Activated macrophages down-regulate podocyte nephrin and podocin expression via stress-activated protein kinases

The development of proteinuria and glomerulosclerosis in kidney disease is associated with podocyte damage, including down-regulation of nephrin and podocin. Macrophages are known to induce renal injury, but the mechanisms involved are not fully understood. This study examined macrophage-mediated podocyte damage. Conditioned media (CM) from activated macrophages caused a 50-60% reduction in nephrin and podocin mRNA and protein expression in cultured mouse podocytes and rat glomeruli. This was abolished by a neutralizing anti-TNFα antibody. The addition of recombinant TNFα to podocytes or glomeruli caused a comparable reduction in podocyte nephrin and podocin expression to that of macrophage CM. Inhibition of c-Jun amino terminal kinase (JNK) or p38 kinase abolished the TNFα-induced reduction in nephrin and podocin expression. This study demonstrates that activated macrophages can induce podocyte injury via a TNFα-JNK/p38-dependent mechanism. This may explain, in part, the protective effects of JNK and p38 blockade in experimental kidney disease.     

Combination of direct renin inhibition with angiotensin type 1 receptor blockade improves aldosterone but does not improve kidney injury in the transgenic Ren2 rat

Enhanced renin-angiotensin-aldosterone system (RAAS) activation contributes to proteinuria and chronic kidney disease by increasing glomerular and tubulointerstitial oxidative stress, promotion of fibrosis. Renin activation is the rate limiting step in angiotensin (Ang II) and aldosterone generation, and recent work suggests direct renin inhibition improves proteinuria comparable to that seen with Ang type 1 receptor (AT(1)R) blockade. This is important as, even with contemporary use of AT(1)R blockade, the burden of kidney disease remains high. Thereby, we sought to determine if combination of direct renin inhibition with AT(1)R blockade in vivo, via greater attenuation of kidney oxidative stress, would attenuate glomerular and proximal tubule injury to a greater extent than either intervention alone. We utilized the transgenic Ren2 rat with increased tissue RAS activity and higher serum levels of aldosterone, which manifests hypertension and proteinuria. Ren2 rats were treated with renin inhibition (aliskiren), AT(1)R blockade (valsartan), the combination (aliskiren+valsartan), or vehicle for 21days. Compared to Sprague-Dawley controls, Ren2 rats displayed increased systolic pressure (SBP), circulating aldosterone, proteinuria and greater urine levels of the proximal tubule protein excretory marker beta-N-acetylglucosaminidase (β-NAG). These functional and biochemical alterations were accompanied by increases in kidney tissue NADPH oxidase subunit Rac1 and 3-nitrotyrosine (3-NT) content as well as fibronectin and collagen type III. These findings occurred in conjunction with reductions in the podocyte-specific protein podocin as well as the proximal tubule-specific megalin. Further, in transgenic animals there was increased tubulointerstitial fibrosis on light microscopy as well as ultrastructural findings of glomerular podocyte foot-process effacement and reduced tubular apical endosomal/lysosomal activity. Combination therapy led to greater reductions in SBP and serum aldosterone, but did not result in greater improvement in markers of glomerular and tubular injury (i.e. β-NAG) compared to either intervention alone. Further, combination therapy did not improve markers of oxidative stress and podocyte and proximal tubule integrity in this transgenic model of RAAS-mediated kidney damage despite greater reductions in serum aldosterone and BP levels.     

Stabilization of hypoxia-inducible factor 1α by cobalt chloride impairs podocyte morphology and slit-diaphragm function

Glomerular podocytes are the major components of the renal filtration barrier, and altered podocyte permselectivity is a key event in the pathogenesis of proteinuric conditions. Clinical conditions such as ischemia and sleep apnea and extreme physiological conditions such as high-altitude sickness are presented with renal hypoxia and are associated with significant proteinuria. Hypoxia is considered as an etiological factor in the progression of acute renal injury. A sustained increase in hypoxia-inducible factor 1α (HIF1α) is a major adaptive stimulus to the hypoxic conditions. Although the temporal association between hypoxia and proteinuria is known, the mechanism by which hypoxia elicits proteinuria remains to be investigated. Furthermore, stabilization of HIF1α is being considered as a therapeutic option to treat anemia in patients with chronic kidney disease. Therefore, in this study, we induced stabilization of HIF1α in glomerular regions in vivo and in podocytes in vitro upon exposure to cobalt chloride. The elevated HIF1α expression is concurrence with diminished expression of nephrin and podocin, podocyte foot-processes effacement, and significant proteinuria. Podocytes exposed to cobalt chloride lost their arborized morphology and cell-cell connections and also displayed cytoskeletal derangements. Elevation in expression of HIF1α is in concomitance with loss of nephrin and podocin in patients with diabetic nephropathy and chronic kidney disease. In summary, the current study suggests that HIF1α stabilization impairs podocyte function vis-à-vis glomerular permselectivity.     

