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.     

Bradykinin inhibits high glucose- and growth factor-induced collagen synthesis in mesangial cells through the B2-kinin receptor

Mesangial matrix expansion is an early lesion leading to glomeruloclerosis and chronic renal diseases. A beneficial effect is achieved with angiotensin I-converting enzyme inhibitors (ACEI), which also favor bradykinin (BK) B2 receptor (B2R) activation. To define the underlying mechanism, we hypothesized that B2R activation could be a negative regulator of collagen synthesis in mesangial cells (MC). We investigated the effect of BK on collagen synthesis and signaling in MC. Inflammation was evaluated by intercellular adhesion molecule-1 (ICAM-1) expression. BK inhibited collagen I and IV synthesis stimulated by high glucose, epithelial growth factor (EGF), and transforming growth factor-β (TGF-β) but did not alter ICAM-1. Inhibition of collagen synthesis was B2R but not B1R mediated. PKC or phosphatidylinositol 3-kinase (PI3K) inhibitors mimicked the BK effect. B2R activation inhibited TGF-β- and EGF-induced Erk1/2, Smad2/3, Akt S473, and EGFR phosphorylation. A phosphatase inhibitor prevented BK effects. The in vivo impact of B2R on mesangial matrix expansion was assessed in streptozotocin-diabetic rodents. Deletion of B2R increased mesangial matrix expansion and albuminuria in diabetic mice. In diabetic rats, matrix expansion and albuminuria were prevented by ACEI but not by ACEI and B2R antagonist cotreatment. Consistently, the lowered BK content of diabetic glomeruli was restored by ACEI. In conclusion, deficient B2R activation aggravated mesangial matrix expansion in diabetic rodents whereas B2R activation reduced MC collagen synthesis by a mechanism targeting Erk1/2 and Akt, common pathways activated by EGF and TGF-β. Taken together, the data support the hypothesis of an antifibrosing effect of B2R activation.     

Genetic deficiency of Smad3 protects the kidneys from atrophy and interstitial fibrosis in 2K1C hypertension

Although the two-kidney, one-clip (2K1C) model is widely used as a model of human renovascular hypertension, mechanisms leading to the development of fibrosis and atrophy in the cuffed kidney and compensatory hyperplasia in the contralateral kidney have not been defined. Based on the well-established role of the transforming growth factor (TGF)-β signaling pathway in renal fibrosis, we tested the hypothesis that abrogation of TGF-β/Smad3 signaling would prevent fibrosis in the cuffed kidney. Renal artery stenosis (RAS) was established in mice with a targeted disruption of exon 2 of the Smad3 gene (Smad3 KO) and wild-type (WT) controls by placement of a polytetrafluoroethylene cuff on the right renal artery. Serial pulse-wave Doppler ultrasound assessments verified that blood flow through the cuffed renal artery was decreased to a similar extent in Smad3 KO and WT mice. Two weeks after surgery, systolic blood pressure and plasma renin activity were significantly elevated in both the Smad3 KO and WT mice. The cuffed kidney of WT mice developed renal atrophy (50% reduction in weight after 6 wk, P < 0.0001), which was associated with the development of interstitial fibrosis, tubular atrophy, and interstitial inflammation. Remarkably, despite a similar reduction of renal blood flow, the cuffed kidney of the Smad3 KO mice showed minimal atrophy (9% reduction in weight, P = not significant), with no significant histopathological alterations (interstitial fibrosis, tubular atrophy, and interstitial inflammation). We conclude that abrogation of TGF-β/Smad3 signaling confers protection against the development of fibrosis and atrophy in RAS.     

