New Insights in Mechanisms of Renal Injury

South Texas Veterans Health Care System, San Antonio TX

Investigators

Linked publications & trials

Abstract

? DESCRIPTION (provided by applicant): Whether hyperinsulinemia directly contributes to kidney injury by insulin receptor (IR) activation in hyperinsulinemic insulin resistant states, e.g., obesity is not known. Our novel pilot data show (1) In C57BL6 mice, high fat diet (HFD) led to renal cortical IR activation, increase in matrix protein, albuminuria, and mTORC1 activation along with weight gain, insulin resistance, hyperinsulinemia but normal blood glucose. We have generated the kidney proximal tubular IR knock out mice (KPTIRKO) in which HFD-induced IR activation, matrix increment, albuminuria and mTORC1 signaling were reduced. (2) Hydrogen sulfide (H2S) is normally synthesized in the kidney by cystathionine ? lyase (CSE) and cystathionine ? synthase (CBS). In WT mice HFD reduced the renal content of CSE and CBS but not in KPTIRKO mice suggesting that IR activation in HFD reduces CBS and CSE as a part of renal injury. (3) HFD decreased renal CSE and CBS content but not their mRNA; in vitro insulin decreased CSE and CBS protein but not mRNA in proximal tubular epithelial (MCT) cells. These data suggest non-transcriptional regulation of CSE and CBS by insulin. Although our data suggest a role for IR in HFD-associated kidney injury, the requirement of reduced H2S generation is not known. Hypothesis: In hyperinsulinemic insulin resistant state induced by high fat diet, renal insulin receptor activation contributes to kidney injury by reduced generation of hydrogen sulfide. Aim 1. To test if H2S administration ameliorates HFD-induced kidney injury in mice. 3-month old male and female C57BL6 mice will be randomized to normal fat diet (NFD) or HFD for 4 months. Once the kidney injury (albuminuria) is established, we will administer sodium hydrosulfide (NaHS), a H2S donor, at 30umoles/l drinking water for 3 months. The following studies will be done: Structural changes: renal hypertrophy, immunohistochemistry, mRNA/protein analysis of matrix proteins. Functional changes: albuminuria, FITC inulin clearance. Signaling studies: IR activation, signaling reactions that control protein synthesis. Aim 2. (A) To test if augmented H2S production protects against HFD-induced kidney injury. We will randomize male and female 3-month old WT and transgenic mice overexpressing human CBS in the proximal tubule (PThCBS tg, generated by us) to NFD or HFD for 4 months. Studies will be done as in Aim 1. (B) To test if HFD-induced kidney injury is amplified in mice deficient in H2S production. We will randomize male and female 3-month old WT and CSEKO mice that are in our possession to NFD or HFD for 4 months. Structural, functional and signaling studies will be done as in Aim 1. Aim 3. To explore the mechanism of insulin-induced reduction in CSE, CBS expression. (A) In vivo studies: Pilot data show that HFD reduced the renal CSE and CBS expression by non-transcriptional regulation. We will explore if this is due to failure to translate their mRNA by performing Polysome assay on renal preparations from C57BL6 mice on NFD and HFD at 4 months of diet. Changes in the expression lysosomal and proteasomal pathway proteins will be measured. (B) In vitro studies: High insulin inhibited CSE and CBS protein in MCT cells in vitro by non-transcriptional mechanism, similar to in vivo data. Employing 1 and 10 nM insulin as high insulin in MCT cells, we will explore if this is due to (i) failure of translation of mRNA by Polysome assay, and/or, (ii) if this is due to increase in degradation by measuring the degradation of radiolabeled CSE and CBS by pulse chase. We will test if augmented degradation is via proteasomal mechanism by measuring proteasomal activity and proteasomal subunit protein and mRNA expression. We will employ bortezomib, MG132 and siRNA for proteasomal subunits. Recent studies show that mTORC1 stimulates protein degradation by proteasome pathway. We will explore role of mTOR in CSE and CBS degradation. We will also test if high insulin stimulates lysosomal pathway to augment CSE and CBS degradation by employing chloroquine or siRNA against Atg7.