BLRD Research Career Scientist Award Application

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Diabetes is the number one cause of end stage kidney disease and accounts for approximately 47% of cases in the US. More than 34 million Americans have diabetes. It is prevalent in the people aged 18 years and older. The demographic of Veteran population falls in this age group. In Veterans aged 65 years and older, approximately 27% are afflicted with diabetes. A recent study demonstrated that diabetic patients with kidney disease had 87% higher risk of cardiovascular mortality. One in three patients with diabetes develop diabetic nephropathy (DN). Early pathologic changes in DN involve renal, especially glomerular hypertrophy and expansion of matrix proteins. The focus of our research is to investigate the signal transduction mechanisms that lead to the progression of DN. To test our concepts, we use both renal glomerular mesangial and proximal tubular epithelial (PTE) cells in culture and, mouse and rat models of diabetes exhibiting kidney pathologies. In kidney, high levels of transforming growth factor-b (TGFb) mediate many pathologic effects of hyperglycemia. Therefore, along with the effects of high glucose, we investigate the signaling mechanisms of TGFb in mesangial and PTE cells. We were the first to discover that high glucose decreases the expression of the tumor suppressor protein PTEN (phosphatase and tensin homolog deleted in chromosome 10) in these cells and in the renal tissues of diabetic mice and rats. We identified that this effect of high glucose is mediated by TGFb. In investigating the mechanisms, we for the first time reported that multiple microRNAs such as miR-21, miR-26 and miR-214 that are significantly increased in the diabetic kidneys regulate the hyperglycemia- and TGFb- induced inhibition of PTEN. In fact, we showed that this inhibition of PTEN expression resulted in sustained activation of Akt kinase that led to activation of mTORC1 (mechanistic target of rapamycin complex 1). mTORC1 contributes to mesangial and PTE cell hypertrophy, and expression of matrix proteins fibronectin and collagen I a2 causing renal hypertrophy and fibrosis in DN. Indeed, we showed that rapamycin ameliorated complications of DN including albuminuria in type 1 and type 2 diabetic mice. Since increased expression of above-mentioned microRNAs contribute to PTEN inhibition/Akt kinase-mediated mTOR activation, our studies opened the door to the novel application of anti-miR therapy for DN. Rapamycin-mediated complete inhibition of mTORC1 causes deleterious clinical outcome. Proximal tubular loss of mTORC1 in mice showed progressive renal fibrosis. Therefore, more recently we have focused on a novel protein, called deptor, which is a component of mTOR and is a negative regulator of both mTORC1 and mTORC2 activities. For the first time, we showed that the renal expression of deptor was significantly reduced in humans with diabetes and in diabetic rodents. This reduction contributed to enhanced mTOR activity. We also found that both high glucose and TGFb decrease the expression of deptor in mesangial and PTE cells. We identified a microRNA, miR-181a, which is increased in response to high glucose or TGFb, regulates the downregulation of deptor. More recently, we identified an independent epigenetic mechanism involving the PRC2 (polycomb repressor complex 2) component enhancer of zeste homolog 2 for high glucose-induced deptor suppression. We plan to use both these mechanisms to target the complications of DN in rodent models. Furthermore, we have identified a novel cross-talk between high glucose/TGFb and PDGFRb (platelet-derived growth factor receptor-b) activation in mesangial and PTE cells. PDGFRb inhibitor blocked hypertrophy and matrix protein expression, indicating that this can be utilized therapeutically for amelioration of DN. A strong correlation between diabetes and renal cell carcinoma (RCC) has been established. We have identified two microRNAs, miR-21 and miR-214, which are involved in DN, also contribute to the activation of mTORC1 and, proliferation and invasion of renal carcinoma cells. Thus, the goal of our studies is to investigate the molecular mechanisms of the progression of DN and RCC, and identify signaling molecules that can be targeted by small molecular drugs and anti-miR based therapies.