Expression and functional role of p38 MAPK in the kidney after unilateral ureteral obstruction in rats

AIM: To investigate the expression and functional role of p38MAPK in the kidney after unilateral ureteral obstruction in rats. METHODS: Unilateral ureteral obstruction (UUO) models were induced by ligating the left ureter. Rats were sacrificed at 1 h, 3 h, 6 h, 12 h, 1, 3, 5, 7, 14, 21, and 28 days after UUO was initiated. p38MAPK activity was assayed by immunohistochemical staining and specific substrate phosphorylation with immunoprecipitation and Western blotting. TGFβ mRNA and protein expression were analyzed with in situ hybridization and immunohistochemical stainning. RESULTS: A basic p38MAPK activity was detectable in the normal kidney(0.22±0.06).p38MAPK pathway was rapidly act ivated at 1 hour(0.45±0.14 vs control,P<0.05)and this was steadily in creased by 12 hours(0.91±0.07 vs control,P<0.01)after UUO.Afterwards,the activity of p38MAPK reduced grad ually,then increased again from 3 days and this was steadily increased by 7 days(0.93±0.06 vs control,P<0.01). Upregulation of TGFβ₁ was markedly tested at 3 days(13.55±6.33 vs control,P<0.05)and this was steadily in creased by 7 days(26.78 8.77 vs control,P<0.01).The activation of p38MAPK preceded markedly the expression of TGF 1.The early activity of p38 MAPK was positively related to the amount of TGFβ₁ expression.The amount of TGFβ₁ expressed in obstructed kidney also related significant ly to the late activity of p38MAPK(r=0.84,P<0.01). CONCLUSION: The activity of p38MAPK is increased significantly in the obstructed kidney, which may cause renal fibrosis via inducing the expression of TGFβ₁.     

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.     

Pancreatic kininogenase protects against renal fibrosis in rat model of unilateral ureteral obstruction

AIM To assess the beneficial effects of pancreatic kininogenase （PK） on renal fibrosis in rat model of unilateral ureteral obstruction （UUO）. METHODS Male Sprague-Dawley rats were randomly assigned to 5 groups and treated daily with PK for 7 d and 14 d. Masson trichrome and HE staining were used to assess the degree of tubulointerstitial fibrosis, and immunohistochemistry and Western blot were employed to evaluate the expression of profibrotic and proinflammatory cytokines, and apoptosis- and autophagy-related proteins. RESULTS PK treatment significantly decreased expression of profibrotic and proinflammatory cytokines, and these were paralleled with attenuation of tubulointerstitial inflammation ［monocyte chemoattractant protein-1 （MCP-1） and Toll-like receptor 2 （TLR-2）］ and fibrosis ［transforming growth factor β1 （TGF-β1） and connective tissue growth factor （CTGF）］ in a time-dependent manner. Oxidative stress induced by UUO manifested by augment of oxidant enzymes ［NADPH oxidase-2 （NOX-2） and NOX-4］ and decrease in antioxidant enzymes ［superoxide dismutase 1 （SOD1） and SOD2］, which was closely associated with dysregulation of apoptosis- and autophagy-related proteins subsequent excessive apoptotic （Bcl-2/Bax and cleaved caspase-3） and autophagy （LC3B, beclin-1 and P62）, and all of these were eliminated by administration of PK. CONCLUSION PK treatment protects against the progression of renal fibrosis in obstructed kidneys probably by interfering oxidative stress and programmed cell death.     

Effects of p38 MAPK gene knockout on arsenite-induced cell apoptosis

AIM: To investigate the role of p38 mitogen-activated protein kinase (MAPK) in the cell apoptosis induced by arsenite.METHODS: After the treatment with or without arsenite, p38+/+ and p38-/- cells were fixed with paraformaldehyde and stained with DAPI. The nuclear morphological changes were observed under a fluorescence microscope. p38+/+ and p38-/- cells were double-stained with Annexin V-FITC and PI, and the ratios of apoptotic cells were detected by flow cytometry.RESULTS: With the stimulation of arsenite, typical apoptotic morphological changes appeared in most p38-/- cells, yet only a few p38+/+ cells showed nucleus condensation. Consistently, the ratio of apoptotic p38-/- cells induced by arsenite significantly increased in comparison with that in the untreated cells (P<0.01), and also much higher than that in p38+/+ cells stimulated with arsenite (P<0.01).CONCLUSION: p38 gene knockout leads to a decrease in cellular resistance to stress of arsenite, which produces a proapoptotic state. p38 MAPK may play an important role on the enhancement of cell adaptive capability to harmful stresses.     

