RESUMO
ObjectiveTo evaluate the effects of 1,25(OH)zD3 on podocyte apoptosis in kidney of puremyein aminonueleoside nephropathy (PAN) rats. Methods Seventy-two male Sprague-Dawley rats were randomly divided into three groups: PAN model group(PAN), 1,25 (OH)2D3 treated group (T, 0.2 μg·kg-1d-1 by garages) and normal control group (NC). PAN rat model was constructed by a single intravenous injection of 100 mg/kg body weight. Renal function and 24hour urinary protein were measured at day 3, 7, 14, 21 after PAN injection. The renal tissue morphology was observed by light and electron microscope. Podocyte apeptosis was evaluated by TUNEL. Protein expressions of nephrin, TGF-β1 and p-Smad2/3 were examined by immunofluoreacence, immunohistochemistry and Western blot, respectively. Results(1)The levels of serum creafinine, BUN and 24-h urinary protein [(20.26±4.87) mg vs (1.01±0.41) mg at day 7, P <0.01] were significantly higher and the number of glomerular pedocyte was significantly lower [(10.9±4.2)/glomerular volume vs (31.9±6.2)/glomerular volume at day 14, P<0.01] in PAN group compared with NC group. T group rats had less urinary protein excretion [(9.95±3.82) mg/24 h, P<0.01] and more glomerular podocytes compared with PAN group. (2) Distribution of nephrin expression was changed from linear to granular pattern in PAN rats on day 7, nephrin mRNA and protein expressions were markedly decreased(P<0.01), while the number of apoptotic podocyte was increased in PAN group(P<0.01). However, higher nephrin expression and less apoptotic podocytes were found in T group (P<0.01). (3) Compared with NC group, the mRNA and protein expression of TGF-β1 and p-Smad2/3 were higher in PAN group (P<0.01), while 1,25 (OH)2D3 treatment abrogated PAN-induced changes in the expression of TGF-β1 and p-Smad2/3 (P<0.01). Conclusions 1,25 (OH)2D3 can significantly suppress PAN-induced podocyte apoptosis and ameliorate proteinnuria. The beneficial effect of 1,25(OH)2D3 on podocyte may contribute to direct suppression of TGF-β signaling.
RESUMO
Objective To study the effects of the interaction of advanced glycation end products (AGEs) and the receptor of AGEs (RAGE) on apoptosis of mice podocytes. Methods Podocytes were exposed to soluble AGEs such as bovine serum albumin (BSA), carboxymethyl-lysin (CML)-BSA, AGE-BSA and matrix-bound AGEs (AGE-modified collagen Ⅳ ), and to different concentrations of AGE, such as 10 mg/L, 50 mg/L, 100 mg/L. Apoptosis was assessed by TUNEL staining. Fluorescence-activated cell sorting (FACS) was used for the quantification of apoptotic andnecrotic podocytes after Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling. Apoptosis was described as the ratio of apoptotic cells to the total number cells under the high-power field, siRNA was transfected into podocytes through combining Dharmacon on Targetplus SMART pool siRNA reagents and Amaxa RNAi nucleofection kit. Results The apoptosis rate was higher in podoeytes exposed to either CML-BSA or AGE-BSA than that exposed to BSA. There was a two- to three-fold increase in apoptosis when podocytes were cultured in AGE-modified collagen Ⅳ as compared with native collagen Ⅳ. The apoptotic response of podocytes to AGE-BSA exposure occurred in a dose-dependent manner. Podocyte necrosis occurred only at the highest concentration of AGE-BSA(100 mg/L). AGE-BSA failed to induce apoptosis in podocytes transfected with RAGE siRNA. RAGE-specific gene knockdown did not significantly reduce the apoptosis of podocytes cultured in AGE-modified collagen IV. Conclusions The AGE-RAGE interaction plays a major role in the apoptosis of podocytes triggered by soluble AGEs, but not by matrix-bound AGEs. Reduction of AGE burden and RAGE expression may be important therapeutic approaches to prevent the progression of kidney disease.