Inhibition of mitochondrial fission protects podocytes from albumin-induced cell damage in diabetic kidney disease

AimsIdentifying the mechanisms that underlie progression from endothelial damage to podocyte damage, which leads to massive proteinuria, is an urgent issue that must be clarified to improve renal outcome in diabetic kidney disease (DKD). We aimed to examine the role of dynamin-related protein 1 (Drp1)-mediated regulation of mitochondrial fission in podocytes in the pathogenesis of massive proteinuria in DKD.MethodsDiabetes- or albuminuria-associated changes in mitochondrial morphology in podocytes were examined by electron microscopy. The effects of albumin and other diabetes-related stimuli, including high glucose (HG), on mitochondrial morphology were examined in cultured podocytes. The role of Drp1 in podocyte damage was examined using diabetic podocyte-specific Drp1-deficient mice treated with neuraminidase, which removes endothelial glycocalyx.ResultsNeuraminidase-induced removal of glomerular endothelial glycocalyx in nondiabetic mice led to microalbuminuria without podocyte damage, accompanied by reduced Drp1 expression and mitochondrial elongation in podocytes. In contrast, streptozotocin-induced diabetes significantly exacerbated neuraminidase-induced podocyte damage and albuminuria, and was accompanied by increased Drp1 expression and enhanced mitochondrial fission in podocytes. Cell culture experiments showed that albumin stimulation decreased Drp1 expression and elongated mitochondria, although HG inhibited albumin-associated changes in mitochondrial dynamics, resulting in apoptosis. Podocyte-specific Drp1-deficiency in mice prevented diabetes-related exacerbation of podocyte damage and neuraminidase-induced development of albuminuria. Endothelial dysfunction-induced albumin exposure is cytotoxic to podocytes. Inhibition of mitochondrial fission in podocytes is a cytoprotective mechanism against albumin stimulation, which is impaired under diabetic condition. Inhibition of mitochondrial fission in podocytes may represent a new therapeutic strategy for massive proteinuria in DKD.     

Emerging role of podocyte autophagy in the progression of diabetic nephropathy

Glomerular podocytes are pivotal in maintaining glomerular filtration barrier function. As severe podocyte injury results in proteinuria in patients with diabetic nephropathy, determining the pathogenesis of podocyte injury may contribute to the development of new treatments. We recently showed that autophagy is involved in the pathogenesis of diabetes-related podocyte injury. Insufficient podocyte autophagy and podocyte loss are observed in diabetic patients with massive proteinuria. Podocyte loss and massive proteinuria occur in high-fat diet-induced diabetic mice with podocyte-specific autophagy deficiency, with podocytes of these mice and of diabetic rats having huge damaged lysosomes. Sera from diabetic patients and from rodents with massive proteinuria cause autophagy insufficiency, resulting in lysosome dysfunction and apoptosis of cultured podocytes. These findings suggest the importance of autophagy in maintaining lysosome homeostasis in podocytes under diabetic conditions. Impaired autophagy may be involved in the pathogenesis of podocyte loss, leading to massive proteinuria in diabetic nephropathy.     

Advanced oxidation protein products decrease expression of nephrin and podocin in podocytes via ROS-dependent activation of p38 MAPK

Accumulation of plasma advanced oxidation protein products(AOPPs) promotes progression of proteinuria and glomerulo-sclerosis.To investigate the molecular basis of AOPPs-induced proteinuria,normal Sprague-Dawley rats were treated with AOPPs-modified rat serum albumin.The expression of glomerular podocyte slit diaphragm(PSD)-associated proteins,nephrin and podocin,was significantly decreased coincident with the onset of albuminuria in rats treated with AOPPs.Chronic inhibi-tion of NADPH oxidase by apocynin p...     

Increased urinary excretion of nephrin, podocalyxin, and βig-h3 in women with preeclampsia

Emerging evidence has shown that podocyte injury and reduced specific podocyte protein expressions contribute to proteinuria in preeclampsia. We collected urine specimens from women with preeclampsia to study whether podocyte-specific protein shedding is associated with renal barrier dysfunction. Urine specimens from women with normal pregnancies and from pregnant women complicated by chronic hypertension were used for comparison. We determined soluble podocyte slit protein nephrin levels in the urine specimens. Podocalyxin, βig-h3, and VEGF concentrations were also measured. We found that nephrin and podocalyxin were barely detectable in the urine specimens from normal pregnant women and from women with chronic hypertension. In preeclampsia, urinary nephrin and podocalyxin concentrations were significantly increased and highly correlated to each other, r(2) = 0.595. Nephrin and podocalyxin were also correlated with urine protein concentrations. βig-h3 was detected in the urine specimens from women with preeclampsia, and it is highly correlated with nephrin and podocalyxin concentrations in preeclampsia. βig-h3 was undetectable in normal pregnancy and pregnancy complicated by chronic hypertension. Elevated VEGF levels were also found in women with preeclampsia compared with those of normal pregnancy and pregnancy complicated by chronic hypertension. These results provide strong evidence that podocyte protein shedding occurs in preeclampsia, and their levels are associated with proteinuria. The finding of urinary βig-h3 excretion in preeclampsia suggests that increased transforming growth factor activity might also be involved in the kidney lesion in this pregnancy disorder.     

