Ameliorates Podocyte Injury Research Articles

Fine-tuning of NFκB by glycogen synthase kinase 3β directs the fate of glomerular podocytes upon injury

NFκB is regulated by a myriad of signaling cascades including glycogen synthase kinase (GSK) 3β and plays a Janus role in podocyte injury. In vitro, lipopolysaccharide or adriamycin elicited podocyte injury and cytoskeletal disruption, associated with NFκB activation and induced expression of NFκB target molecules, including pro-survival Bcl-xL and podocytopathic mediators like MCP-1, cathepsin L and B7-1. Broad range inhibition of NFκB diminished the expression of all NFκB target genes, restored cytoskeleton integrity, but potentiated apoptosis. In contrast, blockade of GSK3β by lithium or TDZD-8, mitigated the expression of podocytopathic mediators, ameliorated podocyte injury, but barely affected Bcl-xL expression or sensitized apoptosis. Mechanistically, GSK3β was sufficient and essential for RelA/p65 phosphorylation specifically at serine 467, which specifies the expression of selective NFκB target molecules, including podocytopathic mediators, but not Bcl-xL. In vivo, lithium or TDZD-8 therapy improved podocyte injury and proteinuria in mice treated with lipopolysaccharide or adriamycin, concomitant with suppression of podocytopathic mediators but retained Bcl-xL in glomerulus. Broad range inhibition of NFκB conferred similar but much weakened antiproteinuric and podoprotective effects accompanied with a blunted glomerular expression of Bcl-xL and marked podocyte apoptosis. Thus, the GSK3β dictated fine-tuning of NFκB may serve as a novel therapeutic target for podocytopathy.

The novel role of TRPC6 in vitamin D ameliorating podocyte injury in STZ-induced diabetic rats.

Podocyte injury plays a critical role in the development and progression of diabetic nephropathy (DN). Over expression of TRPC6 on the podocytes has been revealed to cause podocyte injury in non-diabetic states. Besides, the emerging evidence from clinic revealed that vitamin D could reduce albuminuria and improve renal function, which was associated with podocyte protection. Our study aimed to investigate whether calcitriol ameliorating podocyte impairment is associated with regulation of the expression of TRPC6 in STZ-induced rats. Sprague-Dawley rats were randomly divided into three groups: normal control, DN, and DN treated with calcitriol (DN+VD); VD rats were treated with 0.1μg/kg/d calcitriol by gavage. DN model rats were established by intraperitoneal injections of streptozocin. The rats were sacrificed after 18weeks treatment. DN rats exhibited increased proteinuria accompanied by elevated TRPC6 expression. Treatment with calcitriol not only reduced proteinuria, but also normalized TRPC6 expression. Meanwhile, in DN rats, the expression of podocyte specific markers including nephrin and podocin was significantly decreased, accompanied by increased desmin, a marker of podocyte injury. Treatment with calcitriol reversed above changes. In addition, vitamin D receptor (VDR) was significantly decreased, whereas this reduction was attenuated by the calcitriol treatment. Moreover, TRPC6 was positively correlated with both 24h urinary protein and desmin. In contrast, TRPC6 was negatively correlated with both VDR and nephrin expression in podocytes. Calcitriol can ameliorate podocyte injury, which is contributed by the inhibition of enhanced TRPC6 expression in the early stages of DN rats.

The PI3K/p-Akt signaling pathway participates in calcitriol ameliorating podocyte injury in DN rats

ObjectivesThe present study aimed to investigate the relationship between PI3K/p-Akt signaling pathway and podocyte impairment in DN rats as well as the protective effect of calcitriol. MethodsSD rats were randomly divided into four groups: normal control (NC), normal treated with calcitriol (NC+VD), diabetic nephropathy (DN) and DN treated with calcitriol (DN+VD); all VD rats were treated with 0.1μg/kg/d calcitriol by gavage. DN model rats were established by intraperitoneal injections of streptozotocin (STZ). Rats were sacrificed after 18weeks of treatments. ResultsIn the present study, increased albuminuria was observed as early as 3weeks of diabetes and continued to increase more than six-fold throughout the length of the study (18weeks). Expectedly, animals receiving the treatment with calcitriol was protected from this increase, lower about one third. Meanwhile, the expression of podocyte specific markers, including nephrin and podocin, together with PI3K/p-Akt was significantly decreased in DN rats, whereas calcitriol reversed these above changes accompanied by elevated the expression levels of VDR. Additionally, a positive correlation was observed between the expression levels of nephrin and VDR (r=0.776, P<0.05). Likewise, the expression of nephrin was positively correlated with both PI3K-p85 and p-Akt (r=0.736, P<0.05; r=0.855, P<0.05, respectively). ConclusionPI3K/p-Akt signaling pathway participates in calcitriol ameliorating podocyte injury in DN rats. The manipulation of calcitriol might act as a promising therapeutic intervention for diabetic nephropathy.

