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

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.^3–6 Although sources of ROS are numerous, the NADPH oxidase (Nox) family of enzymes yields significant superoxide production in the kidney.^7–10 Nox-induced ROS production has been closely linked to various glomerular pathologies. In animal models of minimal change disease, membranous nephropathy, and FSGS, inhibition of Nox activity is associated with decreased podocyte effacement and amelioration of albuminuria.^11–14 In models of diabetic nephropathy, treatment with the Nox inhibitor apocynin, as well the antioxidant vitamin E, reduces oxidative stress, podocyte effacement and loss, and albuminuria.^6,15,16 Noxs are regulated by many factors, including the renin angiotensin aldosterone system.^5,9 Several studies have linked increased renin angiotensin aldosterone system activity to enhanced renal Nox activity and ROS generation.^5,17 Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers slow progression of proteinuria in models of diabetes, and these effects may be, in part, independent of their effects on systemic BP,^17–20 because direct activation of Nox enzymes through the angiotensin II (AngII)/AT1 receptor (AT1R) pathway leads to oxidative stress. In vitro studies in both human and rodent cell lines have also shown that Nox family member expression and activity are regulated by disease-associated factors, including AngII, ET-1, TGF-β, high glucose, mechanical stretch, and PDGF (factors that are upregulated in the diabetic milieu).^3,21–23The roles of Nox4, and to a lesser extent, Nox1 and -2, in the kidney have been examined, but nothing is known regarding the role of the most recently identified member of the Nox family, Nox5. The Nox5 gene is absent from the mouse and rat genomes, making the use of conventional animal models unfeasible. Unlike other Nox family members, Nox5 does not require membrane-bound or cytosolic components, such as p22phox or p47phox, for its activity, but is tightly regulated by changes in intracellular calcium levels.^24,25 Nox5 has a large amino terminal EF hand-containing domain that plays a critical role in its calcium-dependent activation along with several phosphorylation sites that alter the sensitivity of Nox5 to intracellular calcium.^26–29 Because AngII increases intracellular calcium concentrations, it seems to induce renal Nox5-dependent ROS generation, which was shown in human endothelial cells.²³ Here, we show that (1) Nox5 is upregulated in human diabetic glomeruli; (2) AngII stimulates ROS generation in human podocytes in a Nox5-dependent manner, a process associated with actin cytoskeletal reorganization and activation of Rac GTPase, which promotes podocyte motility in vitro; (3) mice that express human Nox5 in a podocyte-specific manner (Nox5β^pod+ mice) exhibit renal dysfunction, including albuminuria, podocyte effacement, glomerular basement membrane (GBM) thickening, interstitial fibrosis, and hypertension; and (4) Nox5^pod+ mice subjected to streptozotocin (STZ)-induced diabetes develop a more severe kidney phenotype than nontransgenic littermates. These novel data indicate the potential importance of podocyte Nox5 in human renal pathologies, such as diabetic nephropathy.