Hypoxia induces myofibroblast formation and stimulates production of collagen I in myofibroblasts through ERK1/2 pathway

AIM: To investigate the effect of hypoxia on the myofibroblast transdifferentiation from fibroblasts,and associated signaling of hypoxia on the production of collagen I in cultured rat renal cortical myofibroblasts.METHODS: The study is composed of two relevant parts.In the first part,a normal rat renal interstitial fibroblast cell line NRK-49F was treated with hypoxia (1% O2) or normoxia (21% O2) for 6 h,12 h and 24 h.The expression of hypoxia inducible factor-1α (HIF-1α) was examined by Western blotting in order to make sure the hypoxic condition is reliable.The myofibroblast transformation from fibroblasts induced by hypoxia was assayed by detecting the protein levels of α-smooth muscle actin (α-SMA).In the second part,the object was done on the primary cultured rat renal cortical myofibroblasts.Myofibroblasts were subjected to hypoxic or normoxic conditions for variety of times.The levels of HIF-1α in cell lysates and collagen I protein in supernatant culture medium and the activation of extracellular signal-regulated kinase (ERK)1/2 MAPK pathway were analyzed by Western blotting.RT-PCR was carried out to measure the levels of collagen I mRNA at different time points (2 h,4 h and 6 h).The distribution of HIF-1α in myofibroblasts was demonstrated by immunocytochemistry.The changes of collagen I production were detected after PD98059,a specific inhibitor of ERK1/2 activation pretreatment and during the hypoxia incubation.The activity of gelatinase matrix metalloproteinase-2(MMP-2) and MMP-9 in the supernatant medium from the cultured cells were assayed by gelatin zymography.RESULTS: Significant increased levels of HIF-1α protein appeared in cell lysates under hypoxia for 6 h.Furthermore,HIF-1α was translocated into nuclei of myofibroblasts after 6 h exposure of myofibroblasts to hypoxia.The levels of α-SMA protein increased in NRK-49F under hypoxia for 12 h (187%±32%,P<0.05).The level of collagen I protein in culture medium was increased in hypoxia treated myofibroblasts at 6 h (171%±27%,P<0.05) and 12 h (256%±61%,P<0.05).Collagen I mRNA expression was increased in cells under hypoxia condition for 4 h (189%±28%,P<0.05) and 6 h (221%±44%,P<0.05).The activities of MMP-2 and MMP-9 in the supernatant medium were not significantly changed at different experimental time points between the normoxic and hypoxic conditions.Activation of ERK1 /2 occurred as early as 15 min,sustained the high level at 30 min and 60 min and was back to the baseline level at 2 h.Blockade of ERK activation with PD98059 abolished hypoxia-induced expressions of collagen I protein.CONCLUSION: Hypoxia contributes to the renal interstitial fibrosis through inducing formation of myofibroblasts and stimulating the production of collagen I in myofibroblasts.     

Relationship between peritubular capillary injury and renal interstitial fibrosis in mice with unilateral ureteral obstruction

AIM: To observe the injury of peritubular capillary (PTC), hypoxia and interstitial fibrosis after unilateral ureteral obstruction (UUO), and to explore the effects of PTC injury and hypoxia on interstitial fibrosis in mouse model of UUO. METHODS: Forty-eight male KM mice were randomly divided into control group and UUO group. On the 1st, 3rd, 7th and 14th days, 6 mice in each group were sacrificed. The changes of pathomorphism in the kidney were observed by HE and Masson staining. The expression levels of thrombospondin-1 (TSP-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), and PTC density were detected by immunohistochemistry. The protein expression of VEGF was also determined by Western blotting. RESULTS: No histological abnormalities of the kidneys were observed in sham-operated mice. The expression of TSP-1 was increased 1 day after UUO, and significantly increased on the 3rd, 7th and 14th days(P<0.05). The expression of VEGF was obviously decreased(P<0.05). PTC density was gradually decreased. The expression of HIF-1α was gradually increased, and renal interstitial area was gradually expanded. PTC density was negatively correlated with the expression of TSP-1 and HIF-1α (r=-0.874 and r=-0.930, respectively). VEGF expression was positively correlated with PTC density (r=0.745). PTC density was negatively correlated with the area of renal fibrosis (r=-0.787). HIF-1α expression was positively correlated with the area of renal fibrosis (r=0.835, P<0.05). CONCLUSION: In mouse UUO model, the expression of TSP-1 is increased. The expression of VEGF is reduced. The peritubular capillary injury and tissue hypoxia are aggravated, and renal interstitial fibrosis area is expanded. Ischemia and hypoxia may play an important role in the progression of UUO.     