Monocyte/macrophage chemokine receptor CCR2 mediates diabetic renal injury

Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN). C-C chemokine receptor (CCR)2 regulates monocyte/macrophage migration into injured tissues. However, the direct role of CCR2-mediated monocyte/macrophage recruitment in diabetic kidney disease remains unclear. We report that pharmacological blockade or genetic deficiency of CCR2 confers kidney protection in Ins2(Akita) and streptozotocin (STZ)-induced diabetic kidney disease. Blocking CCR2 using the selective CCR2 antagonist RS504393 for 12 wk in Ins2(Akita) mice significantly attenuated albuminuria, the increase in blood urea nitrogen and plasma creatinine, histological changes, and glomerular macrophage recruitment compared with vehicle. Furthermore, mice lacking CCR2 (CCR2(-/-)) mimicked CCR2 blockade by reducing albuminuria and displaying less fibronectin mRNA expression and inflammatory cytokine production compared with CCR2(+/+) mice, despite comparable blood glucose levels. Bone marrow-derived monocytes from CCR2(+/+) or CCR2(-/-) mice adoptively transferred into CCR2(-/-) mice reversed the renal tissue-protective effect in diabetic CCR2(-/-) mice as evaluated by increased urinary albumin excretion and kidney macrophage recruitment, indicating that CCR2 is not required for monocyte migration from the circulation into diabetic kidneys. These findings provide evidence that CCR2 is necessary for monocyte/macrophage-induced diabetic renal injury and suggest that blocking CCR2 could be a novel therapeutic approach in the treatment of DN.     

Diabetic kidney disease in FVB/NJ Akita mice: temporal pattern of kidney injury and urinary nephrin excretion

Akita mice are a genetic model of type 1 diabetes. In the present studies, we investigated the phenotype of Akita mice on the FVB/NJ background and examined urinary nephrin excretion as a marker of kidney injury. Male Akita mice were compared with non-diabetic controls for functional and structural characteristics of renal and cardiac disease. Podocyte number and apoptosis as well as urinary nephrin excretion were determined in both groups. Male FVB/NJ Akita mice developed sustained hyperglycemia and albuminuria by 4 and 8 weeks of age, respectively. These abnormalities were accompanied by a significant increase in systolic blood pressure in 10-week old Akita mice, which was associated with functional, structural and molecular characteristics of cardiac hypertrophy. By 20 weeks of age, Akita mice developed a 10-fold increase in albuminuria, renal and glomerular hypertrophy and a decrease in the number of podocytes. Mild-to-moderate glomerular mesangial expansion was observed in Akita mice at 30 weeks of age. In 4-week old Akita mice, the onset of hyperglycemia was accompanied by increased podocyte apoptosis and enhanced excretion of nephrin in urine before the development of albuminuria. Urinary nephrin excretion was also significantly increased in albuminuric Akita mice at 16 and 20 weeks of age and correlated with the albumin excretion rate. These data suggest that: 1. FVB/NJ Akita mice have phenotypic characteristics that may be useful for studying the mechanisms of kidney and cardiac injury in diabetes, and 2. Enhanced urinary nephrin excretion is associated with kidney injury in FVB/NJ Akita mice and is detectable early in the disease process.     

Smad3 mediates ANG II-induced hypertensive kidney disease in mice

Although Smad3 is a key mediator for fibrosis, its functional role and mechanisms in hypertensive nephropathy remain largely unclear. This was examined in the present study in a mouse model of hypertension induced in Smad3 knockout (KO) and wild-type (WT) mice by subcutaneous angiotensin II infusion and in vitro in mesangial cells lacking Smad3. After angiotensin II infusion, both Smad3 KO and WT mice developed equally high levels of blood pressure. However, disruption of Smad3 prevented angiotensin II-induced kidney injury by lowering albuminuria and serum creatinine (P < 0.01), inhibiting renal fibrosis such as collagen type I and IV, fibronectin, and α-SMA expression (all P < 0.01), and blocking renal inflammation including macrophage and T cell infiltration and upregulation of IL-1β, TNF-α, and monocyte chemoattractant protein-1 in vivo and in vitro (all P < 0.001). Further studies revealed that blockade of angiotensin II-induced renal transforming growth factor (TGF)-β1 expression and inhibition of Smurf2-mediated degradation of renal Smad7 are mechanisms by which Smad3 KO mice were protected from angiotensin II-induced renal fibrosis and NF-κB-driven renal inflammation in vivo and in vitro. In conclusion, Smad3 is a key mediator of hypertensive nephropathy. Smad3 promotes Smurf2-dependent ubiquitin degradation of renal Smad7, thereby enhancing angiotensin II-induced TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation. Results from this study suggest that inhibition of Smad3 or overexpression of Smad7 may be a novel therapeutic strategy for hypertensive nephropathy.     

