Nephropathy and Elevated BP in Mice with Podocyte-Specific NADPH Oxidase 5 Expression

Abstract

NADPH oxidase (Nox) enzymes are a significant source of reactive oxygen species, which contribute to glomerular podocyte dysfunction. Although studies have implicated Nox1, -2, and -4 in several glomerulopathies, including diabetic nephropathy, little is known regarding the role of Nox5 in this context. We examined Nox5 expression and regulation in kidney biopsies from diabetic patients, cultured human podocytes, and a novel mouse model. Nox5 expression increased in human diabetic glomeruli compared with nondiabetic glomeruli. Stimulation with angiotensin II upregulated Nox5 expression in human podocyte cultures and increased reactive oxygen species generation. siRNA-mediated Nox5 knockdown inhibited angiotensin II-stimulated production of reactive oxygen species and altered podocyte cytoskeletal dynamics, resulting in an Rac-mediated motile phenotype. Because the Nox5 gene is absent in rodents, we generated transgenic mice expressing human Nox5 in a podocyte-specific manner (Nox5 pod+ ). Nox5 pod+ mice exhibited early onset albuminuria, podocyte foot process effacement, and elevated systolic BP. Subjecting Nox5 pod+ mice to streptozotocin-induced diabetes further exacerbated these changes. Our data show that renal Nox5 is upregulated in human diabetic nephropathy and may alter filtration barrier function and systolic BP through the production of reactive oxygen species. These findings provide the first evidence that podocyte Nox5 has an important role in impaired renal function and hypertension. Albuminuria is a clinical marker of kidney dysfunction that arises in most glomerulopathies and is associated with poor prognoses for ESRD, hypertension, and cardiovascular mortality. Changes to the podocyte (e.g., foot process effacement, hypertrophy, detachment, and loss) underlie the development and progression of albuminuria and thereby highlight the critical role for these cells in upholding the glomerular filtration barrier. 1,2 Therefore, identifying factors that induce podocyte injury and loss is essential to understanding the mechanisms of filtration barrier dysfunction. Of the many factors implicated in podocyte dysfunction, excessive production of reactive oxygen species (ROS; oxidative stress) may be particularly important. [3][4][5][6] Although sources of ROS are numerous, the NADPH oxidase (Nox) family of enzymes yields significant superoxide production in the

NADPH Oxidase 5 Is a Pro‐Contractile Nox Isoform and a Point of Cross‐Talk for Calcium and Redox Signaling‐Implications in Vascular Function

“…Nox5 expression is increased in human atherosclerotic and aneurysm lesions26, 27 and in kidneys from diabetic patients 28. We demonstrated that mice expressing human Nox5 in a podocyte‐specific manner exhibit podocyte injury and renal dysfunction,28 and that human Nox5 expression in mesangial cells causes renal fibrosis, nephropathy, and exacerbated atherosclerosis in diabetic mice 29. Findings from recent genome wide‐association studies of 475 000 individuals identified Nox5 as a candidate blood pressure–associated gene 30.…”

“…Nox5 is expressed in all vascular cell types, including endothelial cells, VSMCs, and perivascular adventitial fibroblast,12, 14, 24 and plays a role in angiotensin II (Ang II)‐ and endothelin‐1 (ET‐1)‐mediated redox signaling 25. Nox5 expression is increased in human atherosclerotic and aneurysm lesions26, 27 and in kidneys from diabetic patients 28. We demonstrated that mice expressing human Nox5 in a podocyte‐specific manner exhibit podocyte injury and renal dysfunction,28 and that human Nox5 expression in mesangial cells causes renal fibrosis, nephropathy, and exacerbated atherosclerosis in diabetic mice 29.…”

Podocyte-specific Nox4 deletion affords renoprotection in a mouse model of diabetic nephropathy

“…Urinary 8-isoprostanes is expressed as picograms per 24 h. Urinary 8-hydroxy-2′-deoxyguanosine ELISA A urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) enzyme immunoassay kit (StressMarq Biosciences Victoria, BC, Canada) was used to measure 8-OHdG in urine, as described by the manufacturer. Urinary 8-OHdG is expressed as nanograms per 24 h. Lucigenin assays Glomerular fractions obtained from the frozen renal cortices of the different experimental groups were harvested in 100 μl ice-cold phosphate buffer (50 mmol/l KH 2 PO 4 , 1 mmol/l EGTA and 150 mmol/l sucrose; pH 7.4) with protease inhibitors, as previously described [11]. Baseline activity was measured by adding 50 μl glomerular extract to 175 μl buffer and 2.5 μl 1 mmol/l lucigenin (Sigma-Aldrich).…”

Metabolomics Reveals a Key Role for Fumarate in Mediating the Effects of NADPH Oxidase 4 in Diabetic Kidney Disease

“…Among the seven members of the NOX family, NOX1, NOX2 (also named gp91 phox ), and NOX4 are expressed in kidneys of humans and mice, whereas NOX5 is expressed only in humans. [1][2][3][4][5] We have been examining the role of NOX isoforms in diabetic kidney disease (DKD) and we recently found that NOX2 did not play a major role in mediating DKD with type 1 diabetes. 1,6 However, NOX4 was upregulated in the NOX2 knockout (KO) diabetic kidney, particularly in glomeruli and podocytes, and thus may play a prominent role.…”