccharide, is an mTOR independent autophagy inducer. It is unclear whether trehalose alleviates podocyte injury. Therefore, we investigated the efficacy of trehalose in puromycin aminonucleoside (PAN)-treated podocytes which mimic cell damage in minimal change nephrotic syndrome in vitro. Human conditional immortalized podocytes were treated with trehalose with or without PAN. Autophagy was investigated by immunofluorescence staining for LC3 puncta and Western blotting for LC3, Atg5, p-AMPK, p-mTOR and its substrates. Podocyte apoptosis and necrosis were evaluated by flow cytometry and by measuring lactate dehydrogenase activity respectively. We also performed migration assay to examine podocyte recovery. It was shown that trehalose induced podocyte autophagy in an mTOR independent manner and without reactive oxygen species involvement. Podocyte apoptosis significantly decreased after trehalose treatment, while the inhibition of trehalose-induced autophagy abolished its protective effect. Additionally, the disrupted actin cytoskeleton of podocytes was partially reversed by trehalose, accompanying with less lamellipodias and diminished motility. These results suggested that trehalose induced autophagy in human podocytes and showed cytoprotective effects in PAN-treated podocytes.     

Losartan reverses glomerular podocytes injury induced by AngII via stabilizing the expression of GLUT1

Podocyte impairment is a key pathogenic even in the initiation and development of glomerular diseases associated with proteinuria. The type 2 diabetic patients is characterized by progressive increases in albuminuria which are associated with the development of characteristic histopathological features. Losartan had a benefit in decreasing albuminuria in type 2 diabetic patients,suggesting that losartan may have another effect other than blockade of the traditional renin–angiotensin system (RAS). However, the mechanism has remained undetermined. Glucose transporter 1 (GLUT1) is the predominant basal glucose transporter. In the kidney, GLUT1 was overexpressed predominantly in glomerular mesangial cells and in small vessels, rather than in podocytes. The increased glomerular GLUT1 mimicked diabetes-induced glomerular GLUT1 expression. In this study, we hypothesized that increased GLUT1 expression induced by angiotensinII (AngII) contributes to the progression of podocytes injury, losartan can block the effect of AngII and protect podocytes via stabilizing the expression of GLUT1, our results strongly suggest that losartan has a direct and protective effect on podocytes. This represents a novel mechanism by which losartan may protect podocyte from apoptotic death and improve podocyte function via stabilizing the expression of GLUT1. This finding underlines the crucial role of GLUT1 in the pathogenesis of podocyte injury and proteinuria.     

Cathepsin C is a novel mediator of podocyte and renal injury induced by hyperglycemia

A growing body of evidence suggests a role of proteolytic enzymes in the development of diabetic nephropathy. Cathepsin C (CatC) is a well-known regulator of inflammatory responses, but its involvement in podocyte and renal injury remains obscure. We used Zucker rats, a genetic model of metabolic syndrome and insulin resistance, to determine the presence, quantity, and activity of CatC in the urine. In addition to the animal study, we used two cellular models, immortalized human podocytes and primary rat podocytes, to determine mRNA and protein expression levels via RT-PCR, Western blot, and confocal microscopy, and to evaluate CatC activity. The role of CatC was analyzed in CatC-depleted podocytes using siRNA and glycolytic flux parameters were obtained from extracellular acidification rate (ECAR) measurements. In functional analyses, podocyte and glomerular permeability to albumin was determined. We found that podocytes express and secrete CatC, and a hyperglycemic environment increases CatC levels and activity. Both high glucose and non-specific activator of CatC phorbol 12-myristate 13-acetate (PMA) diminished nephrin, cofilin, and GLUT4 levels and induced cytoskeletal rearrangements, increasing albumin permeability in podocytes. These negative effects were completely reversed in CatC-depleted podocytes. Moreover, PMA, but not high glucose, increased glycolytic flux in podocytes. Finally, we demonstrated that CatC expression and activity are increased in the urine of diabetic Zucker rats. We propose a novel mechanism of podocyte injury in diabetes, providing deeper insight into the role of CatC in podocyte biology.