Grape seed proanthocyanidin extracts ameliorate podocyte injury by activating peroxisome proliferator-activated receptor-γ coactivator 1α in low-dose streptozotocin-and high-carbohydrate/high-fat diet-induced diabetic rats.

Podocytes are part of the glomerular filtration membrane in kidney and serve to prevent the filtration of protein from the blood. Several evidences suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of diabetic nephropathy and it is an early event in podocyte injury. Mitochondrial dysfunction promotes oxidative stress that can favor the development of podocyte injury. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) was considered to be a major regulator of metabolic homeostasis and mitochondrial function. Some studies indicated that polyphenols may improve mitochondrial dysfunction, maintain the podocyte integrity and have therapeutic effects on glomerular diseases by promoting PGC-1α expression. Our study investigated whether grape seed proanthocyanidin extracts (GSPE), a strong antioxidant, ameliorate podocyte injury by activating PGC-1α in low-dose streptozotocin-and high-carbohydrate/high-fat diet-induced diabetic rats. After 16 weeks of GSPE treatment, GSPE slightly increased the body weight and decreased plasma glucose, food intake, water intake and urine volume in diabetic rats. Further, GSPE significantly decreased 24 h albumin levels and increased the expression of nephrin and podocalyxin. The antioxidant levels were improved and the cellular damage of kidney in diabetic rats was also relieved effectively after the treatment. Moreover, GSPE increased the mRNA expression of mitochondrial biogenesis factors and mitochondrial DNA content. Finally, GSPE activated the expression of PGC-1α, silent mating type information regulation 2 homolog 1 (SIRT1) and AMP-activated protein kinase (AMPK). These results suggest that GSPE ameliorate podocyte injury in diabetic nephropathy by the activation of AMPK-SIRT1-PGC-1α signalling, which appears to inhibit oxidative stress and mitochondrial dysfunction in the kidney.

Podocyte-specific RAP1GAP expression contributes to focal segmental glomerulosclerosis–associated glomerular injury

Injury to the specialized epithelial cells of the glomerulus (podocytes) underlies the pathogenesis of all forms of proteinuric kidney disease; however, the specific genetic changes that mediate podocyte dysfunction after injury are not fully understood. Here, we performed a large-scale insertional mutagenic screen of injury-resistant podocytes isolated from mice and found that increased expression of the gene Rap1gap, encoding a RAP1 activation inhibitor, ameliorated podocyte injury resistance. Furthermore, injured podocytes in murine models of disease and kidney biopsies from glomerulosclerosis patients exhibited increased RAP1GAP, resulting in diminished glomerular RAP1 activation. In mouse models, podocyte-specific inactivation of Rap1a and Rap1b induced massive glomerulosclerosis and premature death. Podocyte-specific Rap1a and Rap1b haploinsufficiency also resulted in severe podocyte damage, including features of podocyte detachment. Over-expression of RAP1GAP in cultured podocytes induced loss of activated β1 integrin, which was similarly observed in kidney biopsies from patients. Furthermore, preventing elevation of RAP1GAP levels in injured podocytes maintained β1 integrin-mediated adhesion and prevented cellular detachment. Taken together, our findings suggest that increased podocyte expression of RAP1GAP contributes directly to podocyte dysfunction by a mechanism that involves loss of RAP1-mediated activation of β1 integrin.

Carvedilol ameliorates early diabetic nephropathy in streptozotocin-induced diabetic rats.

Diabetic nephropathy results in end-stage renal disease. On the other hand, carvedilol has been reported to have various pharmacological properties. The aim of this study therefore is to evaluate the possible protective effect of carvedilol on streptozotocin-induced early diabetic nephropathy and various mechanisms underlie this effect in rats. Single i.p. injection of streptozotocin (65 mg/kg) was administered to induce early diabetic nephropathy in Wistar rats. Oral administration of carvedilol at a dose level of 1 and 10 mg/kg daily for 4 weeks resulted in nephroprotective effect as evident by significant decrease in serum creatinine level, urinary albumin/creatinine ratio, and kidney index as well as renal levels of malondialdehyde, nitric oxide, tumor necrosis factor-α, and cyclooxygenase-2 with a concurrent increase in creatinine clearance and renal reduced glutathione level compared to diabetic untreated rats. The protective effect of carvedilol was confirmed by renal histopathological examination. The electron microscopic examination indicated that carvedilol could effectively ameliorate glomerular basement membrane thickening and podocyte injury. In conclusion, carvedilol protects rats against streptozotocin-induced early diabetic nephropathy possibly, in part, through its antioxidant as well as anti-inflammatory activities, and ameliorating podocyte injury.