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.     

Restoration of Podocyte Structure and Improvement of Chronic Renal Disease in Transgenic Mice Overexpressing Renin

Background

Proteinuria is a major marker of the decline of renal function and an important risk factor of coronary heart disease. Elevated proteinuria is associated to the disruption of slit-diaphragm and loss of podocyte foot processes, structural alterations that are considered irreversible. The objective of the present study was to investigate whether proteinuria can be reversed and to identify the structural modifications and the gene/protein regulation associated to this reversal.

Methodology/Principal Findings

We used a novel transgenic strain of mouse (RenTg) that overexpresses renin at a constant high level. At the age of 12-month, RenTg mice showed established lesions typical of chronic renal disease such as peri-vascular and periglomerular inflammation, glomerular ischemia, glomerulosclerosis, mesangial expansion and tubular dilation. Ultrastructural analysis indicated abnormal heterogeneity of basement membrane thickness and disappearance of podocyte foot processes. These structural alterations were accompanied by decreased expressions of proteins specific of podocyte (nephrin, podocin), or tubular epithelial cell (E-cadherin and megalin) integrity. In addition, since TGFβ is considered the major pro-fibrotic agent in renal disease and since exogenous administration of BMP7 is reported to antagonize the TGFβ-induced phenotype changes in kidney, we have screened the expressions of several genes belonging in the TGFβ/BMP superfamily. We found that the endogenous inhibitors of BMPs such as noggin and Usag-1 were several-fold activated inhibiting the action of BMPs and thus reinforcing the deleterious action of TGFβ.Treatment with an AT1 receptor antagonist, at dose that did not decrease arterial pressure, gradually reduced albuminuria. This decrease was accompanied by re-expression of podocin, nephrin, E-cadherin and megalin, and reappearance of podocyte foot processes. In addition, expressions of noggin and Usag-1 were markedly decreased, permitting thus activation of the beneficial action of BMPs.

Conclusions/Significance

These findings show that proteinuria and alterations in the expression of proteins involved in the integrity and function of glomerular and renal epithelial phenotype are reversible events when the local action of angiotensin II is blocked, and provide hope that chronic renal disease can be efficiently treated.     

Interaction with podocin facilitates nephrin signaling

Mutations of NPHS1 or NPHS2, the genes encoding for the glomerular podocyte proteins nephrin and podocin, cause steroid-resistant proteinuria. In addition, mice lacking CD2-associated protein (CD2AP) develop a nephrotic syndrome that resembles NPHS mutations suggesting that all three proteins are essential for the integrity of glomerular podocytes. Although the precise glomerular function of either protein remains unknown, it has been suggested that nephrin forms zipper-like interactions to maintain the structure of podocyte foot processes. We demonstrate now that nephrin is a signaling molecule, which stimulates mitogen-activated protein kinases. Nephrin-induced signaling is greatly enhanced by podocin, which binds to the cytoplasmic tail of nephrin. Mutational analysis suggests that abnormal or inefficient signaling through the nephrin-podocin complex contributes to the development of podocyte dysfunction and proteinuria.     

Plasma membrane targeting of podocin through the classical exocytic pathway: effect of NPHS2 mutations

Podocytes are specialized epithelial cells of the glomerulus in the kidney, which interconnect at the top of the glomerular basement membrane through the slit diaphragm, an adherens-like junction that plays a crucial role in the glomerular filtration process. Podocin, a plasma membrane anchored stomatin-like protein, is expressed in lipid rafts at the insertion of the slit diaphragm in podocytes. Mutations in NPHS2 , the gene encoding podocin, are associated with inherited and sporadic cases of steroid-resistant nephrotic syndrome. Here, we show that brefeldin A induces accumulation of newly synthesized podocin in the endoplasmic reticulum, suggesting that podocin biosynthesis follows the classical secretory pathway, and we study the effect of 12 NPHS2 mutations associated with steroid-resistant nephrotic syndrome on the trafficking of the protein. We found that 9 podocin mutants were not targeted to the plasma membrane, 8 being retained in the endoplasmic reticulum and one being localized in late endosomes. Furthermore, by screening our database of patients with NPHS2 mutations, we found that podocin mutants retained in the endoplasmic reticulum are associated with earlier onset of the disease than those correctly targeted to the cell membrane. Our data suggest that most of NPHS2 mutations lead to retention of podocin in the endoplasmic reticulum and therefore provide a rationale for devising therapeutic approaches aimed at correcting the protein processing defect.     