Expression of low density lipoprotein receptor-related protein in renal interstitial fibrosis

AIM: To understand the expression of low density lipoprotein receptor-related protein (LRP) in tubulointerstitial fibrosis of unilateral ureter obstruction (UUO) rats. METHODS: The localization of LRP within kidneys were assessed by immunohistochemical staining, the protein level of LRP and connective tissue growth factor (CTGF) in kidney were analysised by Western blot. RESULTS: The location of LRP positive-staining and the protein level of LRP were in the same tendency with CTGF, the level of LRP had strong correlationship with the level of CTGF (r=0.786, P＜0.01); The expression of LRP was positive correlated with tubular-interstitial injury index (r=0.800, P＜0.01). Enalapril treatment downregulated LRP level at 9 days time point. CONCLUSION: The expression of LRP was increased according to the progression of interstitial fibrosis in UUO rats, implicating that LRP may act as receptor of CTGF to mediate the profibrotic effect of CTGF.     

Increased fucosyltransferase 8 expression in rat kidney with unilateral ureteral obstruction

AIM: To investigate the effect of fucosyltransferase 8 (FUT8) on rat renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). METHODS: Ninety male Wistar rats were randomly divided into normal control group (control), sham operation group (sham) and UUO group, and sacrificed on day 1, 3, 7, 14 and 21 after operation. Serum creatinine and urea nitrogen were detected to assess renal function. PAS and Masson staining were used to observe histological changes of the rat kidneys. The time-correlated expression of FUT8 in the kidney was monitored by RT-PCR and Western blotting. The protein expression levels of FUT8 and activin receptor-like kinase 5(ALK5) were determined by immunofluorescence double-staining. Immunohistochemical staining was also used to assess the protein expression of fibronectin (FN), type I collagen (Col I) and α-smooth muscle actin (α-SMA). RESULTS: Compared with control group, serum creatinine and urea nitrogen in UUO group elevated on day 3 after operation (P<0.05) and reached the peak value on day 21 after operation (P<0.01). Renal tubule atrophy and renal interstitial fibrosis were observed in UUO group 7 and 14 days after operation. The mRNA and protein expression levels of FUT8 increased markedly in UUO group on day 3 (P<0.05) and reached its peak value on day 14 and 21 after operation (P<0.01). The results of immunofluorescence and immunohistochemistry showed that FUT8 and ALK5 were coexpressed in renal tubulointestitium. FN, Col I and α-SMA were significantly elevated in UUO group (P<0.05), and were positively correlated with the expression of FUT8 (P<0.05).CONCLUSION: The expression of FUT8 influences the progress of renal interstitial fibrosis, tubule atrophy and inflammatory cell infiltration in the kidney.     

Effects of HIF-1α silencing on expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919

AIM: To study the effect of hypoxia-inducible factor 1α(HIF-1α) silencing on the expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919 under hypoxia. METHODS: Hypoxic condition was induced by CoCl₂ and the expression of HIF-1α was silenced by small interference RNA. HIF-1α-specific RNAi lentiviral vector was constructed. Real-time RT-PCR and Western blotting were used to detect the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells under hypoxia. The expression of p27 and Ki67 was observed by Western blotting after HIF-1α silencing was performed. The cell cycle of hepatoma cells was detected by flow cytometry. RESULTS: Under hypoxic condition, the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells increased significantly (P<0.05). HIF-1α silencing significantly reduced the expression of Ki67 but increased the expression of p27 (P<0.05) in CBRH-7919 cells. In transfected cells, the number of cells in G₀/G₁ phase was much higher and that in S phase was much lower than those in the control cells. CONCLUSION: Hypoxia induces the expression of HIF-1α. HIF-1α silencing can regulate the proliferation of hepatoma cells through reducing the expression of Ki67 and increasing the expression of p27.     