Atorvastatin ameliorates podocyte injury in patients with type 2 diabetes complicated with dyslipidemia

We examined the effects of atorvastatin on urinary podocyte excretion. Thirteen patients with type 2 diabetes receiving 2.5mg of rosuvastatin were recruited and the medication was switched to 10mg of atorvastatin for a 24-week period. With the switch to atorvastatin, the urinary excretion of podocytes was significantly reduced.

Mesenchymal Stem Cells Ameliorate Podocyte Injury and Proteinuria in a Type 1 Diabetic Nephropathy Rat Model

Mesenchymal stem cells (MSC) attenuate albuminuria and preserve normal renal histology in diabetic mice. However, the effects of MSC on glomerular podocyte injury remain uncertain. The aim of this study was to evaluate the effects of MSC on podocyte injury in streptozotocin (STZ)-induced diabetic rats. Thirty days after diabetes induction by STZ injection (65 mg/kg, intraperitoneally) in Sprague-Dawley rats, the diabetic rats received medium or 2 × 106 enhanced green fluorescent protein-labeled MSC via the renal artery. In vivo tracking of MSC was followed by immunofluorescence analysis. Diabetes-related physical and biochemical parameters were measured on day 60 after the MSC infusion. The expression of podocyte markers (nephrin and podocin), podocyte survival factors (VEGF and BMP-7), and the ultrastructural pathology of podocytes were also assessed. MSC were only detected in the glomeruli from the left kidney receiving MSC infusion. Compared with medium-treated diabetic rats, rats treated with MSC showed a suppressed increase in kidney weight, kidney to body weight index, creatinine clearance rate, and urinary albumin to creatinine ratio; however, the treatment had no effect on blood glucose or body weight levels. Furthermore, the MSC treatment reduced the loss of podocytes, effacement of foot processes, widening of foot processes, thickening of glomerular basal membrane (GBM), and loss of glomerular nephrin and podocin. Most important, MSC-injected kidneys expressed higher levels of BMP-7 but not of VEGF. Our results clearly demonstrated that intra-arterial administration of MSC prevented the development of albuminuria as well as any damage to or loss of podocytes, though there was no improvement in blood sugar levels. The protective effects of MSC may be mediated in part by increasing BMP-7 secretion.

Genipin Inhibits Mitochondrial Uncoupling Protein 2 Expression and Ameliorates Podocyte Injury in Diabetic Mice

Diabetic nephropathy (DN) is one of the most common causes of end stage renal disease (ESRD) in China, which requires renal replacement therapy. Recent investigations have suggested an essential role of podocyte injury in the initial stage of DN. This study investigated the potential therapeutic role of genipin, an active extract from a traditional Chinese medicine, on progression of DN in diabetic mice induced by intraperitoneally injection of streptozocin (STZ). In diabetic mice, orally administration of genipin postponed the progression of DN, as demonstrated by ameliorating body weight loss and urine albumin leakage, attenuating glomerular basement membrane thickness, restoring the podocyte expression of podocin and WT1 in diabetic mice. The protective role of genipin on DN is probably through suppressing the up-regulation of mitochondrial uncoupling protein 2 (UCP2) in diabetic kidneys. Meanwhile, through inhibiting the up-regulation of UCP2, genipin restores podocin and WT1 expression in cultured podocytes and attenuates glucose-induced albumin leakage through podocytes monolayer. Therefore, these results revealed that genipin inhibited UCP2 expression and ameliorated podocyte injury in DN mice.

