Targeting NADPH oxidase with a novel dual Nox1/Nox4 inhibitor attenuates renal pathology in type 1 diabetes.

Reactive oxygen species (ROS) generated by Nox NADPH oxidases may play a critical role in the pathogenesis of diabetic nephropathy (DN). The efficacy of the Nox1/Nox4 inhibitor GKT137831 on the manifestations of DN was studied in OVE26 mice, a model of type 1 diabetes. Starting at 4-5 mo of age, OVE26 mice were treated with GKT137831 at 10 or 40 mg/kg, once-a-day for 4 wk. At both doses, GKT137831 inhibited NADPH oxidase activity, superoxide generation, and hydrogen peroxide production in the renal cortex from diabetic mice without affecting Nox1 or Nox4 protein expression. The increased expression of fibronectin and type IV collagen was reduced in the renal cortex, including glomeruli, of diabetic mice treated with GKT137831. GKT137831 significantly reduced glomerular hypertrophy, mesangial matrix expansion, urinary albumin excretion, and podocyte loss in OVE26 mice. GKT137831 also attenuated macrophage infiltration in glomeruli and tubulointerstitium. Collectively, our data indicate that pharmacological inhibition of Nox1/4 affords broad renoprotection in mice with preexisting diabetes and established kidney disease. This study validates the relevance of targeting Nox4 and identifies GKT137831 as a promising compound for the treatment of DN in type 1 diabetes.

Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells.

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase ß, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase ß inhibitor, had the same effect. Renal cortical content of cystathionine ß-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

Apelin retards the progression of diabetic nephropathy.

Apelin and its receptor APJ have pleiotropic effects in mice and humans and play a protective role in cardiovascular diseases at least partially by inhibiting oxidative stress. Our objective was to study the effect of apelin on the progression of kidney disease in mice with established type 1 diabetes. Ove26 mice with type 1 diabetes received daily subcutaneous injections of apelin for 2 or 14 wk. APJ localizes in the glomeruli and blood vessels of kidneys. Renal APJ expression was reduced in diabetic mice but increased after treatment with apelin. Apelin treatment did not affect glycemia, body weight, or blood pressure in diabetic mice. Whole kidney and glomerular hypertrophy, as well as renal inflammation, including monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression, NF-κB activation, and monocyte infiltration, was inhibited after short and long treatment with apelin. Apelin administration significantly reduced albuminuria at 6 mo. Short treatment with apelin was sufficient to reverse the downregulation of the antioxidant enzyme catalase. Expression of angiotensin II and angiotensin type 1 receptor (AT1) in kidneys from diabetic mice treated was not affected by apelin. These findings show for the first time that apelin exerts a protective effect on the diabetic kidney. Short administration is sufficient to reduce kidney and glomerular hypertrophy as well as renal inflammation, but prolonged treatment is required to improve albuminuria. This effect was independent of the activation of the renin angiotensin system but correlated with upregulation of the antioxidant catalase. Apelin may represent a novel tool to treat diabetic nephropathy.

Antifibrotic effect of tamoxifen in a model of progressive renal disease.

Tamoxifen, a selective estrogen receptor modulator, has antifibrotic properties; however, whether it can attenuate renal fibrosis is unknown. In this study, we tested the effects of tamoxifen in a model of hypertensive nephrosclerosis (chronic inhibition of nitric oxide synthesis with L-NAME). After 30 days, treated rats had significantly lower levels of albuminuria as well as lower histologic scores for glomerulosclerosis and interstitial fibrosis than untreated controls. Tamoxifen was renoprotective despite having no effect on the sustained, severe hypertension induced by L-NAME. Tamoxifen prevented the accumulation of extracellular matrix by decreasing the expression of collagen I, collagen III, and fibronectin mRNA and protein. These renoprotective effects associated with inhibition of TGF-ß1 and plasminogen activator inhibitor-1, and with a significant reduction in α-smooth muscle actin-positive cells in the renal interstitium. Furthermore, tamoxifen abrogated IL-1ß- and angiotensin-II-induced proliferation of fibroblasts from both kidney explants and from the NRK-49F cell line. Tamoxifen also inhibited the expression of extracellular matrix components and the production and release of TGF-ß1 into the supernatant of these cells. In summary, tamoxifen exhibits antifibrotic effects in the L-NAME model of hypertensive nephrosclerosis, likely through the inhibition of TGF-ß1, suggesting that it may have therapeutic use in CKD treatment.

Metformin prevents renal interstitial fibrosis in mice with unilateral ureteral obstruction.

Unilateral ureteral obstruction causes important tubulo-interstitial fibrosis in the kidney. Metformin reduces fibrosis in mice with diabetic nephropathy. We examined the effects of metformin in a mouse model of unilateral ureteral obstruction (UUO). Expression of inflammation and fibrosis markers was studied by immunohistochemistry, immunoblot and quantitative real-time polymerase chain reaction. Seven days after UUO, kidneys presented dilated tubules, expansion of the tubulo-interstitial compartment, and significant infiltration of inflammatory cells. Macrophage infiltration and inflammation markers expression were increased in obstructed kidneys and reduced by metformin. Metformin reduced expression of extracellular matrix proteins and profibrotic factor TGFß in obstructed kidneys, measured by immunohistochemistry. Interstitial fibroblast activation was evident in obstructed kidneys and ameliorated by metformin. UUO did not affect adenosine monophosphate-activated kinase (AMPK) activity, but metformin activated AMPK. Our results show that metformin prevents or slows down the onset of renal inflammation and fibrosis in mice with UUO, an effect that could be mediated by activation of AMPK.

The potential use of stem cells derived from human amniotic fluid in renal diseases.

Amniotic fluid (AF) contains a variety of cell types derived from fetal tissues that can easily grow in culture. These cells can be obtained during amniocentesis for prenatal screening of fetal genetic diseases, usually performed during the second trimester of pregnancy. Of particular interest, some expanded sub-populations derived from AF cells are capable of extensive self-renewal and maintain prolonged undifferentiated proliferation, which are defining properties of stem cells. These human AF stem cells (hAFSCs) exhibit multilineage potential and can differentiate into the three germ layers. They have high proliferation rates and express mesenchymal and embryonic markers, but do not induce tumor formation. In this study, hAFSCs derived from amniocentesis performed at 16-20 weeks of pregnancy were isolated, grown in culture, and characterized by flow cytometry and by their potential ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. After 4-7 passages, 5 × 105 hAFSCs were inoculated under the kidney capsule of Wistar rats that were subjected to an experimental model of chronic renal disease, the 5/6 nephrectomy model (Nx). After 30 days, Nx rats treated with hAFSCs displayed significant reductions in blood pressure, proteinuria, macrophages, and α-smooth muscle actin expression compared with Nx animals. These preliminary results suggest that hAFSCs isolated and expanded from AF obtained by routine amniocentesis can promote renoprotection in the Nx model. Considering that the AF cells not used for fetal karyotyping are usually discarded, and that their use does not raise ethical issues, they may represent an alternative source of stem cells for cell therapy and regenerative medicine.