ABSTRACT
Objective This study was designed to explore the effect of hyperglycemia on the expression of Toll-like receptor 4 (TLR4 ) in renal tubular epithelial cells and its significance in diabetic nephropathy. Methods In vitro cultured renal tubular epithelial cells ( NRK-52E) were divided into LG group (cultured in 5mmol / L glucose DMEM) and HC group (cultured in 25mmol / L glucose DMEM). Cells were harvested at different time points. Immunohistochemistry, Rt-PCR, Western Blot were used to detect TLR4 protein and mRNA expression, and the levels of IL-6 and TNF-α from the cell culture supernatant were determined by EL1SA assay. Results After 6 hours, there was increased expression TLR4 mRNA in HC group, which appeared to be maintained for 24 hours and began to decrease after 48 hours ( P < 0.05). TLR4 protein expression increased in HC group after 24 hours, and increased even further after 48 hours. Compared with LG groups, the difference had statistical significance ( P <0.05). In HG group, IL-6 and TNF-α expression in the supernatant from the NRK-52E culture were significantly increased ( P < 0.05) , and the expression of IL-6 and TNF-α was positive correlated with the expression of TLR4 protein ( r =0.799,0.820). Conclusion High glucose triggers an increase in expression of TLR4 in NRK-52E cells, itself leading to an increase in expression of inflammatory factors such as TNF-α and IL-6. In this way, TLR4 participates in the progress of diabetic nephropathy.
ABSTRACT
Objectives To investigate the changes of cystatin C in the serum of patients with acute kidney injury ( AKI) or end-stage renal disease ( ESRD), and study its significance in the early diagnosis of AKI and its correlation with the degree of renal function injury. Method The cases in Xiangya hospital were enrolled in this study according to the RIFLE criteria, including 20 cases of slight acute kidney injury, 30 cases of medium-severe acute kidney injury, 48 cases of victims of the 5. 12 wenchuan earthquake, 32 cases of end-stage renal disease and 20 healthy patients. The microparticle-enhanced immunoturbidimetry method was used to detect serum cystatin C, and the colorimetric method was used to detect urine N-acetyl-beta-D-glucosaminidase (NAGase). The enzymic method was used to detect serum creatinine. The correlation between serum cystatin C and serum creatinine was analyzed, and the sensitivity and specificity of serum cystatin C were evaluated with the receiver operator characteristic curve (ROC curve). Results Compared with healthy control group, the serum cystatin C increased obviously in acute kidney injury group and ESRD group( P <0. 05 and P <0. 01). The serum cystatin C was positively correlated with serum creati-nine( P <0.01). The serum cystatin C in the 5. 12 wenchuan earthquake injured group was also higher than that in healthy control group ( P <0.05), an the serum cystatin C had an AUC - ROC of 0.931 ( P <0. 01). Conclusion Compared to the conventional biomarkers, the earlier emergence of serum cystatin C can contribute better to early clinical diagnosis of AKI. The serum cystatin C is positively correlated with renal function, and reflect changes in renal function accurately.
ABSTRACT
Objective To determine the effect of aldosterone and its antagonist, spironolactone on epithelial-mesenchymal transition (EMT) of normal rat kidney epithelial cells (NRK-52E) in a high glucose milieu,and to explore the mechanism of renoprotection in diabetic nephropathy (DN ) in rats involving aldosterone and spironolacton. Methods NRK-52E cells were simultaneously cultured in the serum-free Dulbecco's modification of Eagle's medium Dulbecco (DMEM) for 12 hours. Then the low glucose (LG) group was cultured in LG (1000 mg/L) DMEM:The high glucose (HG) group was cultured in high glucose (4 500 mg/L) DMEM. The aldosterone (Aldo) groups were cultured in high glucose DMEM with the addition of 10,50 and 100 nmol/L aldosterone respectively. The SP group was cultured in high glucose (4 500 mg/L) DMEM plus 10~(-7)mol/L spironolactone. Immunohistochemistry, RT-PCR and Western blot were used to detect E-cadherin and α smooth muscle actin(α-SMA) mRNA expression. Results RT-PCR showed that compared with the LG Group, E-cadherin mRNA expression in the HG group was significantly lower, and α-SMA mRNA expression was significantly increased(P＜0.05). E-cadherin mRNA expression in the 50 nmol/L Aldo group and 100 nmol/L Aldo group was significantly lower than that in the HG group, while the expression of α-SMA mRNA was significantly increased in the HG group(P＜0.05), with a dose-dependent relationship with aldosterone(r=-0.70,P＜0.05;r=0.67, P＜0.05). E-cadherin mRNA in the SP group was significantly higher,while α-SMA mRNA expression was lower than that in the HG group(P＜0.01). Immunohistochemistry and Western blot showed that compared with the LG group, E-cadherin protein expression was significantly reduced, and α-SMA expression was significantly increased in the HG group(P＜0.01). In the 10 nmol/L Aldo, 50 nmol/L Aldo, and the 100 nmol/L Aldo groups, E-cadherin protein expression was significantly lower, and α-SMA protein expression was significantly higher than that in the HG group(P＜0.05), with a dose-dependent relationship with aldosterone(r=-0.83,P＜0.05;r=0.81, P＜0.05). In the SP group, E-cadherin protein expression was higher, and α-SMA protein level was lower than that in the HG group(P＜0.05). Conclusion Aldosterone can promote EMT of tubular epithelial cells in a high sugar milieu, leading to renal interstitial fibrosis in Diabetic nephropathy. Spironolactone can inhibit high glucose-induced renal tubular epithelial cells EMT, which may be an important mechanism for the inhibition of renal interstitial fibrosis.