Retinoic acid inhibits renal interstitial myofibroblast proliferation in a rat unilateral ureteral obstruction model

AIM: To investigate the effect of retinoic acid on proliferation of renal interstitial myofibroblasts in a rat model of the left unilateral ureteal obstruction (UUO). METHODS: UUO model was established by unilateral ligation of ureter in 36 SD rats. Rats were divided into 2 groups: Retinoic acid-treated group and control group, each with 18 rats. UUO rats were treated with either daily subcutaneous injection of 10 mg/kg body weight of all trans-retinoic acid or vehicle alone two days before the operation until being sacrificed. Groups of 6 rats were killed on day 3, 7 and 12 after ligation of the left ureter. The percentage of renal tubular lesion, interstitial fibrosis score, the number of interstitial myofibroblasts, the number of proliferating myofibroblasts and the expression of TGFβ-1 mRNA were determined. RESULTS: There were significant accumulation and local proliferation of myofibroblasts in the interstitium of the UUO rats. On day 7 of the UUO model, the percentage of tubular lesions and interstitial fibrosis score were significantly lower in the retinoic acid-treated group than those in control group[(15.9±2.0)%vs(27.3±2.2)%和(0.47±0.12)vs(1.65±0.18),P<0.01]. The numbers of interstitial myofibroblasts and proliferating myofibroblasts in the retinoic acid-treated group were significantly lower than those in the control group[(12.2±2.2)cells/HPF vs(29.5±1.8)cells/HPF和(1.4±0.6)cells/HPF vs(4.3±0.8)cells/HPF,P<0.01]. Furthermore, the expression of TGFβ-1 mRNA was significantly lower in the retinoic acid-treated group than that of the control group (P<0.01). CONCLUSION: Retinoic acid suppresses interstitial fibrosis in a rat UUO model by inhibiting the accumulation and local proliferation of myofibroblasts in the renal interstitium.     

Effect of p38 MAPK on cisplatin-induced renal tubular cell apoptosis

AIM:To investigate the role of p38 MAPK in cisplatin-induced rat renal proximal tubular cell (RPTC) apoptosis. METHODS:To determine the optimal concentration of cisplatin to induce RPTC apoptosis, the cells were treated with 0, 5, 10 and 20 μmol/L cisplatin for 24 h, and then the cell lysates were collected for Western blot analysis of cleaved PARP, p38 and phosphor ylated p38 (p-p38). To determine the role of p38 MAPK in cisplatin-induced RPTC apoptosis, the cells were divided into control group, cisplatin group (the cells were treated with cisplatin for 24 h) and cisplatin+p38 MAPK inhibitor group (the cells were treated with p38 MAPK inhibitor SB203580 for 1 h, and then treated with cisplatin for another 24 h). The morphological changes of apoptotic cells were observed under phase-contrast fluorescence microscope. The apoptotic rate of the cells were analyzed by flow cytometry. The caspase activity of RPTC lysates was examined using Ac-DEVD-AFC kit. The protein levels of p-p38, p38, cleaved PARP and cleaved caspase-3 were determined by Western blot. The pH value of extracellular environment of the cells was measured by pH meter. RESULTS:Cisplatin at 20 μmol/L obviously induced apoptosis of RPTC. The p38 MAPK was phosphorylated and its phosphorylation peaked at 15 min after cisplatin treatment. The apoptotic rate of RPTC was 12.08% after cisplatin induction. Cisplatin treatment also enhanced caspase activity, and increased cleavage of PARP and caspase-3 proteins (P<0.05). The p38 MAPK inhibitor SB203580 effectively inhibited the phosphorylation of p38 MAPK, down-regulated the RPTC apoptosis rate and caspase activity, and reduced the cleavage of PARP and caspase-3 proteins. The pH value change in RPTC culture medium was also inverted by SB203580. CONCLUSION:The phosphorylation of p38 MAPK is involved in cisplatin-induced apoptosis of RPTC. The apoptosis induced by cisplatin results in the change of acidic extracellular environment, which is inhibited by p38 MAPK inhibitor SB203580.     