Natriuretic Peptide Receptor Guanylyl Cyclase-A Protects Podocytes from Aldosterone-Induced Glomerular Injury

Natriuretic peptides produced by the heart in response to cardiac overload exert cardioprotective and renoprotective effects by eliciting natriuresis, reducing BP, and inhibiting cell proliferation and fibrosis. These peptides also antagonize the renin-angiotensin-aldosterone system, but whether this mechanism contributes to their renoprotective effect is unknown. Here, we examined the kidneys of mice lacking the guanylyl cyclase-A (GC-A) receptor for natriuretic peptides under conditions of high aldosterone and high dietary salt. After 4 weeks of administering aldosterone and a high-salt diet, GC-A knockout mice, but not wild-type mice, exhibited accelerated hypertension with massive proteinuria. Aldosterone-infused GC-A knockout mice had marked mesangial expansion, segmental sclerosis, severe podocyte injury, and increased oxidative stress. Reducing the BP with hydralazine failed to lessen such changes; in contrast, blockade of the renin-angiotensin-aldosterone system markedly reduced albuminuria, ameliorated podocyte injury, and reduced oxidative stress. Furthermore, treatment with the antioxidant tempol significantly reduced albuminuria and abrogated the histologic changes. In cultured podocytes, natriuretic peptides inhibited aldosterone-induced mitogen-activated protein kinase phosphorylation. Taken together, these results suggest that renoprotective properties of the endogenous natriuretic peptide/GC-A system may result from the local inhibition of the renin-angiotensin-aldosterone system and oxidative stress in podocytes.

Induction of Podocyte-Derived VEGF Ameliorates Podocyte Injury and Subsequent Abnormal Glomerular Development Caused by Puromycin Aminonucleoside

Our previous studies using puromycin aminonucleoside (PAN) established that podocyte damage leads to glomerular growth arrest during development and glomerulosclerosis later in life. This study examined the potential benefit of maintaining podocyte-derived VEGF in podocyte defense and survival after PAN injury using conditional transgenic podocytes and mice, in which human VEGF-A (hVEGF) transgene expression is controlled by tetracycline responsive element (TRE) promoter and reverse tetracycline transactivator (rtTA) in podocytes. In vitro experiments used primary cultured podocytes harvested from mice carrying podocin-rtTA and TRE-hVEGF transgenes, in which hVEGF can be induced selectively. Induction of VEGF in PAN-exposed podocytes resulted in preservation of intrinsic VEGF, α-actinin-4 and synaptopodin, antiapoptotic marker Bcl-xL/Bax, as well as attenuation in apoptotic marker cleaved/total caspase-3. In vivo, compared with genotype controls, PAN-sensitive neonatal mice with physiologically relevant levels of podocyte-derived VEGF showed significantly larger glomeruli. Furthermore, PAN-induced up-regulation of desmin, down-regulation of synaptopodin and nephrin, and disruption of glomerular morphology were significantly attenuated in VEGF-induced transgenic mice. Our data indicate that podocyte-derived VEGF provides self-preservation functions, which can rescue the cell after injury and preempt subsequent deterioration of the glomerulus in developing mice.

Blockade of Wnt/β-Catenin Signaling by Paricalcitol Ameliorates Proteinuria and Kidney Injury

Recent studies implicate Wnt/β-catenin signaling in podocyte dysfunction. Because vitamin D analogs can inhibit β-catenin in other tissues, we tested whether the vitamin D analog paricalcitol could ameliorate podocyte injury, proteinuria, and renal fibrosis in adriamycin (ADR) nephropathy. Compared with vehicle-treated controls, paricalcitol preserved expression of nephrin, podocin, and WT1; prevented proteinuria; and reduced glomerulosclerotic lesions induced by ADR. Paricalcitol also inhibited expression of proinflammatory cytokines, reduced renal infiltration of monocytes/macrophages, hampered activation of renal myofibroblasts, and suppressed expression of the fibrogenic TGF-β1, CTGF, fibronectin, and types I and III collagen. Selective suppression of renal Wnt4, Wnt7a, Wnt7b, and Wnt10a expression after ADR accompanied these renoprotective effects of paricalcitol. Significant upregulation of β-catenin, predominantly in podocytes and tubular epithelial cells, accompanied renal injury; paricalcitol largely abolished this induction of renal β-catenin and inhibited renal expression of Snail, a downstream effector of Wnt/β-catenin signaling. Administration of paricalcitol also ameliorated established proteinuria. In vitro, paricalcitol induced a physical interaction between the vitamin D receptor and β-catenin in podocytes, which led to suppression of β-catenin-mediated gene transcription. In summary, these findings suggest that paricalcitol prevents podocyte dysfunction, proteinuria, and kidney injury in adriamycin nephropathy by inhibiting Wnt/β-catenin signaling.

