Acute Renal Failure And Cell Cycle Regulatory Proteins Cyclin E/CDK2 And P27～(kip1)

Objective:Acute renal failure (ARF) is one of common severe renal diseases in clinical practice It may be caused by acute renal ischemia or receiving aniinoglycoside antibiotics and traditionally the pathological manifestation is regarded as acute renal tubular necrosis (ATN)Recent studies reviewed necrosis, apoptosis and regeneration of renal tubular epithelial cells occur and the balance between cell apoptosis and cell proliferation determines the outcome of ARF. Therefore, understand its molecular mechanisms may help to accelarate the speed of renal function recovery and derease the mortality of the disease. Studies on non-renal cells have shown that cell proliferation and apoptosis are controlled by cell cycle regulatory proteins, among which cyclin E and cyclin-dependent kinase 2 are the key positive proteins and p27 is the key negative protein in phase G 1 to regulate the progression from phase 01 to phase S. Therefore our studies attempt to gain a better insight into the process of proliferation and apoptosis of renal tubular epithelial cells and the expressions of cyclin E and CDK2 and 27KiPI in these cells, and to probe into the possible associations between the pathological features and the expressions of above-mentioned cell cycle regulatory proteins in the cells.Methods:The studies were divided into two parts: in vivo and in vitro study. The in vivo study was conducted by establishing rat model of acute renal failure through renal ischemia reperfusion and sacrifacing the rats 2,7,10 and I 4days after the left kidneys were clamped for 60 minutes and then removed the clamps. The serum creatitine of rats were measured using biochemistry method. By using immunohistochemical staining technique, the degree of proliferation in renal cells and distributions of cyclin E and CDK2 and p27 were analyzed in the renal tissues of rats. To observe cell apoptosis, agarose gel electrophoresis of DNA extracted from ischemia-reperfusion kidney was performed. The terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) technique was used to identif nuclei with DNA strand breaks at a cellular level and apoptosis index (Al) were measured. Western Blotting were utilized to determine the protein levels of cyclin E and CDK2 and p27KIPI in the renal tissues of ischemia-reperfiision rats. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to measured p27PmRNA level in the renal tissue of rats. The in vitro study used a kind of renal proximal tubular cell strain called LLC-PKI cells to characterize and quantitate the apoptosis of cells induced by gentamicin. The studies were initiated by incubation of LLC6 PK1 cells with gentamicin of 0.2~3.2 mg/ml fOr 24 ~96 hours, followed by observing grOwth andaPoPtOsis Of cells through routne HE staining. Then the grOwth inhibiting role of geIhaicin oncells were measured by 3 -(4, 5 d imethylthiazo l -2 -y l )-2, 5 - d ipheny ltetraZo l ium brotnide (Mrnmethod and agarose gel electrophoresis of DNA extuted frOm gentamicinotated cells wasperformed to observe aPoptOsis of cells. Flow cytomeny was used to determine the aPoptOsisindex (AI) of cells and distribution of cell cycle. The contentS of N-acety l - 6-Dilucosaminidase(NAG) in the supernnd of cells after coincubation wdri gemtamicin were d6t6rmined byeope method. WeStem Blotting were done to determine the protein levels of cyclin E andCDK2 and p27'.P' in ceIls treated with gentamicin. Reverse transcriPtion-poIymerase chainreaction or-PCR) was conducted to measure p27"p'mRNA of cells.ResultS: 1. The serum creatitine value increased markedly 2 days after ischemic renaIrepedsion and peaked at day 7. The value decreased at day l0 and retUmed to normai at day l4.The proliferating index (PI) which refiects degree of cell proliferation in control rats and renalischemia-repeffosion rats at dny 2,7,10 and l4 were 0.7%, l4.8%, 30.9%, 23.6% and l2.4%,respectively Agarose...