Role of p38MAPK activation in high glucose-induced collagen Ⅲ synthesis in normal rat kidney tubular epithelial cells NRK52E

AIM: To study the role of p38 mitogen-activated protein kinase (p38MAPK) activation in high glucose-induced collagen Ⅲ synthesis in NRK52E cells. METHODS: Normal rat tubular epithelial cell line NRK52E was cultured in D-glucose of different concentrations, pretreated with SB203580 and collected at different time points. The levels of phospho-p38MAPK and extracellular matrix collagen Ⅲ were examined by Western blotting. RESULTS: The activation of p38MAPK was shown to be dependent upon D-glucose concentration and the time-course. Pretreatment with SB203580 blocked p38MAPK activation induced by high concentration of D-glucose in NRK52E cells. CONCLUSIONS: The activation of p38MAPK induced by high concentration of glucose may play a role in diabetic interstital renal fibrosis. SB203580 has a potential value of clinical applications in the prevention and treatment of diabetic nephropathy.     

Effects of 11, 12-EET and ischemic preconditioning on phosphorylated ERK and p38 MAPK during ischemia and reperfusion in rat myocardium

AIM: In order to study the relationship between the ERK and p38 MAPK activation and the protection of 11, 12-epoxyeicosatrienoic acid (11, 12-EET) and ischemia preconditioning (IP), the effects of 11, 12-EET and ischemic preconditioning on phosphorylated ERK and p38 MAPK during ischemia and reperfusion in rat myocardium were examined. METHODS: The rat heart was subjected to ischemia for 5 min by ligating the left anterior descending coronary artery followed by reperfusion for 5 min (two times) to undergo ischemia preconditioning. The rats were divided into 5 groups: (1) control; (2) sham group; (3) ischemia/reperfusion (I/R) group, in which the rat heart suffered from 60 min ischemia followed by 30 min reperfusion; (4) IP plus I/R group; (5) EET plus I/R group, in which 6.28×10^-8 mol/L 11, 12-EET was injected intravenously 20 min before I/R. The heart function was examined, and phosphorylated ERK and p38 MAPK were detected by Western blot. RESULTS: At 30 min reperfusion, +dp/dt_max, -dp/dt_max and LVDP decreased significantly in I/R group compared with sham group, IP plus I/R group and EET plus I/R group; Phosphorylated ERK1/2 level was higher in I/R group than sham group, but was lower in I/R group than IP plus I/R group and EET plus I/R group; Phosphorylated p38 MAPK level was lower in control, sham, IP plus I/R and EET plus I/R group than I/R group. CONCLUSION: 11,12-EET protects rat heart against ischemia/reperfusion injury, the mechanism may be related to activation of ERK1/2 and inhibition of p38 MAPK.     

Association among COX2, iNOS and p38MAPK in late phase of preconditioning in rat cardiomyocytes

AIM:To explore the association among COX₂ , iNOS and p38MAPK in late phase of preconditioning. METHODS: The expression of COX₂ , iNOS and p38MAPK activity were determined by western blotting and the injury of cardiomyocyte was assessed by LDH release and trypan blue exclusion in four groups: control group, group IPC (ischemic preconditioning), group SMT (iNOS inhibitor), group NS₃₉₈ (COX₂ inhibitor) and group SB (p38MAPK inhibitor).RESULTS:The expression of COX₂ in group IPC increased markedly in comparision with group SMT and group SB.The expression of iNOS in group SB was lower than that in group IPC and group NS₃₉₈.The difference of the amount of iNOS was not significant between group IPC and group NS₃₉₈.The difference of the amount of phospho-p38MAPK was not significant among group IPC, group SMT and group NS₃₉₈(P>0.05).The LDH was lower, and cell viability was higher in group IPC than those in control group.The LDH was higher, and cell viability was lower in group SMT, group NS₃₉₈ and group SB than those in group IPC. CONCLUSIONS:In late phase of preconditioning , the p38MAPK activity and expression of iNOS and COX₂ increase significantly in rat cardiomyocytes, activited p38MAPK mediates iNOS, then promotes COX₂ expression.     