Effect of Kang Xianling decoction on TGF-β1-Smad pathway in a unilateral ureteral obstruction rat model

AIM: To study the effect of Kang Xianling decoction,comprised of dahuang,danshen,taoren,niuxi and danggui,on TGF-β₁-Smad pathway in unilateral ureteral obstruction rat model.METHODS: Eighteen male SD rats were divided into 3 groups,sham group,model group and model group treated with Kang Xianling decoction randomly.Renal interstitial fibrosis model was established in rats by unilateral ureteral obstruction (UUO).After treatment for additional 14 d,parameters of hydroxyproline in obstructed kidney from 3 groups were analyzed.Rats were sacrificed and the pathological statuses of their kidneys were checked by HE staining and electron microscopy.Transforming growth factor-β₁ (TGF-β₁) mRNA in kidney tissue was determined by RT-PCR.TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),phosphorylated Smad2 and Smad2 protein were determined by Western blotting.RESULTS: Parameters of hydroxyproline in animals of model group were significantly increased than those in sham operation group (P<0.05).The mRNA expression of TGF-β₁ and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2 in kidney tissue of animals in model group were significantly up-regulated.After intervention with Kang Xianling decoction,the above-mentioned up-regulated parameters,except TGF-β₁,were all significantly inhibited.Compared to model group,the pathological changes in renal tissues in treatment group were remarkable improved.CONCLUSION: Kang Xianling decoction inhibits the TGF-β₁-Smad pathway and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2,so as to decrease the level of collagen in obstructed kidney and to alleviate the renal interstitial fibrosis in UUO rats.     

Phosphatidylinositol 3-kinase regulates hypoxia-inducible factor 1α in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension

AIM:To investigate relationship among phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in lung of rats with hypoxia-inducible pulmonary hypertension. METHODS:Forty male adult Wistar rats were randomly divided into five groups (eight rats in each group):control group (C group) and groups with hypoxia for 3, 7, 14 and 21 days (H3, H7, H14 and H21 group). Mean pulmonary arterial pressure (mPAP), right ventric hypertrophy index (RVHI) and vessel morphometry were measured. The levels of HIF-1α mRNA expression in lung tissue was measured by in siteu hybridization (ISH). The protein expression of HIF-1α，VEGF and phosphorylated protein kinase β (P-AKT) were observed by immunohistochemistry or Western blotting. RESULTS:mPAP increased significantly 7 days after hypoxia ［(23.53±1.78) mmHg］, peaked 14 days after hypoxia, then remained on the high level. Pulmonary artery remodeling index (extern diameter 100 μm) and RVIH became evident 14 days after hypoxia. Expression of P-AKT protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima and tunica media in all hypoxia rats. HIF-1α mRNA staining was poorly positive in control，hypoxia for 3 days and hypoxia for 7 days, but began to increase significantly 14 days after hypoxia (0.305±0.104, P<0.05), then remained stable. Expression of HIF-1α protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima in all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein was markedly up-regulated after 3 days (0.029±0.019, P<0.05 ), reached its peak 7 days after hypoxia (0.232±0.008, P<0.05), then tended to decline 14 days and 21 days after hypoxia. Expression of VEGF protein began to increase 7 days after hypoxia (0.188±0.018, P<0.05), reached its peak 14 days after hypoxia (0.238±0.017, P<0.05), then remained on the high level in pulmonary arterial tunica intima. Linear correlation analysis showed that P-AKT, HIF-1α mRNA, VEGF and mPAP were correlated with vessel the morphometry and RVHI (P<0.01). P-AKT was positively correlated with HIF-1α and VEGF (tunica intima). CONCLUSION:P-AKT, HIF-1α and VEGF are all involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.     

