Early detection of cysteine rich protein 61 (CYR61, CCN1) in urine following renal ischemic reperfusion injury.

BACKGROUND: Acute renal failure (ARF) has a high morbidity and mortality. Many therapies have worked in animals but were unsuccessful in clinical trials. The inability to diagnose ARF early may have impaired the success of these trials. METHOD: We screened a subtraction library to search for potential disease markers that would be induced rapidly after renal injury. Mice and rats were subjected to 30 to 40 minutes of bilateral ischemia. RESULTS: mRNA for Cyr61, a secreted growth factor-inducible immediate early gene, was markedly up-regulated at two hours in the kidney but not other organs following renal ischemia. In situ hybridization studies suggested Cyr61 was synthesized in the proximal straight tubule. Cyr61 protein was analyzed by capture with heparin beads followed by Western blotting. Induction of Cyr61 protein could be detected in the kidney within one hour, peaked at four to eight hours, and remained elevated for at least 24 hours following ischemia. Cyr61 protein was detected in urine at three to six hours and peaked at six to nine hours after renal injury. Cyr61 was not detected after volume depletion, which is often difficult to differentiate from ARF. CONCLUSIONS: The secreted, cysteine-rich, heparin binding protein Cyr61 is rapidly induced in proximal straight tubules following renal ischemia, and excreted in the urine where it might serve as an early biomarker of renal injury.

Electroporation-mediated gene transfer that targets glomeruli.

Electroporation has been applied to introducing DNA into several organs; however, gene expression was localized around the injected area. Examined was the efficiency of intrarenal injection of DNA followed by in vivo electroporation, using FITC-labeled oligodeoxynucleotides (FITC-ODN) and plasmid DNA expressing beta-galactosidase or luciferase. FITC-ODN or expression vectors were injected into the left renal artery; thereafter, the left kidney was electroporated between a pair of tweezer-type electrodes. FITC-ODN were transferred into all glomeruli, and transfected cells were identified as mesangial cells. Four d after transfection of the pCAGGS-LacZ gene, beta-galactosidase expression was observed in 75% of glomeruli. To compare the transfection efficacy by electroporation with that by the hemagglutinating virus of Japan (HVJ) liposome method, a luciferase reporter gene, pActLuc, was transferred into glomeruli by either electroporation or the HVJ liposome method. On day 4, electroporation resulted in higher glomerular luciferase activity than did the HVJ liposome method. We also observed that co-transfection of pcEBNA, an expression vector for Epstein-Barr virus nuclear antigen, and poriP-cLuc, oriP-harboring vector, resulted in an eightfold higher luciferase gene expression than simple poriP-cLUC: No histologic damages were seen in glomeruli or tubular epithelial cells. In conclusion, gene transfer into renal artery followed by electroporation was an effective and simple strategy for gene transfer that targets glomerular mesangial cells.