FK506 Alleviates Proteinuria In Rats With Adriamycin-induced Nephropathy By Down-regulating TRPC6 And CaN Expressions

Objectives: 1.To observe the expressions and distributions of transient receptor potential cation channel 6(TRPC6) and calcineurin ( CaN ) in normal and adriamycin-induced nephropathy rats′renal tissue for providinging the experimental foundation for prevention and treatment of nephrotic syndrome; 2. To evaluate the protective effect of calcineurin inhibitor tacrolimus(FK506)and study its possible mechanism on adriamycin-induced nephropathy rats.Methods: 1. Male Sprague-Dawley rats were randomly divided into four groups: the control group(Con,n=24), the adriamycin-treated group(ADR,n=24), the low dose of FK506-treated group(ADR+FK0.5,n=6)and the high dose of FK506-treated group(ADR+FK1.0,n=6). Adriamycin nephropathy model was established by two injections of adriamycin through the tail vein. From the 3rd week after the first injection of adriamycin to the day of sacrifice, the rats in ADR+FK0.5 and ADR+FK1.0 groups were given two different doses of FK506 by gastric gavage once a day, whereas the rats in the Con and ADR groups were given equal volume of normal saline; 2. At weeks 2,3,5 and7 after the first injection of adriamycin, 24-hour urine was collected with the metabolic cages. Blood was collected for albumin(ALB),serum creatinine(Scr),blood urea nitrogen(BUN),cholesterol(CHOL)measurement; 3. The rats were sacrificed to obtain the renals at the desired time points, the paraffin and thinnest slices were maked, the pathological and ultrastructure changes were observed under light microscope and transmission electron microscope; 4. The expressions of TRPC6, CaNmRNA in the renal cortex in natural progression and under the intervention of FK506 were detected using Real-time Ploymerase Chain Reaction ; 5. The expressions and distributions of TRPC6, CaN proteins in rat renal tissues were detected by immunohistochemical techniques; 6. The expressions of TRPC6, CaN proteins in the renal cortex were detected by western blot.Results:1. After the first injection of adriamycin, a significant increasement of the 24-hour urinary protein in adriamycin rats was observed at 3rd week and persisted up to 7th week(P<0.01),the adriamycin rats showed fatty substance metabolic disorder,hypoproteinemia and renal functional lesion which are typical performances of nephrotic syndrome .Compared with ADR rats, FK506 could alleviate hyperlipemia, hypoproteinemia and renal insufficiency to some extent.2. The adriamycin rats developed from minimal change disease to focal segmental glomerulosclerosis fom 3rd to 7th week observed by light microscope and the foot processes broadened to a different extent at 3rd week , the diffuse fusion and effacement even disappear of foot processes were observed at 7th week by transmission electron microscope. Compared with ADR rats, FK506 could reduce foot processes fusion subepithelial electron dense deposits obviously.3. Compared with the control rats, mRNA expression of TRPC6 in adriamycin rats began to increase at week3(P<0.05) and persisted up to week7(P<0.01) , mRNA expression of CaN in adriamycin rats was increased(P<0.05~0.01) at any observed time point.4. In normal rat renal, TRPC6 was highly expressed in the glomerular capillary loops, mesangium area, podocytes and renal tubular epithelial cell cytoplasm, and there was likely a few expression in the medulla; CaN was mainly distributed in glomerulus , weakly expressed in tubulointerstitium , and perivascular connective tissue was also seen positive expression. The specific protein bands of TRPC6 and CaN were detected with the size of 106 and 62 kDa, respectively. 5. At 7th week compared with the ADR rats, mRNA and protein expressions of TRPC6 and CaN in ADR+FK0.5 and ADR+FK1.0 rats were significantly decreased (P<0.05~0.01) ,expressions of TRPC6 and CaN between ADR+FK0.5 and ADR+FK1.0 rats showed no significantly change(P>0.05).6. Pearson correlation analysis was performed for the 24-hour urine protein, TRPC6 protein and CaN protein from week2 to week7. The 24-hour urine protein was positively correlated with TRPC6 protein(r=0.867,P=0.001)and positively correlated with CaN protein(r=0.776,P=0.008), TRPC6 protein was positively correlated with CaN protein(r=0.643,P=0.045).Conclusion:1. Establish adriamycin-induced nephropathy model successfully by two injections of adriamycin through the tail vein.2. It is demonstrated that up-regulation of TRPC6 and CaN expressions existed in adriamycin-induced nephropathy rats at mRNA and protein levels, which was closely associated with the development of proteinuria in nephropathy rats. These results will benefit for further exploring the molecular mechanism of proteinuria from the view of ion channel.3. TRPC6 was highly expressed in the glomerular capillary loops, mesangium area, podocytes and renal tubular epithelial cell cytoplasm, and there was likely a few expression in the medulla; CaN was mainly distributed in glomerulus , weakly expressed in tubulointerstitium, and perivascular connective tissue was also seen positive expression.4. FK506 had a reduction effect on proteinuria and renal tissue damage of nephropathy rats by down- regulating the expressions of TRPC6 and CaN.