Hepatocyte growth factor signaling ameliorates podocyte injury and proteinuria

Hepatocyte growth factor (HGF) is a potent antifibrotic protein that inhibits kidney fibrosis through several mechanisms. To study its role in podocyte homeostasis, injury, and repair in vivo, we generated conditional knockout mice in which the HGF receptor, c-met, was specifically deleted in podocytes using the Cre-LoxP system. Mice with podocyte-specific ablation of c-met (podo-met(-/-)) developed normally. No albuminuria or overt pathologic lesions were detected up to 6 months of age, suggesting that HGF signaling is dispensable for podocyte maturation, survival, and function under normal physiologic conditions. However, after adriamycin treatment, podo-met(-/-) mice developed more severe podocyte injury and albuminuria than their control littermates. Ablation of c-met also resulted in more profound suppression of Wilms tumor 1 (WT1) and nephrin expression, and podocyte apoptosis after injury. When HGF was expressed ectopically in vivo, it ameliorated adriamycin-induced albuminuria, preserved WT1 and nephrin expression, and inhibited podocyte apoptosis. However, exogenous HGF failed to significantly reduce albuminuria in podo-met(-/-) mice, suggesting that podocyte-specific c-met activation by HGF confers renal protection. In vitro, HGF was able to preserve WT1 and nephrin expression in cultured podocytes after adriamycin treatment. HGF also protected podocytes from apoptosis induced by a lethal dose of adriamycin primarily through a phosphoinositide 3-kinase (PI3K)/Akt-dependent pathway. Collectively, these results indicate that HGF/c-met signaling has an important role in protecting podocytes from injury, thereby reducing proteinuria.

L‐carnitine ameliorate podocyte injury in diabetic nephropathy by increasing the glucose uptake

High glucose could change podocyte gene expression and subsequently induce podocyte damage through altered glucose metabolism. L‐carnitine is known to play a beneficial role in diabetes, however, there are no studies on the effect of L‐carnitine on podocyte alteration in high glucose condition. This study was to investigate whether L‐carnitine can attenuate diabetic podocyte injury through the prevention of slit diaphragm proteins loss. L‐carnitine treatment group showed an increased glucose uptake compared to control group, suggesting that utilization of glucose in the podocyte increased by L‐carnitine. L‐carnitine treatment also prevented the decreased mRNA expressions of nephrin and podocin in high glucose‐stimulated podocytes. However, the mRNA expression of CD2AP and ¥á‐actinin‐4 were not significantly changed by high glucose condition. Taken together, L‐carnitine could increase glucose uptake of podocyte in high glucose condition and its mechanism may be at least partly related to the upregulation of nephrin and podocin. Our results give information on the understanding the beneficial effects of L‐carnitine in diabetic nephropathy.

Triptolide protects podocytes from puromycin aminonucleoside induced injury in vivo and in vitro

Extracts of Tripterygium wilfordii Hook F have been used to treat glomerulonephritis for more than 30 years in China with dramatic antiproteinuric effects. Triptolide, a diterpene triepoxide, is one of the major active components of these extracts. To clarify its antiproteinuric effects we induced podocyte injury by puromycin aminonucleoside. Triptolide effectively reduced the proteinuria induced by puromycin in nephrotic rats without reducing the glomerular filtration rate. The antiproteinuric effect was associated with improvement in the foot process effacement, a decrease in the podocyte injury marker desmin as well as the restoration of nephrin and podocin expression and distribution. In cultured mouse podocytes triptolide pretreatment prevented the puromycin-induced disruption of the actin cytoskeleton and microfilament-associated synaptopodin while protecting nephrin and podocin expression. Triptolide suppressed reactive oxygen species generation and p38 mitogen-activated protein kinase activation while restoring RhoA signaling activity. These results show that triptolide ameliorates puromycin aminonucleoside-mediated podocyte injury in vivo and in vitro.

Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

Angiotensin II Type 1 Receptor Blockade Inhibits the Development and Progression of HIV-Associated Nephropathy in a Mouse Model

