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“Role of NADPH oxidases Nox1 and Nox4 in diabetic nephropathy: genetic deletion and pharmacological inhibition studies”
thesisposted on 01.03.2017, 02:33 by Jha, Jay Chandra
Diabetic nephropathy (DN) is a major microvascular complication of diabetes, representing the most common cause of end stage renal failure. DN is considered to contribute to the increased morbidity and mortality observed in patients with diabetes. In diabetes a range of hemodynamic and metabolic factors interact leading to the development and progression of DN. The early stages of DN are clinically characterised by excessive urinary excretion of albumin with a subsequent decline in glomerular filtration rate (GFR) in more advanced stages of DN leading to end stage renal failure. It has been suggested that microalbuminuria in diabetic patients may be associated with intra-glomerular hyperfiltration early in DN and possibly also reduced tubular reabsorption, although the exact cause remains to be determined. The characteristic histopathological changes in DN include renal hypertrophy, the subsequent development of thickened glomerular and tubular basement membranes and the progressive accumulation of extracellular matrix (ECM) proteins in the glomerular mesangium and tubulointerstitium resulting in glomerulosclerosis and tubulointerstitial fibrosis ultimately leading to end stage renal insufficiency. The underlying mechanisms responsible for diabetic nephropathy remain poorly understood. Therefore, more effective and mechanism-based therapies are needed. In recent years, it has been postulated that diabetes mellitus is associated with increased renal oxidative stress, more specifically, increased levels of reactive oxygen species (ROS), resulting in renal damage. Accordingly, oxidative stress is increasingly considered to be a major contributor to the development and progression of DN. Various renal sources of ROS have been suggested to be relevant to the diabetic kidney. However, NADPH oxidases (Nox) are the only known dedicated source of the ROS forming enzyme family. Nox are suggested to play a pivotal role in the development and progression of renal injury in animal models of type 1 and type 2 diabetic nephropathy and hence, they represent a potentially important novel target. In the rodent kidney three isoforms of the catalytic subunit (Nox1, Nox2 and Nox4) of NADPH oxidase are expressed. However, the relative importance of the Nox1, 2 and 4 isoforms in the development and progression of diabetic nephropathy remains unclear. With respect to Nox2, our own studies in streptozotocin-induced diabetic Nox2 knockout (KO) mice have shown increased susceptibility to infections and 100% mortality at week 20 of diabetes. Thus, it was not considered that Nox2 blockade should be a priority in the studies presented in this thesis which address strategies to reduce diabetic nephropathy. Nox4, originally termed Renox, is highly expressed in renal tissues. The role of Nox4 in DN remains controversial. Nox4 downregulation by systemic administration of antisense oligonucleotides, albeit for a short period of only 2 weeks, reduced renal and glomerular hypertrophy and attenuated the increased expression of fibronectin in renal cortex and glomeruli in streptozotocin-induced diabetic rats. However, the Nox4 antisense oligonucleotide may not be absolutely specific for Nox4. On the other hand, a study by Babelova et al has found no upregulation of Nox4 in diabetic C57/bl6 mice compared to its non-diabetic counterpart, and Nox4 deficiency did not attenuate nephropathy. In the non-diabetic context using unilateral ureteric obstruction, an experimental model of renal injury leading to interstitial fibrosis, Nox4 deficiency in C57/bl6 mice was associated with increased renal fibrosis, tubular atrophy and increased ROS formation, consistent with a potential protective role for Nox4. With respect to Nox1, this isoform appears to play a major role in diabetic macrovascular disease but not much is known about the role of Nox1 in diabetic nephropathy. Thus, it remains to be determined which Nox isoform plays the most important role in diabetic kidney disease. We hypothesized that genetic deletion of the NADPH oxidase isoforms, Nox1 or Nox4 or specific pharmacological inhibition of these isoforms would confer various degrees of reno-protection in an animal model of diabetes associated renal disease. Furthermore, it is hypothesized that Nox4 deletion specifically in podocytes, a cell type in the kidney implicated with the pathogenesis of glomerular albumin leakage would attenuate albuminuria and renal injury in DN in vivo. Furthermore, it was postulated that in vitro Nox4 silencing in human podocytes would attenuate pro-inflammatory and pro-fibrotic pathways characteristic of the diabetic milieu. In this light, the overall objective of this thesis was to examine the role of the Nox1 and Nox4 isoforms of NADPH oxidase in a model of diabetic nephropathy in vivo as well as to investigate