Effect of LPS on intracellular localization of p38 protein kinase in Raw264.7 cells

AIM: To investigate the activation dynamics and the intracellular localization of p38 protein kinase in Raw264.7 cells after lipopolysaccharide (LPS) stimulation.METHODS: Protein kinase assay and immunogold electron microscope technique were used to check the activation dynamics and distribution of p38 MAPK in Raw264.7 cells before and after LPS stimulation. RESULTS: The kinase assay results showed that a marked increase in p38 activity was detected 15 min after LPS treatment, and reached maximal activity 30 min post stimulation, then dropped down and got closed to the pre-stimulated level 2 h later. The optimal LPS concentration for treatment was 100 μg/L. The immunogold electron microscope data showed that p38 spread evenly in every part of the cytosol of the non-stimulated and EGF stimulated Raw264.7 cells, such as endoplasmic reticulum, mitochondria, lysosome, while the golden granules intensity in the cytosol area decreased and in the nuclear area increased significantly after LPS stimulation.CONCLUSION: p38 MAPK moves to the nuclei of Raw264.7 cells on account of stimulation by LPS.     

CREG prevents human umbilical vein endothelial cells from apoptosis by inhibiting p38 MAPK activation

AIM: To study the effect of cellular repressor of E1A-stimulated genes(CREG) and its mechanism on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by etoposide (VP-16).METHODS: Primary HUVECs were cultured. RetroviraI eukaryotic expression vectors pLNCX-CREG and pLXSN-shRNA-CREG were transfected into HUVECs. The stable cell clone was selected and obtained by screening with G418 (800 mg/L) and the puromycin (2.5 mg/L), respectively. CREG expression was detected by Western blotting. The cells with overexpression of CREG (H-C) and those with CREG down-regulation (H-S) were pretreated with apoptotic inducer VP-16 at 100 μmol/L for 6 h. The apoptotic rates of the 3 kinds of cells were analyzed by TUNEL and flow cytometry with annexin V/PI dualstaining. Furthermore, the protein levels of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in the 3 kinds of cells were analyzed by Western blotting. The p38-specific inhibitor SB203580(20 μmol/L)was used to investigate the effects of p-p38 expression on apoptosis. RESULTS: Western blotting showed that CREG expression was obviously increased up to 160% in H-C compared to HUVECs. However, CREG expression in H-S cells was identified to be down-regulated to 70% compared with HUVECs. TUNEL assay and annexin V/PI dual-color FACS showed that the apoptotic rate was dramatically increased in H-S cells,but decreased in H-C cells. Subsequently, Western blotting exhibited that p-p38 expression was increased in H-S cells compared to HUVECs and H-C cells. When the H-S was pretreated with SB203580, the apoptotic rate was decreased. CONCLUSION: CREG overexpression might prevent HUVECs from apoptosis by inhibiting p38 MAPK activition.     

Recent progresses on the study of endotoxin-induced p38 MAPK signal transduction

The diseases caused by endotoxin have seriously affected human health. Previous studies have shown that p38 MAPK pathway is involved in the intracellular signal transduction induced by lipopolysaccharide (LPS), which plays an important role in the activation of inflammation-related cells to release inflammation mediator. Recently there have been some progresses in the isoforms distribution, substrate, molecular mechanism of regulating the release of inflammatory mediators, cellular specific activation and levels of p38 MAPK.     

Manumycin inhibits the activity of breast cancer cell line SK-BR-3 via inducing apoptosis

AIM: To investigate the anticancer effect of manumycin on abdominal metastatic breast cancer cell line - SK-BR-3 and its relationship with p38 MAPK. METHODS: The test of anticancer effect was performed by the method of MTT, apoptosis induced by manumycin and affected by SB203580, a specific p38 MAPK inhibitor, were examined by caspase-3 activity assay kit, and the protein expression was detected by immunoblotting assay. RESULTS: The inhibition rates at 24 h after treatment with manumycin of 6 μmol/L, 18 μmol/L, 54 μmol/L were (7.4±3.9)%, (21.0±4.4)% and (64.7±4.1)%, respectively and showed dosage-effect relationship. Compared with the control group, the survival rates of the last two treatment groups were decreased significantly (P<0.01). The value of IC₅₀ 24 h after treatment with manumycin was 42.5 μmol/L. Manumycin simultaneously activated caspase-3 protein, which was partly blocked by p38 MAPK inhibitor, SB203580. The results of immunoblotting showed that manumycin increased p38 MAPK protein phosphorylation. CONCLUSION: Manumycin exerts anticancer effect on SK-BR-3 cell line via inducing cell apoptosis, which is partly regulated by p38 MAPK.     

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.