MDM2 controls NRF2 antioxidant activity in prevention of diabetic kidney disease

Oxidative stress and P53 contribute to the pathogenesis of diabetic kidney disease (DKD). Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of cellular antioxidant defense system, is negatively regulated by P53 and prevents DKD. Recent findings revealed an important role of mouse double minute 2 (MDM2) in protection against DKD. However, the mechanism remained unclear. We hypothesized that MDM2 enhances NRF2 antioxidant signaling in DKD given that MDM2 is a key negative regulator of P53. The MDM2 inhibitor nutlin3a elevated renal P53, inhibited NRF2 signaling and induced oxidative stress, inflammation, fibrosis, DKD-like renal pathology and albuminuria in the wild-type (WT) non-diabetic mice. These effects exhibited more prominently in nutlin3a-treated WT diabetic mice. Interestingly, nutlin3a failed to induce greater renal injuries in the Nrf2 knockout (KO) mice under both the diabetic and non-diabetic conditions, indicating that NRF2 predominantly mediates MDM2's action. On the contrary, P53 inhibition by pifithrin-α activated renal NRF2 signaling and the expression of Mdm2, and attenuated DKD in the WT diabetic mice, but not in the Nrf2 KO diabetic mice. In high glucose-treated mouse mesangial cells, P53 gene silencing completely abolished nutlin3a's inhibitory effect on NRF2 signaling. The present study demonstrates for the first time that MDM2 controls renal NRF2 antioxidant activity in DKD via inhibition of P53, providing MDM2 activation and P53 inhibition as novel strategies in the management of DKD.     

Increase in AMPK brought about by cocoa is renoprotective in experimental diabetes mellitus by reducing NOX4/TGFβ-1 signaling

The aims of the present study were to investigate, in diabetes mellitus (DM), the mechanism of NOX4 up-regulation, its link with 5′ adenosine monophosphate-activated protein kinase (AMPK) inactivation and transforming growth factor (TGF) ß-1 signaling in determining the accumulation of kidney extracellular matrix (ECM), and the possible action of cocoa enriched with polyphenols (CH) in these events. After 16 weeks of DM, spontaneously hypertensive rats showed increased kidney TGFβ-1 levels and expression of phosphorylated smad2, collagen IV and fibronectin in parallel with elevated NOX4 expression and reduced phosphorylated AMPK. CH treatment in diabetic rats prevented all of these abnormalities. In immortalized human mesangial cells exposed to high glucose (HG), or TGFβ-1, CH, nicotinamide adenine dinucleotide phosphate blocker, or silencing NOX4 ameliorated enhanced phosphorylated smad2 and collagen IV. Reduction in phosphorylated AMPK induced by HG or TGFβ-1 was ameliorated by CH or activation of AMPK, which reduced phosphorylation of smad2 and collagen IV via reduction in NOX4 expression. The effects of CH were abolished by AMPK blockade. These results suggest that inactivation in AMPK leads to NOX4 up-regulation, activation of TGFβ-1 signaling and increased ECM accumulation. Additionally, increased TGF-ß1 per se leads to the amplification of ECM production by reducing AMPK and promoting the activation of NOX4. It is suggested that the activation of AMPK by CH followed by reduction in NOX4/TGFβ-1 signaling may have a therapeutic potential in diabetic nephropathy.     

