Multiple lines of evidence indicate genetic susceptibility and oxidative stress (ROS) arecritical factors in development of diabetic nephropathy (DN). The p66ShcA protein playsa pivotal role in hyperglycemic (HG) ROS signals that drive the pathobiology of DN andinflict irreversible cell injury. The central hypothesis of this application ishomozygous mutation at the p66ShcA locus, in mice genetically at risk for DN,will attenuate or prevent biomarkers of incipient DN and ROS inducedphenotypes of apoptosis and cell senescence. Accordingly, Sv129p66ShcA-/- micewill be backcrossed to; i) Akita mice bearing the Ins2+/C96Y mutation, which in the inbredC57BL/6J strain, confers a phenotype of mild DN; ii) Akita (Ins2+/C96Y) mice bearing asecond mutation at the bradykinin 2 receptor locus (Bdkr2-/-), that markedly enhancesDN and senescent associated phenotypes (SAP).
Specific Aim 1 will determine ifmesangial cells (MC) and podocytes of p66ShcA-/- Akita mice exhibit in-vivoresistance to HG-ROS signals, DNA damage and apoptosis. The experimentalapproach will include entry of p66ShcA-/- Akita mice into chronic (12 mo) protocols, withdetailed morphometric analysis, at multiple time points, using confocal imaging to detectROS, ROS-induced DNA damage and cell death. In-vitro protocols, using conditionallyimmortalized human podocytes, will examine mechanism(s) of cell survival e.g. DNArepair and slit diaphragm (SD) protein signaling.
Specific Aim 2 will determine ifp66ShcA-/- Akita mice exhibit life long resistance to DN and whether SAP areattenuated in resident glomerular cells. During chronic protocols (Specific Aim 1) wewill perform serial determinations of UAE and GFR in conscious mice; detailedglomerular morphometry and Immuno-gold EM to detect SD localization of nephrin;confocal imaging with immunocytochemistry to detect senescent associated proteinsand telomere shortening.
Specific Aim 3 will determine how inhibition of p66ShcAsuppresses HG-ROS signals in cytosol and mitochondria. Based on our preliminarydata, we hypothesize a key role for the Forkhead homolog, FOXO3a in orchestrating theROS resistant gene program. We will rely on in vitro systems, using molecular probesand p66ShcA deficient cell lines, established in our laboratory. The long-termobjective(s) will be novel gene based strategies, targeting mutant genes or dysfunctionalgene products, to arrest or prevent DN. Multiple lines of evidence indicate genetic susceptibility and oxidative stress (ROS) arecritical factors in development of diabetic nephropathy. The p66ShcA protein plays apivotal role in the generation of hyperglycemic ROS signals that drive the pathobiologyof diabetic nephropathy and inflict irreversible cell injury. The central hypothesis of thisapplication is homozygous mutation at the p66ShcA locus, in mice genetically at risk fordiabetic nephropathy, will attenuate or prevent biomarkers of incipient diabeticnephropathy and ROS phenotypes of apoptosis and cell senescence.
| Vashistha, Himanshu; Singhal, Pravin C; Malhotra, Ashwani et al. (2012) Null mutations at the p66 and bradykinin 2 receptor loci induce divergent phenotypes in the diabetic kidney. Am J Physiol Renal Physiol 303:F1629-40 |
| Husain, Mohammad; Meggs, Leonard G; Vashistha, Himanshu et al. (2009) Inhibition of p66ShcA longevity gene rescues podocytes from HIV-1-induced oxidative stress and apoptosis. J Biol Chem 284:16648-58 |
| Malhotra, Ashwani; Vashistha, Himanshu; Yadav, Virendra S et al. (2009) Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis. Am J Physiol Heart Circ Physiol 296:H380-8 |
