Résumé
Objective To investigate the expression of nestin, a type Ⅵ intermediate filament protein in the glomeruli with foot process effacement and the potential relationship between nestin expression in the kidney and the degree of proteinuria. Method Immunohistochemistry was used to determine the localization of nestin in the kidney samples obtained from needle biopsies of normal human and patients with minimal change disease (MCD). Puromycin aminonucleoside (PAN) nephrosis rat models were established by a single intraperitoneal injection of PAN. Both real time quatitative reverse PCR and Western blot methods were applied to evaluate the levels of nestin expression at day 1, 4, 10 and 20 after PAN injection. Results Immunohistochemistry showed that the expression of nestin in glomeruli of MCD patients was significantly reduced compared with normal samples (0.93±0.08 vs 1.65±0.12, P<0.05) . The mRNA and protein expressions of nestin in the rat kidney were transitorily increased by 1.23 folds and 1.48 folds of control group (NC) after 1 day of PAN injection (P<0.05), then decreased quickly in the following days. The mRNA levels of nestin in the kidney were 35.8% and 12.1% of NC after 4 days and 10 days of PAN injection, respectively, (P<0.01) as determined by real time PCR. After 20 days of PAN injury, nestin mRNA expression partly recovered to 65.8% of NC (P< 0.05 ). The protein levels of nestin detected by Western blot presented the similar trend, which were 77.0%, 58.0% and 83.4% of NC after 4 days, 10 days and 20 days of PAN injection, respectively (P<0.05). The degree of proteinuria in puromycin aminonucleoside nephrosis rats was negatively correlated with both mRNA and protein levels of nestin in the kidney(r=-0.667,P<0.05 and r=-0.621 ,P<0.05, respectively). Conclusions The expression of intermediate filament protein nestin is down-regnlated in the kidney characterized with foot process effacement and negatively correlated with the degree of proteinuria in puromycin aminonucleoside nephrosis rats. Nestin may play a potential role in modulating the structure and function of podocyte.
Résumé
AIM A&A treated PAN rats, and to observe the effects of A&A on MAPK signaling pathway. METHODS Rats were divided into control, PAN, A&A treated PAN (A&A) and enapril treated PAN (ACEI) groups. The pathological lesion was observed under a light microscope. Immunohistochemistry combined with semi quantitive method was used to investigate the following parameters: cell number, ? SMA expression and extracellular matrix deposition. Expression and phosphorylation of protein kinases ERK, JNK and p38 were assayed. RESULTS In PAN rats, A&A suppressed ? SMA expression, which was closely correlated to cell proliferation, and extracellular matrix accumulation in glomerular mesangium. A&A significantly attenuated ? SMA expression in the tubulo interstitial area which was also parallel to the renal interstitial fibrosis.In this study, expression of all subtypes of MAPK had no difference between control and PAN groups. Compared with the inactivation of ERK and p38, phosphorylation of JNK was observed in glomeruli, renal tubules and interstitial cells in PAN rats, which was also inhibited by A&A treatment. CONCLUSION The inhibitory effect of A&A on phenotypic changes of renal resident cells, especially glomerular mesangial cell, may participate in its renal protective mechanisms. This effect, at least partially, was mediated by down regulated JNK activation.
Résumé
Captopril, an orally active ACE inhibitor, has been known to attenuate the protein excretion in animal model such as puromycin-aminonucleoside(PAN)- induced nephrotic rats or partially nephrectomized rats and clinical study, but the exact mechanism of its action has not been elucidated yet. Captopril interacts with several important vasoactive agents such as angiotensin, bradykinin, prostaglandin, etc., and then can alter the intrarenal hemodynamics. But it is unclear whether the antiproteinuric effect of captopril is mediated by its action on intrarenal hemodynamics. Because it has been found that the depletion of anionic sites(AS) on glomerular basement membrane (GBM) is the important pathophysiologic mechanism in PAN nephrosis, it can be postulated that captopril may reduce the proteinuria by the action on GBM anionic sites. But it needs the morphological evidence. To find out the mechanism of action of captopril on reducing proteinuria and the relationship with GBM anionic sites in PAN nephrosis, the author induced PAN nephrosis by intraperitoneal injection of PAN, (10mg/100g of body weight) in Sprague- Dawley rats and administered captopril per oral route(25mg/day/100g of body weight). GBM anionic sites were stained with polyethyleneimine(PEI) and cuprolinic blue, the cationic markers, and morphometry was done under the electronmicroscope. In PAN nephrotic rats(group A), systemic blood pressure was significantly elevated compared with normal control rats of group C, (157+/-17 vs. 128+/-14 mmHg, p<0.01). Captopril did not lower the systolic blood pressure significantly, but incresed the creatinine clerance(0.37+/-0.17 vs. 0.13+/-0.04ml/min/100g of body weight, P<0.05). In PAN+captopril group (group B), daily urinary excretion of protein began to attenuate from the sixth day compared with PAN group. On the final day of experiment(the 12nd day), captopril reduced the urinary protein excretion below the half of the amount of group A (20.3+/-5.3 vs. 44.1+/-17.1mg/24hr, P<0.01), but which did not decreased to that of normal group(9.7+/-2.7mg/24hr, P<0.01). The anionic sites on lamina rara externa showed reduction in number in group A (11.8+/-1.1/1000nm GBM) compared with normal group(19.3+/-0.7/1000nm, P<0.01), and in group B the loss of GBM anionic sites was significantly attenuated (16.1+/-1.1/1000nm, P<0.01). Captopril acts to reduce urinary protein excretion in PAN nephrotic rats, and which is associated with inhibition of depletion of GBM anionic sites, so that the action on the GBM anionic sites is thought to be one of the important mechanisms of antiproteinuric effect of captopril.