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.
More than a third of Veterans suffer from obesity and are at risk for developing chronic kidney disease. Justifiably the VA has declared metabolic disorders such as obesity a priority area for research. The mechanism by which obesity damages kidney function is not well understood. Although circulating insulin level is elevated in obesity, whether it activates the insulin recepto in the kidney leading to damage is not known. We have evidence that while high fat diet induces obesity associated kidney injury in normal mice, it is ameliorated in mice genetically lacking the kidney insulin receptor. We also noted that insulin receptor activation in the kidney of high fat diet fed mice was associated with reduced production of hydrogen sulfide. We will test if insulin receptor activation mediated reduction in production of H2S by the kidney is required for obesity associated kidney injury by employing state of the art approaches. Data obtained from this project will help understand kidney injury in obesity, and may lead to novel therapies benefiting the Veterans and others.