Expression of Sonic hedgehog pathway-related genes in kidney tissues of rats with unilateral ureteral obstruction

AIM: To investigate the role of Sonic hedgehog (Shh) signaling pathway in renal interstitial fibrosis in the rats with unilateral ureteral obstruction (UUO). METHODS: Forty-eight male Sprague-Dawley rats were divided randomly into sham operation group and UUO model group with 24 rats each. The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fiber in the kidneys was detected with HE and Masson staining. Immunohistochemical analysis was performed to evaluate the expression of Shh signaling pathway-related proteins, including Shh, Smo,Ptch1 and Gli1. The contents of TGF-β₁ and Shh in the kidney tissues were determined by ELISA. Real-time RT-PCR was used to detect the mRNA expression of TGF-β₁, Col I, Col III and Shh signaling-related genes.RESULTS: Fibrosis observed with HE and Masson staining was obviously increased in UUO kidneys, and aggravated as time prolonged. The contents of TGF-β₁, Col I and Col III were also increased. In addition, the expression of Shh, Smo and Gli1 was markedly increased in obstructive kidneys, and the expression of Ptch1 was decreased (P<0.01), suggesting that Shh signaling was activated. The level of Shh in UUO rats was associated with the content of TGF-β₁. CONCLUSION: Shh signaling is activated in the progress of renal interstitial fibrosis in UUO rats, and the possible mechanism triggering the fibrogenic response is that Shh signaling promotes the expression of TGF-β₁.     

Mechanisms of hypoxia-induced tumor necrosis factor-α production in rat alveolar macrophages

AIM: To study the effect of hypoxia inducible factor-1 alpha (HIF-1α) on tumor necrosis factor alpha (TNF-α) production in rat alveolar macrophages cultured under hypoxic condition. METHODS: Using HIF-1α decoy inhibiting its function, Immunohistochemistry, Western blot, semiquantitative RT-PCR and ELISA were used to determine the expression of HIF-1α protein and mRNA and the production of TNF-α in rat alveolar macrophages cultured under hypoxic condition (3% O2, 5% CO2, 92% N2), respectively. RESULTS: Expression of HIF-1α was positive in cultured macrophage nucleoli in hypoxia group and HIF-1α decoy group but it was negative in nomoxic control group. The content of HIF-1α protein in hypoxia group and HIF-1α decoy group were significantly higher than that in nomoxic control group (P<0.05). The content of HIF-1α mRNA in hypoxia group and HIF-1α decoy group were markedly higher than that in nomoxic control group (P<0.05), respectively. The content of TNF-α in hypoxia group (115±17 ng/L) was higher than that in control group ［（69±13） ng/L, P<0.05］ and HIF-1α decoy group ［（81±15） ng/L, P<0.05］. CONCLUSION: Hypoxia can increase significantly expression and activity of HIF-1α， which can promote the production of TNF-α in rat alveolar macrophages. It suggests that HIF-1α plays an important role in the pathogenesis of chronic inflammation-related diseases that can give rise to lung hypoxia such as COPD.     