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.     

Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice

To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4–5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease.     

Regulation of Podocyte Injury by CircHIPK3/FUS Complex in Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a major microvascular complication of diabetes mellitus and is one of the leading causes of end-stage kidney disease. Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that play important roles in various diseases, yet their roles in DKD are poorly understood. CircRNA HIPK3 (circHIPK3), a highly conserved circRNA, is closely related to various cellular functions, including cell proliferation and apoptosis. The association between circHIPK3 and diabetic complications has been well demonstrated in multiple previous studies. However, the role of circHIPK3 in podocyte injury in DKD remains unclear. Herein, we discovered that circHIPK3 expression is markedly elevated in cultured podocytes under high-glucose (HG) conditions and glomeruli of diabetic mice, which is closely associated with podocyte injury in DKD. Functionally, lentivirus-mediated knockdown of circHIPK3 dramatically suppresses HG-induced podocyte apoptosis in vitro. Therapeutically, silencing circHIPK3 by adeno-associated virus-mediated RNA interference ameliorates podocyte injury and albuminuria in STZ-induced diabetic mice. Mechanistically, circHIPK3 facilitates the enrichment of fused in sarcoma (FUS) on the ectodysplasin A2 receptor (EDA2R) promoter, resulting in the upregulation of EDA2R expression and activation of apoptotic signaling. Taken together, these results indicate circHIPK3/FUS/EDA2R axis as a therapeutic target for podocyte injury and DKD progression.     

Podocyte-associated proteins FAT, alpha-actinin-4 and filtrin are expressed in Langerhans islets of the pancreas

Nephrin is a crucial podocyte molecule in the kidney glomerular filtration barrier and it is also expressed in Langerhans islet beta cells of the pancreas. Recently, genetic mapping of proteinuric kidney disease genes and animal models have revealed further important molecules for the kidney filtration function including alpha-actinin-4, podocin, FAT, and NEPH1. This study was addressed to explore the pancreatic expression of the podocyte molecules podocin, FAT, alpha-actinin-4, NEPH1, NEPH2, filtrin/NEPH3, synaptopodin and CD2 associated protein (CD2AP). The mRNA and protein expressions were studied by RT-PCR and immunoblotting, and localization in the pancreas was investigated by immunofluorescence. Of the nephrin-associated podocyte proteins, filtrin/NEPH3, FAT, and alpha-actinin-4 were found to be expressed in the pancreas at the gene and protein level and localized to Langerhans islets. Immunoreactivity with the podocin antibody was detected mostly in the exocrine pancreas. NEPH1 and synaptopodin expression was detected only at the mRNA level. Further studies are needed to unravel the functional role of these podocyte-associated molecules in the pancreatic Langerhans islets.     

The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1

Blood filtration in the kidney glomerulus is essential for physiological homeostasis. The filtration apparatus of the kidney glomerulus is composed of three distinct components: the fenestrated endothelial cells, the glomerular basement membrane, and interdigitating foot processes of podocytes that form the slit diaphragm. Recent studies have demonstrated that podocytes play a crucial role in blood filtration and in the pathogenesis of proteinuria and glomerular sclerosis; however, the molecular mechanisms that organize the podocyte filtration barrier are not fully understood. In this study, we suggest that tight junction protein 1 (Tjp1 or ZO-1), which is encoded by Tjp1 gene, plays an essential role in establishing the podocyte filtration barrier. The podocyte-specific deletion of Tjp1 down-regulated the expression of podocyte membrane proteins, impaired the interdigitation of the foot processes and the formation of the slit diaphragm, resulting in glomerular dysfunction. We found the possibility that podocyte filtration barrier requires the integration of two independent units, the pre-existing epithelial junction components and the newly synthesized podocyte-specific components, at the final stage in glomerular morphogenesis, for which Tjp1 is indispensable. Together with previous findings that Tjp1 expression was decreased in glomerular diseases in human and animal models, our results indicate that the suppression of Tjp1 could directly aggravate glomerular disorders, highlights Tjp1 as a potential therapeutic target.     

Acute podocyte vascular endothelial growth factor (VEGF-A) knockdown disrupts alphaVbeta3 integrin signaling in the glomerulus

Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGF(KD)) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ~20% of non-induced controls and urine VEGF-A to ~30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alpha(V)beta(3) integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta(3) integrin and neuropilin-1 in the kidney in vivo and in VEGF(KD) podocytes. Podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGF(KD) podocytes downregulates fibronectin and disrupts alpha(V)beta(3) integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alpha(V)beta(3) integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure.