【Abstract】 Objective To observe whether bone marrow stem cells can differentiate into renal tubular epithelial cells in physiology and pathology conditions. Methods Transgenic C57BL/6 mice labelled with green flouresent protein(GFP) were bone marrow donors. C57BL/6 mice without fluorescence label were divided into normal group, total body irradiation group,ischemia/reperfusion (IR) group, bone marrow transplantation (BMT) group, BMT+IR group. Bone marrow reconstruction of the recipients was proved by blood routine and flow cytometry of bone marrow cells after transplantation, and the distribution and the amount of green fluorescence in kidneys of recipients were observed by fluorescence histochemistry and immunohistochemistry. Results Lethally total body irradiation made no significant change in renal histological structure and function. Green fluorescence was seen in renal tubular epithelium of the recipients on day 56 and 84(positive cell,78.75%±5.99%,79.54%±4.60% after bone marrow transplantation. These positive cells locating in tubular epithelium were further proved by laser scanning confocal microscope, and fluorescence histochemistry showed these cells expressed renal tubular epithelium specific protein megalin. Conclusion Bone marrow stem cells can differentiate into renal tubular epithelial cells and participate in regeneration of renal tubular epithelium in physiology and pathology conditions, and the number of epithelial cells derived from the bone marrow is associated with the degree of the injury in acute tubular necrosis.

Objective To investigate the role of mitochondrial Smac/Diablo and XIAP in ATP depletion/recovery-induced apoptosis in renal tubular epithelial cells. Methods To induce apoptosis, HK-2 cells were transiently exposed to metabolic inhibitor for 90 min followed by recovery. Apoptosis was detected by Hoechst33342 staining. The release of Smac/Diablo was examined by indirect immunofluorescence. Cytosolic fraction was extracted to detect the level of Smac/Diablo by Western blot. Total protein was extracted to measure the protein level of XIAP and pro-caspase-3. Results After ATP depletion/recovery in HK-2 cells, Hoechst33342 staining showed nucleus condensation and apoptotic body conformation. Indirect immunofluorescence revealed the release of Smac/Diablo. Western blot showed the increased level of Smac/Diablo in cytosolic fraction and the decreased level of XIAP and pro-caspase-3 after ATP depletion/recovery. Conclusion Smac/Diablo release from mitochondria and decreased level of XIAP in HK-2 cells after ATP depletion/recovery may play an important role in apoptosis through caspase-dependent pathway.

Objective To explore whether hypoxia inducible factor-1(HIF-1) activation by prolyl 4-hydroxylase-2 gene silencing attenuates the apoptosis of human renal proximal tubular cells(HKCs). Methods The plasmid encoding small interfering RNA (siRNA) to silence human HIF-1α-prolyl-4 hydroxylase-2 (PHD2) was constructed, then transfected into HKCs. The efficiency of PHD2 siRNA was identified by detecting PHD2 mRNA with RT-PCR and HIF-1α protein with Western blot. HKCs were divided into three groups: control, model and siRNA treatment. HKCs pretreated with or without PHD2 siRNA were exposed to antimycin. The apoptosis of HKCs was investigated by flow cytometer. Results The PHD2 siRNAs produced a significant reduction in PHD2 mRNA (P < 0.01)and a significant increase in HIF-1α protein expression compared with control(P < 0.05). Antimycin could induce significant up-regulation of HIF-1α protein expression in model and siRNA treatment group compared with control group (P < 0.01). Meanwhile,HIF-1α protein expression was more higher in PHD2 siRNA treatment group than that in model group (P < 0.05).The apoptosis of HKCs in siRNA treatment group was significantly reduced as compared to model group (P < 0.05). Conclusion PHD2 silencing by RNA interference (RNAi) can attenuate hypoxia-induced apoptosis mediated by HIF-1 activation.