Role of miR-219 and TGFBR2 in pathogenesis of renal fibrosis

AIM:To study the role of microRNA-219 (miR-219) in regulation of transforming growth factor-β receptor type 2 (TGFBR2) in renal fibrosis. METHODS:The renal fibrosis patients (n=70) were selected in this stu-dy, and 20 cases of healthy people were selected as control group. RT-qPCR was used to detect the expression of miR-219 in the serum of the patients with renal fibrosis and control group, and the expression of miR-219 in NRK49F cells after stimulation with angiotensin Ⅱ(AngⅡ) was detected. The protein expression of α-smooth muscle actin (α-SMA) in the NRK49F cells transfected with miR-219 mimics after stimulation with AngⅡ was determined by Western blot. The potential target gene TGFBR2 of miR-219 was screened and verified by the method of luciferase reporter gene. RT-qPCR and Western blot were used to detected the effect of miR-219 mimics on the expression of TGFBR2 at mRNA and protein levels, and the mRNA expression of α-SMA, connective tissue growth factor (CTGF), type I collagen α1 (COL1A1) and COL3A1 in the NRK49F cells was also detected, respectively. The unilateral ureteral occlusion (UUO) mouse model was established and the expression of miR-219 in the renal tissue was monitored. The morphological change of renal fibrosis was observed in the UUO mice after injection of miR-219, and the mRNA expression levels of COL1A1 and COL3A1 were detected. RESULTS:The expression level of miR-219 in the patients with renal fibrosis was significantly lower than that in control group, and the expression of miR-219 in the UUO mice was decreased significantly (P<0.01). The expression level of miR-219 was significantly decreased in the NRK49F cells after AngⅡ stimulation, and miR-219 mimics inhibited the protein expression of α-SMA(P<0.01). miR-219 mimics had a targeted regulatory effect on TGFBR2 gene, which inhibited the mRNA and protein expression of TGFBR2. miR-219 mimics inhibited the mRNA expression of α-SMA, CTGF, COL1A1 and COL3A1. miR-219 also down-regulated the mRNA expression of COL1A1 and COL3A1 in the UUO mice and inhibited the process of renal fibrosis. CONCLUSION:miR-219 inhibits the development of renal fibrosis by inhibiting the expression of TGFBR2, which may become a new target for the diagnosis and treatment of renal fibrosis.     

The mechanism of lipopolysaccharide-activated rat alveolar macrophages producing tumor necrosis factor alpha

AIM: To investigate the expression of hypoxia inducible factor-1alpha (HIF-1α) and the role of HIF-1α in tumor necrosis factor alpha (TNF-α) production in rat alveolar macrophages activated by lipopolysaccharide (LPS). METHODS: HIF-1α function was inhibited by using the method of HIF-1α decoy. Western blotting and semiquantitative RT-PCR were applied to determine the expression of HIF-1α protein and mRNA, respectively. The production of TNF-α was determined with ELISA. RESULTS: The content of HIF-1α protein in LPS group (1.95±0.57) and HIF-1α decoy group (1.89±0.59) were 4.8 times and 4.6 times higher than that in control group (0.41±0.14), respectively. The expression of HIF-1α mRNA showed no difference among three groups (F=3.14，P>0.05). The production of TNF-α in LPS group was higher than that in control group (61 ng/L vs 156 ng/L, q=5.12, P<0.05) and HIF-1α decoy group (90 ng/L vs 156 ng/L, q=4.63, P<0.05), respectively. However, the content of TNF-α in HIF-1α decoy group was still higher than that in control group (61 ng/L vs 94 ng/L, q=4.47, P<0.05). CONCLUSION: The enhanced stability of HIF-1α protein results in the marked upregulation of its protein and HIF-1α is contributed to the production of TNF-α in LPS-stimulating rat alveolar macrophages. It is indicated that HIF-1α plays important role in the pathogenesis of chronic inflammation involved in diseases such as COPD.     

Cell cycle arrest induced by hypoxia inducible factor-1 alpha in SW626 cell line of human ovarian cancer

