Zhu Zhonghua

Effects of mycophenolate mofetil on renal interstitial fibrosis after Unilateral ureteral obstruction in rats.

SummaryTo investigate the effects of mycophenolate mofetil (MMF) on the process of renal interstitial fibrosis, unilateral ureteral obstruction (UUO) model was established in rats. Twenty Sprague-Dawley rats underwent UUO and received vehicle (n=10) or MMF (20 mg. kg−1, d−1, by daily gastric gavage,n=10) during a period of 5 days following surgery, and the additional 10 rats were served as sham-operated group. The rats were killed 5 days after surgery. Immunohistochemistry was performed on renal tissue for proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA) and type I and III collagen (col I, col III). Histological studies were also done by MASSON staining. Five days after surgery, proliferating cells in tubules, interstitium as well as interstitial myofibroblast (MyoF) infiltration and interstitial col I, col III deposition were all significantly reduced by MMF treatment. MMF also alleviated the histological changes of UUO rats. These results suggested that the reduction of interstitial MyoF infiltration may be an important event by which MMF prevents renal injury caused by UUO and MMF could be used to limit the progression of renal fibrosis.

Significance and expression of Serum and Glucocorticoid-inducible Kinase in kidney of mice with diabetic nephropathy

SummaryTo investigate the expression and the role of three isoforms of Serum and Glucocorticoid-inducible Kinase (SGK) in experimental diabetic nephropathy (DN), 12 male C57BL/6 mice of 8-weeks-old were divided into two groups. Streptozotocin (STZ)-induced diabetic nephropathy and normal controls were analyzed at the end of the 4th week after the induction of diabetes. Renal hemodynamics and histological studies were performed. The expression of SGK1 mRNA, SGK2 mRNA and SGK3 mRNA of kidney cortex were measured by RT-PCR, and the cortical SGK1 protein was detected with Western blotting. Our results showed that the blood glucose, blood HbA1c, 24-h urinary protein, creatinine clearance and the renal index were all increased in DN group. More extracellular matrix (ECM) accumulation was observed. The level of cortical SGK1 mRNA and protein were up-regulated in DN group in comparison with control group. SGK2 and SGK3 mRNA were elevated in DN mice. In DN, mRNA level of three SGK isoforms and SGK1 protein were increased significantly. It is concluded that SGKs may contribute to the early renal injury of DN.

Different expressions of protein kinase C-α, βI and βII in glomeruli of diabetic nephropathy patients

In current study, the expressions of protein kinase C (PKC)-α, βI and βII as well as their correlation to the expression of transforming growth factor-βI (TGF-βI) and vascular endothelial growth factor (VEGF) were investigated in glomeruli of normal renal tissues taken from human kidney tumors and kidney tissues from patients with diabetic nephropathy (DN). The accumulation of glomerular extracelluar matrix (ECM) was determined by PAS staining, the expressions of PKC-α, PKC-βI, PKC-βII, TGF-βI and VEGF were measured by semi-quantitative immunohistochemistry. Our results showed that in glomeruli of normal renal tissues, PKC-α and βII had a strong expression whereas the expression of PKC-βI was weak; in glomeruli of DN patients, the expressions of PKC-α, PKC-βI, VEGF and TGF-βI and the accumulation of ECM increased significantly, but the expression of PKC-βII decreased markedly. Meanwhile, the expressions of PKC-α and βI had a positive correlation to the expressions of VEGF and TGF-βI respectively, whereas PKC-βII showed no correlation to VEGF and TGF-βI. It is concluded that the expressions of PKC-α, βI and βII in glomeruli of normal subjects and DN patients are different. PKC-α seems to play a critical role in human DN by up-regulating VEGF expression, whereas PKC-βI is relatively important for the up-regulation of TGF-βI and the accumulation of ECM under diabetic conditions.

Effect of lipo-prostaglandin E1 on mesangial proliferative glomerulonephritis in rats.

SummaryThe antinephritic effect of lipo-prostaglandin E1, prostaglandin E1 incorporated in lipid microspheres was investigated using an experimental model of mesangial proliferative glomerulonephritis (MsPGN). Twenty-two female rats were randomly divided into nephritic group (N,n=6), lipo-prostaglandin E1 treated group (NL,n=8) and control group (C,n=6). Lipo-prostaglandin E1 was given intravenously at 40 μg·kg−1·d−1 from the 6th week to the 8th week. Twenty-four h urinary protein contents and blood creatinine (Cr) were determined and the pathological changes were observed in the experiment. The expression of proliferating cell nuclear antigen (PCNA), extracellular matrix (fibronectin, FN; collagen type IV, Col IV) and transforming growth factorβ1 (TGFβ1) was detected by using immunohistochemistry. The results showed that lipo-prostaglandin E1 significantly inhibited the glomerular histopathological changes as well as the elevation of plasma Cr (P<0.05). The overexpression of PCNA, FN, Col IV and TGFβ1 were also obviously inhibited in group NL as compared with the group N (P<0.01). It was suggested that lipo-prostaglandin E1 could improve renal function, inhibit the proliferation of glomerular cells and reduce the deposition of extracellular matrix, which may be related to the down-regulation of the TGFβ1 expression.

The study on the relationship between serum vascular endothelial growth factor and proteinuria in adriamycin-induced nephrotic rats

SummaryTo study the relationship between serum vascular endothelial growth factor (VEGF) and proteinuria in adriamycin-induced nephrotic rats, a rat model of adriamycin-induced nephrotitis was developed by injection of adriamycin into a tail vein in a rat. At different time points, 24-h urinary protein excretion was measured by using Coomassie brilliant blue method and the serum VEGF levels detected by using ELISA assay. The interventional effect of VEGF on this model was observed. The results showed that: (1) The adriamycin-induced nephrotic syndrome rat model was developed successfully; (2) Serum VEGF levels and proteinuria were significantly increased at 7th day after intravenous injection of adriamycin. There was a positive correlation between serum VEGF levels and 24-h urinary protein excretion (r=0. 67,P<0. 05). (3) The 24-h urinary protein excretion was significantly increased in the rats receiving administration of VEGF (P<0. 05). It was concluded that VEGF might play an important role in the pathogenesis of proteinuria in adriamycin-induced nephrotic rats.

