The Endoplasmic Reticulum Stress Protein Calreticulin in Diabetic Chronic Kidney Disease
Annual rept. 1 Jul 2014-30 Jun 2015
ALABAMA UNIV IN BIRMINGHAM
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We hypothesize that ER stress induced by glucose in diabetes promotes diabetic CKD through CRT stimulation of TGF-beta-dependent calciumNFAT signaling in renal proximal tubule cells. In Aim 1 we will determine the role of CRT in mediating the fibrogenic effects of TGF-beta and glucose in renal cells. In Aim 2, we will determine the role of CRT in mouse models of diabetic nephropathy. To date, we have established cultures of mouse proximal tubule cells MPTC isolated from CRT-floxed mice and shown that TGF-beta and glucose stimulate ECM fibronectin, collagen in both MPTC and in human PT cells HK- 2. In HK-2 cells, we showed that siRNA for CRT reduces TGF-beta stimulation of ECM. Cre-recombinase plasmid reduced CRT expression in MPTC. We are developing lentiviral mouse and human CRT shRNA approaches to establish stably transduced cell lines and for use in long-term assays. For aim 2, we established that 0.7 mPa of ultrasound to deliver crerecombinase plasmid to the kidney is safe no urinary AKI markers and reduces CRT protein in renal tubules by 80-65 over days 3-14 by IHC and blot of protein lysate. CRT is expressed primarily in the tubules. These results suggest the feasibility of the proposed studies.
- Medicine and Medical Research