AIM: To investigate the cell cycle arrest induced by hypoxia, hypoxia inducible factor-1 and their possible mechanism in human ovarian cancer cell line SW626. METHODS: CoCl2, a chemical inducer of hypoxia and hypoxic cell culture chamber were used to induce chemical and physical hypoxia in human ovarian cancer cell line SW626. The method of ‘decoy’ was used to block the function of HIF-1α because it acts as the core sequence of the target gene as a competitor combined to the HIF-1α. The cells were divided into group A1 (normal oxygen), A2 (normal oxygen plus HIF-1α decoy), B1 (CoCl2), B2 (CoCl2 plus HIF-1α decoy), C1 (hypoxia) and C2 (hypoxia plus HIF-1α). The expression of the HIF-1α protein, mRNA and cell cycle analysis were detected by Western blotting, RT-PCR and flow cytometry (FCM). RESULTS: The expression level of HIF-1α protein in group B1 (3.75±1.31) and group C1 (3.48±1.01) was significantly higher than that in group A1 (0.97±0.31) (P<0.05). The expression levels of HIF-1α mRNA in group A1 (0.65±0.32) and group B1 (0.64±0.34) were significantly lower than that in group C1 (1.28±0.62) (P<0.05). Decoy had no effect in the expression of HIF-1α protein and mRNA level (P>0.05). FCM showed that the G0/G1 phase was markedly increased in group B1 (81.78±24.33) and group C1 (77.62±22.76) and was significantly higher than that in group A1 (49.49±18.54) (P<0.05), group B2 (61.54±20.84) was lower than that in group B1 with statistical significance (P<0.05) and group C2 (56.03±21.42) was lower than that in group C1 with statistical significance (P<0.05) , but the difference between group A1 and group A2 (51.77±16.45) had no statistical significance (P>0.05). CONCLUSION: Both CoCl2 and physical hypoxia could distinctly induce cell cycle arrest in G0/G1 phase and the expression of HIF-1α in human ovarian cancer cell line SW626. HIF-1α plays an important role in cell cycle arrest induced by hypoxia in human ovarian cancer cell line SW626.     

Changes of hypoxia inducible factor 1 alpha and heme oxygenase 1 in the process of hypoxic pulmonary hypertension development in rats

AIM: To observe the expression of hypoxia inducible factor-1α (HIF-1α) gene and heme oxygenase-1 (HO-1) gene in pulmonary arteries in hypoxic rats. METHODS: Forty male Wistar rats were exposed to hypoxia for 0, 3, 7, 14 or 21 days. Mean pulmonary pressure (mPAP), vessel morphometry, right ventricle hypertrophy index (RVHI) were measured. Lungs were either inflation fixed for immunohistochemistry and in situ hybridization or frozen for later measurement of HO-1 enzyme activity. RESULTS: During hypoxia, mPAP increased to significantly higher values than the control values after 7-day of hypoxia，reaching its peak after 14-day of hypoxia, then remained on the high level. Pulmonary artery remodeling developed significantly after 14-day of hypoxia. Expression of HIF-1α protein in control was poorly positive, but was up-regulated in pulmonary arterial tunica intimae of all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein were markedly up-regulated after 3-day and 7-day of hypoxia, then tended to decline after 14-day and 21-day of hypoxia. HIF-1α mRNA staining was poorly positive in control, hypoxia for 3 days and hypoxia for 7 days, but began to enhance significantly after 14-day of hypoxia, then remained stable. Expression of HO-1 protein began to increase after 7-day of hypoxia, reaching its peak after 14-day of hypoxia, then remained stable. Expression of HO-1 mRNA began to increase after 3-day of hypoxia, reaching its peak after 7-day of hypoxia, then declined. CONCLUSION: HIF-1α and HO-1 are both involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats. Furthermore, HIF-1α may inter-regulate with HO-1 gene in this process.     

p38MAPK expression and apoptosis in the rat unilateral ureteral obstruction model

AIM: To investigate the expression of p38 mitogen-activated protein kinase (p38MAPK) in the kidney after unilateral ureteral obstruction (UUO) in rats and the functional role of it on apoptosis and fibrosis.METHODS: Eighteen Wistar rats underwent UUO were killed at 3, 7, 14 days. Additional 7 rats were sham operated. Histological changes were observed by HE and Masson staining. Immunohistochemistry study was performed on renal tissue for proliferating cell nuclear antigen (PCNA). Apoptotic cells were determined by terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) and the electrophoresis analysis of genomic DNA. Western blotting of cysteinyl aspartate specific proteinase-3 (caspase-3), p38MAPK and p-p38MAPK were measured.RESULTS: UUO induced a significant increase in renal tubular and interstitial cell apoptosis, immunohistochemistry of PCNA and Western blotting of caspase-3, p-p38MAPK as well as severe morphology changes. However, there was no significant difference between UUO and the control in Western blotting of p38MAPK.CONCLUSION: An in vivo model of renal fibrosis after UUO demonstrates that activated or phosphorylated p38MAPK plays a role in apoptosis of renal tubulointerstitial cells.     

