SUN-302 The Relative Roles of Pro-oxidant Ezymes Nox4 versus Nox5 in Diabetic Kidney Disease

Abstract

Renal oxidative stress plays an important role in mediating kidney injury in diabetes. There is increasing evidences that recently discovered pro-oxidant enzyme, Nox5 plays a significant role in human diabetic kidney disease (DKD). Nox5 is present in humans and rabbits but not in mice or rats. Thus, there is a paucity of information about Nox5 in conventional mice/rats models of DKD. We examined the role of Nox5 in human diabetic nephropathy, in human renal cell populations as well as in a high fat fed rabbit model of kidney disease. In addition, in vivo, we also examined the contribution of Nox5 in the pathogenesis of DN in the absence of Nox4 expression. Protein expression of Nox4 and Nox5 and their localization in glomerular (podocytes and mesangial cells) and tubular cells were examined by immunostaining in human kidney biopsies obtained from non-diabetic and diabetic individuals. In vitro, human mesangial cells, podocytes and proximal tubules were exposed to high glucose, TGF-β and AngII and Nox4 and Nox5 were knockdown in these renal cells. Cell morphology, gene and protein expression of markers of fibrosis and inflammation as well as putative signalling pathways and the level of ROS were assessed in these human renal cells. In addition, we have developed a novel mode of diabetic complications which is more similar to the human context, the New Zealand White rabbit which expresses Nox5 and the other Nox isoforms as in humans. We have exposed the rabbits to high fat feeding (as a model of type 2 diabetes) as well as inducing insulin deficient diabetes (type 1 diabetes) using alloxan. As a world first, we have generated a Nox5 knockout rabbit using CRISP/Cas9.We have also generated a unique humanised Nox5 transgenic mouse with concomitant Nox4 KO with the aim to definitively delineate the relative effects of Nox4 versus Nox5 in diabetic complications. Expression of Nox5 was increased in both glomerular and tubular compartments of kidney biopsies obtained from diabetic individuals when compared to non-diabetic individuals. Nox5 is the Nox isoform which shows the highest upregulation in response to high glucose. Silencing of Nox5 reduces ROS formation and expression of proinflammatory and profibrotic cytokines and growth factors as well as putative elements that are implicated in DKD. Our data also suggest that Nox5 is upstream of Nox5 and that Nox5 inhibition also downregulates Nox4, but not vice versa. High fat feeding and alloxan induced diabetes rabbits develop increased mesangial area, increased Nox5 expression in renal cortex and upregulation of a range of pro-inflammatory (MCP-1) and pro-fibrotic genes (CTGF) as analysed by RNAseq and confirmed by RT-PCR. In addition, over expression of Nox5 in mesangial cells of Nox4 KO diabetic mice demonstrated 30% increase in albuminuria, mesangial expansion, increased renal injury and inflammation via enhanced ROS production. These studies will for the first time explore in a comprehensive manner the role of Nox5 in recently generated rabbit models of diabetes (type 1 and type 2) as well as delineate the relative role of Nox4 versus Nox5 in diabetic complications. These studies will provide evidence if Nox5 blockade alone or combined with Nox4/Nox5 blockade is the optimal approach to treat and prevent diabetic complications, in particular nephropathy.