the mechanisms providing renoprotection using in vitro approaches. The first chapter of this thesis represents the introduction and literature review (Chapter 1), which collectively cover the following central themes addressed in this study. ‘Part A’ of the literature review addresses the general introduction of diabetes, the pathophysiology of diabetic nephropathy, oxidative stress, ROS, NADPH oxidase including its various isoforms Nox1, Nox4, Nox2 and Nox5 and their respective role and regulation in diabetic nephropathy. ‘Part B’ of the literature review is a published review paper 1 entitled “NAD(P)H oxidase isoforms as therapeutic targets for diabetic complications” which addresses the pathophysiological role of the different Nox isoforms in vascular complications of diabetes including diabetic nephropathy. This manuscript also summarises the most recent and relevant Nox specific therapeutic agents including GKT137831 (Genkyotex, Geneva, Switzerland) for the prevention and treatment of diabetic nephropathy. GKT137831 is a novel, first in class Nox1/Nox4 specific inhibitor which has been used for pharmacological intervention in my research project. Furthermore, ‘Part C’ of the literature review is a published review paper 2 entitled “Identifying and interpreting novel targets that address more than one diabetic complication: a strategy for optimal end organ protection in diabetes”. This review paper provides an overview of diabetic complications including diabetic nephropathy together with various therapeutic agents currently available or in development including Nox specific inhibitors for the prevention and treatment of DN in different experimental settings. Chapter 2 describes the expanded methodology used for laboratory experimental investigations. The succeeding chapters 3 and 4 describe the outcome of my laboratory experimental investigations. Chapter 3 is a published original research paper titled “Genetic Targeting or Pharmacologic Inhibition of NADPH Oxidase Nox4 Provides Renoprotection in Long-Term Diabetic Nephropathy”. This manuscript addresses the main questions of my PhD project. I have used global genetically modified animal models as well as pharmacological inhibition approaches to explore the role of Nox1 and Nox4 NADPH oxidase isoforms in an experimental model of diabetic nephropathy in vivo and in conditionally immortalized human podocyes in vitro. I have been able to investigate for the first time the long-term effects of Nox1 and Nox4 deletion in the development and progression of diabetic nephropathy by directly comparing renal injury in streptozotocin (STZ)-induced diabetic Nox1¯/? ApoE-/- and Nox4-/-ApoE-/- double knockout mice and their respective wild type (WT) control mice. In addition, the genetic deletion studies were complemented by a pharmacological intervention study using the currently most specific Nox inhibitor, GKT137831. Key findings in the in vivo studies were also addressed in vitro by using conditionally immortalized human renal epithelial cells also known as podocytes which are a distinct cell population implicated in the regulation of albuminuria. The laboratory findings which I have described are novel and demonstrate that Nox4 is the main source of renal ROS in a mouse model of diabetic nephropathy induced by streptozotocin administration in ApoE-/- mice. Deletion of Nox4 but not Nox1 resulted in renal protection, specifically reduced glomerular injury as evidenced by attenuated albuminuria, preserved renal structure, reduced glomerular accumulation of extracellular matrix proteins, attenuated glomerular macrophage infiltration and reduced renal expression of inflammatory markers such as monocyte chemoattractant protein-1 and the transcription factor NF-kB. Importantly, administration of the specific Nox1/4 inhibitor, GKT137831 to diabetic ApoE-/- mice, replicated these renoprotective effects of Nox4 deletion. In human podocytes, silencing of the Nox4 gene resulted in reduced production of ROS and downregulation of proinflammatory (MCP-1 and NF-kB) and profibrotic proteins (collagen IV and fibronectin) which are implicated in diabetic nephropathy. In addition, chapter 4 represents an unpublished manuscript entitled “Podocyte specific Nox4 deletion attenuates albuminuria in diabetic nephropathy”. I have successfully demonstrated the detrimental role of Nox4 in the development and progression of diabetic nephropathy using a global Nox4KO mouse model. Albuminuria is a key feature of diabetic nephropathy and it is considered that injury to podocytes is associated with the development of albuminuria. The podocyte, a glomerular epithelial cell, is a critical cell type in the slit pore diaphragm and protects from protein leakage. Podocyte injury is characterised by foot process effacement, increased VEGF and decreased nephrin expression, as well as loss of podocytes in the urine ultimately resulting in the development of albuminuria. In this light, I have explored the role of Nox4 in podocytes in a model of diabetic nephropathy. esis.