Expression of BMP-7 and its receptors in rats with unilateral ureteral obstruction

AIM:To explore the expression changes of b one morphogenetic protein-7 (BMP-7) and its receptors (BMPR-Ⅱ,ALK-2,ALK-3,A LK-6) in the renal tubulo-interstitial lesions induced by unilateral ureteral ob struction (UUO).METHODS:Rats were divided into normal control,sham operation a nd UUO groups,and sacrificed at postoperative day 1,3,7 and 14.The mRNA leve ls of BMP-7,BMPR-Ⅱ,ALK-2,ALK-3 and ALK-6 were examined by RT-PCR.The protei n expression site and level of BMP-7 was detected by immunohistochemistry staini ng.RESULTS:Compared to sham operation group,the mRNA levels of BM P-7,BMPR-Ⅱ,ALK-2 and ALK-3 were significantly decreased,but the change of AL K-6 mRNA was not marked in UUO rats.The reduction of mRNA expression levels of BMP-7,BMPR-Ⅱ,ALK-2 and ALK-3 in the kidney tissue aggravated along with the d elayed post-operated days.The results of immunohistochemistry staining indicate d that BMP-7 mainly expressed in renal tubular and interstitial,rarely in glome ruli.In UUO rats,the protein expression of BMP-7 decreased in varying degrees according to the post-operated days.CONCLUSION:The loss of BMP-7 and its receptors (BMPR-Ⅱ,ALK-2,ALK-3) were observed in the early phase of fibrotic process and this may play a very important role in mediating the renal tubulointerstital fibrosis.     

Ginsenoside Rh1 attenuates unilateral ureteral obstruction-induced renal interstitial fibrosis in rats

AIM:To observe the effects of ginsenoside Rh1 (G-Rh1) on unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis and to investigate the underlying mechanisms. METHODS:Male Sprague-Dawley (SD) rats (n=40) were divided into the following 4 groups:UUO-operated group (UUO group), sham-operated group (sham group), UUO-operated plus a low dose (50 mg·kg^-1·d^-1) of G-Rh1 treatment (low G-Rh1 group) and UUO-operated plus a high dose (100 mg·kg^-1·d^-1) of G-Rh1 treatment group (high G-Rh1 group). The G-Rh1 treatment was carried out by gastric gavage from the next day after the UUO operation once a day for 2 weeks (14 d). Immediately after the final dose of G-Rh1, 24 h urine was collected for the urine protein test, and then the rats were euthanized. The blood was collected for the blood urea nitrogen (BUN) and serum creatinine (SCr) assays, and the kidney was removed for pathological and biochemical evaluations. RESULTS:The levels of 24 h urine protein did not show any significant diffe-rence among the groups, while significantly increased levels of BUN and SCr in UUO group were observed (P<0.05), which was prevented by the treatment with G-Rh1 at both doses in a dose-dependent manner. Pathological evaluation showed the renal tissue damage was obvious in UUO group, which was improved by the treatment with G-Rh1 at both doses. Immunohistochemcial analysis exhibited that UUO increased renal interstitial transforming growth factor-β₁ (TGF-β₁) expression, which was also inhibited by the treatment with G-Rh1 at both doses(P<0.05). Significantly increased protein expression of renal interstitial collagen type I, α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) in UUO group was detected, which was suppressed by the treatment with G-Rh1 at both doses. CONCLUSION:G-Rh1 improves UUO-induced renal dysfunction and attenuates interstitial fibrosis, which is mediated via modulation of TGF-β₁-related pro-fibrogenic signaling pathway.