HIV-associated nephropathy (HIVAN) is characterized by a collapsed glomerular capillary tuft with hyperplasia and hypertrophy of podocytes. Recently generated were conditional transgenic mice (podocin/Vpr) that express one of the HIV-1 accessory genes, vpr, selectively in podocytes using podocin promoter and Tet-on system. These transgenic mice developed renal injury similar to HIVAN when treated with doxycycline for 8 to 12 wk. This study demonstrated that nephron reduction by heminephrectomy markedly enhanced phenotypic changes of podocytes and led to severe FSGS within 4 wk. Nephrotic-range proteinuria was observed already at 2 wk, together with dedifferentiation and dysregulation of podocytes, indicated by decreased expression of nephrin, synaptopodin, and Wilms' tumor 1 protein and increased expression of Ki-67. The acceleration of phenotypic changes of podocytes, proteinuria, and subsequent glomerulosclerosis by heminephrectomy was almost completely inhibited by angiotensin II type 1 receptor (AT1R) blocker olmesartan. In contrast, the renoprotective effect of the calcium channel antagonist azelnidipine was minimal, although it lowered systemic BP to the same level as olmesartan, demonstrating that the inhibitory effect of AT1R blocker was independent of systemic BP. Olmesartan also reduced proteinuria and prevented glomerulosclerosis even by the delayed treatment, which was initiated after the podocyte injury appeared. These data suggest that nephron reduction exaggerates podocyte injury and subsequent glomerulosclerosis, possibly through glomerular hypertension, in the mouse model of HIVAN. AT1R blockade could be beneficial in the treatment of HIVAN by ameliorating podocyte injury by avoiding the vicious cycle of nephron reduction and glomerular hypertension.

Fluvastatin Ameliorates Podocyte Injury in Proteinuric Rats via Modulation of Excessive Rho Signaling

Statins have been reported to confer renoprotection in several experimental models of renal disease through pleiotropic actions. The roles of statins in glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of fluvastatin on podocyte and tubulointerstitial injury in puromycin aminonucleoside (PAN)-induced nephrosis. PAN induced massive proteinuria and serum creatinine elevation on day 7, which were significantly suppressed by fluvastatin. Immunofluorescence studies of podocyte-associated proteins nephrin and podocin revealed diminished and discontinuous staining patterns in rats with PAN nephrosis, indicating severe podocyte injury. Fluvastatin treatment dramatically mitigated the abnormal staining profiles. Reduction of nephrin expression by PAN and its reversal by fluvastatin were confirmed by quantitative analyses. By electron microscopy, effacement of foot processes was ameliorated in fluvastatin-treated rats. Fluvastatin also mitigated tubulointerstitial damage in PAN nephrosis, with the repression of PAN-induced NF-kappaB and activator protein-1 activation in the kidneys. In addition, expression of activated membrane-bound small GTPase RhoA was markedly increased in the glomeruli of PAN nephrosis, which was inhibited by fluvastatin treatment. In cultured podocytes, fluvastatin suppressed PAN-evoked activation of RhoA and actin cytoskeletal reorganization. Furthermore, fasudil, a specific Rho-kinase inhibitor, successfully ameliorated PAN-induced podocyte damage and proteinuria. In summary, fluvastatin alleviated podocyte and tubulointerstitial injury in PAN nephrosis. The beneficial effects of fluvastatin on podocytes can be attributable to direct modulation of excessive RhoA activity. Our data suggest a therapeutic role for statins in clinical conditions that are relevant to podocyte injury.

Induction of antioxidant enzymes in murine podocytes precedes injury by puromycin aminonucleoside

An imbalance between the generation of reactive oxygen species (ROS) and antioxidant defense mechanisms has been suggested to play an important role in podocyte injury in nephrotic syndrome. Experimental nephrotic syndrome induced by injection of puromycin aminonucleoside (PAN) into rats is a well-established model of nephrotic syndrome, and can be largely prevented by pretreatment with antioxidant enzymes (AOE), suggesting that podocyte injury may be mediated by ROS. To test the hypothesis that PAN-induced podocyte injury is modulated in part by podocyte antioxidant defenses, we analyzed AOE activities, lipid peroxidation products, and relative ROS levels in podocytes using our recently reported in vitro model of PAN-induced podocyte injury. PAN treatment induced early increases in both podocyte hydrogen peroxide and superoxide and later increases in lipid peroxidation products. Compared to baseline activities, PAN also induced significant changes in the major cellular AOE activities (maximum increases of 151% for catalase, 134% for superoxide dismutase, and 220% for glutathione peroxidase vs. time-matched controls). These changes largely preceded the development of extensive podocyte process retraction and actin filament disruption, which was maximal at 7 days. These results demonstrate that (1) PAN treatment induces significant early changes in podocyte ROS, (2) podocytes can mount an antioxidant defense against oxidant stress, and (3) this protective response is initiated prior to the development of extensive oxidant-induced podocyte structural injury. These findings suggest that enhancement of podocyte AOE activities represent a potential therapeutic target to protect from or ameliorate podocyte injury during nephrotic syndrome.