Expression of serum and glucocorticoid-inducible kinase1 in diabetic rats and its modulation by fluvastatin.

SummaryThe expression of serum and glucocorticoid-induced protein kinase in the renal cortex of diabetic rats was examined, and the function of signal transduction mediated by SGK1 in diabetic nephropathy and its modulation by fluvastatin were also investigated. 24 male Wistar rats were randomly divided into normal control group (n=8), diabetic nephropathy group (n=8) and fluvastatin-treated diabetic nephropathy group (15 mg/kg/d,n=8). The metabolic parameters were measured at the 8th week. The expression of transforming growth factorβ1 (TGF-β1) and fibronectin (FN) was immunohistochemically examined. The expression of SGK1 was detected by RT-PCR and Western blot, and CTGF mRNA was assessed by RT-PCR. As compared to DN, blood glucose, 24-h urinary protein, Cer and kidney weight index were all decreased and the weight was increased obviously in group F. At the same time, mesangial cells and extracellular matrix proliferation were relieved significantly. The levels of cortex SGK1 mRNA and protein were up-regulated, and both TGF-β1 and FN were down-regulated by fluvastatin. The mRNA of SGK1 was positively correlated with the CTGF, TGF-β1, and FN. SGK1 expression is markedly up-regulated in the renal cortex of DN group and plays an important role in the development and progress of diabetic nephropathy by means of signal transduction. Fluvastatin suppressed the increased SGK1mRNA expression in renal cortex and postponed the development of diabetic nephropathy.

Expression of connective tissue growth factor in renal tubulointerstitial fibrosis in rats and its pathogenic role.

SummaryIn order to explore the role of connective tissue growth factor (CTGF) in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction (UUO) group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor-β1 (TGF-β1), collagen I (col I), and plasminogen activator inhibitor-1 (PAI-1) were detected using reverse transcriptional-polymerase chain reaction (RT-PCR). Immunohistochemistry was performed to evaluate the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO (P<0.01). With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index (r=0.62,P<0.01), the expression of TGF-β1 (r=0.85,P<0.01), col I (r=0.78,P<0.01), and PAI-1 (r=0.76,P<0.01). Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course (P<0.01, as compared with sham-operated group). It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix (ECM) synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

A potential pathological role of angiopoietins expression in glomeruli during progressive glomerulisclerosis related to podocyte injury.

SummaryA potential pathological role of angiopoietins (Ang) in glomeruli following podocyte injury-induced progressive glomerulosclerosis was explored. Eighty male Wistar rats were randomly allocated into sham operation group (Sham,n=25), Uninephrectomy group (UPHT,n=25) and Uninephrectomy+Daunorubicin group (DRB,n=30). In DRB group, daunorubicin (5 mg/kg) was injected via tail vein on the 7th and 14th day after uninephrectomy. At week 1, 2, 4, 6 and 8 respectively following establishment of the animal model, 5 rats in Sham group and UPHT group, and 6 in DRB group were taken respectively for determining 24-h urinary protein excretion rate (24hUPER), blood urea nitrogen (BUN) and serum creatinine (Scr). The sections of kidneys were examined by an electric microscope, PAS staining, immunohistochemical staining and in situ hybridization histochemistry. The results showed that 24hUPER, BUN and Scr in DRB group were more than those in Sham group and UPHT group at the same time points, and there was a trend towards an increase on level of GSI in DRB group from week 2 to week 8. Electric microscopy revealed that podocyte injury presented in DRB group. The expression of Angl mRNA and protein in glomeruli of DRB group was decreased, while the expression of Ang2 protein in glomeruli of DRB group increased, Meanwhile, the expression of Angl mRNA had a negative correlation with expression of Ang2 mRNA, and the expression of Angl protein had a positive correlation with the expression of Ang1 mRNA, and had a negative correlation with 24hUPER, BUN, Scr, glomerular sclerotic index (GSI), the expression of Ang2 protein and CoIV protein. The expression of Ang2 protein had a positive correlation with the expression of Ang2 mRNA, and had a positive correlation with 24hUPER, BUN, Scr, GSI, the expression of CoIV protein. It was concluded that podocyte injury might lead to an alteration in the expression of Ang1 and Ang2 within glomeruli. Ang2 may get rid of inhibition from Angl for downregulation of the Ang1 expression, which facilitate upregulation of the Ang2 expression in glomeruli to promote progressive glomerulosclerosis in the rats.

The effect of connective tissue growth factor on human renal tubular epithelial cell transdifferentiation

SummaryTo investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC),in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose CTGF-treated group (rh CTGF, 2.5 ng/ml) and high dose CTGF-treated (rhCTGF, 5. 0 ng/ml). Then the expression of α-smooth muscle actin (α-SMA) were assessed by indirect immuno-fluorescence, and the percentage of α-SMA positive cells were assessed by flow cytometry. RT-PCR were also performed to examine the mRNA level of α-SMA. Upon the stimulation of different concentrations of rhCTGF, the expression of α-SMA were markedly stronger than that in negative controls. The percentages of α-SMA positive cells were significantly higher in the stimulated groups than that of negative controls (38.9%, 65.5% vs 2.4%,P<0.01). α-SMA mRNA levels were also up-regulated by the stimulation of rhCTGF (P<0.01). These results suggest that CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (Myo-F).