Salvianolate ameliorates oxidative stress and podocyte injury through modulation of NOX4 activity in db/db mice

Podocyte injury is associated with albuminuria and the progression of diabetic nephropathy (DN). NADPH oxidase 4 (NOX4) is the main source of reactive oxygen species (ROS) in the kidney and NOX4 is up-regulated in podocytes in response to high glucose. In the present study, the effects of Salvianolate on DN and its underlying mechanisms were investigated in diabetic db/db mice and human podocytes. We confirmed that the Salvianolate administration exhibited similar beneficial effects as the NOX1/NOX4 inhibitor GKT137831 treated diabetic mice, as reflected by attenuated albuminuria, reduced podocyte loss and mesangial matrix accumulation. We further observed that Salvianolate attenuated the increase of Nox4 protein, NOX4-based NADPH oxidase activity and restored podocyte loss in the diabetic kidney. In human podocytes, NOX4 was predominantly localized to mitochondria and Sal B treatment blocked HG-induced mitochondrial NOX4 derived superoxide generation and thereby ameliorating podocyte apoptosis, which can be abrogated by AMPK knockdown. Therefore, our results suggest that Sal B possesses the reno-protective capabilities in part through AMPK-mediated control of NOX4 expression. Taken together, our results identify that Salvianolate could prevent glucose-induced oxidative podocyte injury through modulation of NOX4 activity in DN and have a novel therapeutic potential for DN.     

Geldanamycin derivative ameliorates high fat diet-induced renal failure in diabetes

Diabetic nephropathy is a serious complication of longstanding diabetes and its pathogenesis remains unclear. Oxidative stress may play a critical role in the pathogenesis and progression of diabetic nephropathy. Our previous studies have demonstrated that polyunsaturated fatty acids (PUFA) induce peroxynitrite generation in primary human kidney mesangial cells and heat shock protein 90β1 (hsp90β1) is indispensable for the PUFA action. Here we investigated the effects of high fat diet (HFD) on kidney function and structure of db/db mice, a widely used rodent model of type 2 diabetes. Our results indicated that HFD dramatically increased the 24 h-urine output and worsened albuminuria in db/db mice. Discontinuation of HFD reversed the exacerbated albuminuria but not the increased urine output. Prolonged HFD feeding resulted in early death of db/db mice, which was associated with oliguria and anuria. Treatment with the geldanamycin derivative, 17-(dimethylaminoehtylamino)-17-demethoxygeldanamycin (17-DMAG), an hsp90 inhibitor, preserved kidney function, and ameliorated glomerular and tubular damage by HFD. 17-DMAG also significantly extended survival of the animals and protected them from the high mortality associated with renal failure. The benefit effect of 17-DMAG on renal function and structure was associated with a decreased level of kidney nitrotyrosine and a diminished kidney mitochondrial Ca(2+) efflux in HFD-fed db/db mice. These results suggest that hsp90β1 is a potential target for the treatment of nephropathy and renal failure in diabetes.     

Paracrine role for TGF-β-induced CTGF and VEGF in mesangial matrix expansion in progressive glomerular disease

Transforming growth factor-β (TGF-β) is a key regulator of extracellular matrix (ECM), and may mediate the development of glomerulosclerosis with accumulation of mesangial matrix. Mesangial cells secrete TGF-β in response to common in vitro fibrogenic stimuli. Yet mesangial immunostaining for active TGF-β1 is frequently negative in chronic glomerular disease. TGF-β is rather expressed and/or activated by podocytes in both mesangial and podocyte diseases. Activated TGF-β/Smad signaling by podocytes may induce connective tissue growth factor (CTGF or CCN2) and vascular endothelial growth factor (VEGF) expression. Podocyte CTGF seems to have paracrine effects on mesangial cells to stimulate CTGF expression. CTGF appears to stimulate the fibronectin-matrix assembly via enhanced cell-surface expression of α5β1 integrin in the mesangium of diseased glomeruli. Podocyte VEGF-A overexpression also seems to play a paracrine role on mesangial cells to upregulate VEGF/VEGF receptor systems and to overproduce matrix proteins. Thus, paracrine CTGF and VEGF may contribute to mesangial matrix accumulation in chronic glomerular disease, culminating in the development of glomerulosclerosis. Together, these data bring new mechanistic insights into our understanding of the pathogenic role of TGF-β-induced CTGF and VEGF in mesangial matrix expansion in chronic progressive glomerular disease.