Objective To study the apoptosis of renal tubular cells in acute renal failure induced by cisplatin and the effect of carvedilol on such apoptosis. Methods Male wistar rats were randomly divided into four groups: group 1 received 0.85% saline 1 ml/200 g ip. single dose; group 2 received cisplatin 10 mg/kg ip; group 3 received carvedilol 15 mg/kg qd ig; group 4 received carvedilol 15 mg/kg qd ig. for 6 days, and cisplatin 10 mg/kg ip at day 4. Nephrotoxicity was assessed by measuring BUN, Scr, urinary acetylglucosaminidase(NAG) and histopathology of kidney 3 days after the cisplatin treatment. Tubular cell apoptosis was examined by TUNEL and DNA agarose gel electrophoresis. Protein expression of caspase-3 as well as malondialdehyde(MDA) content and superoxided dismutase(SOD) activity were detected in kidneys. Results Cisplatin induced a significant elevation in Scr, BUN, urinary NAG, renal tubular cell apoptosis, expression of caspase-3 as well as MDA content and severe morphology changes, while decreased the renal SOD activity. However, carvedilol improved above status of nephrotoxicity. Conclusions Renal tubular cell apoptosis is one of the important causes of cisplatin-induced ARF. Pretreatment with carvedilol can protect the rats from cisplatin-induced nephrotoxicity by reducing ROS, decreasing the apoptosis of renal tubulan cells,and partly inhibiting the caspase-dependent way.

Objective To investigate the effect and mechanism of irbesartan on the apoptosis of renal tubular cells induced by contrast media. Methods NRK-52E cells were exposed to different concentrations (25, 50, 100, 150 mgI/ml) of ioversol (a non-ionic contrast media)for 1 h. Cells of other groups were incubated with ioversol(100 mgI/ml) for 0.5 h, 1 h, 2 h, 4 h, respectively. Mannitol with the same osmolality as ioversol (420 mmol/L) was used to treat NRK-52E cells as control. In separate experiments, irbesartan (0.01,0.1,1 mmol/L) was added 1 h before incubation with ioversol (100 mgI/ml). Apoptosis was determined by Hoechst stains and flow cytometry AnnexinV-FITC/PI double stains. The intracellular ROS was detected by confocal microscopy with fluorescent probe CM-H2DCFDA. Bax and Bcl-2 mRNA expression was examined by reverse transcription-polymerase chain reaction (RT-PCR). Results Ioversol induced NRK-52E cells apoptosis in a concentration- and time-dependant manner. Mannitol could not induce cell apoptosis. The intracellular ROS generation was markedly increased following ioversol treatment. Furthermore, ioversol induced a decrease in the expression of Bcl-2 mRNA and an increase in the expression of Bax mRNA. Irbesartan attenuated the ioversol-induced apoptosis of NRK-52E cells in a dose-dependent manner, in which the protective effect of irbesartan was dependent on decreasing intracellular ROS formation. In addition, irbesartan reversed the ioversol-induced increase in Bax mRNA and decrease in Bcl-2 mRNA. There was a positive correlation between ROS level and apoptosis rate. Conclusions Ioversol induces NRK-52E cells apoptosis in a concentration- and time-dependant manner via trigerring oxidative stress and up-regulating the expression of Bax mRNA and down-regulating the expression of Bcl-2 mRNA. Irbesartan can attenuate the above effects of ioversol.

Objective To observe the effects of TIMP-1 overexpression on renal tubulointerstitial inflammation. Methods Human TIMP-1 transgenic mice and wild-type mice were randomly divided into UUO groups and sham groups respectively and sacrificed at 3 and 14 days after UUO or sham operation(n=6, each group). Masson staining was applied to observe the renal tubulointerstitial pathological changes,and indirect immunofluorescence was applied to detect F4/80 positive cells. Western blot was applied to analyze the protein expression of TIMP-1, TIMP-2, MMP-2, MMP-9 and ICAM-1. The activities of gelatinase and TIMP-1 were detected by gelatin zymography and reverse zymography respectively. Results The degree of renal tubulointerstitial fibrosis[fibrosis area:trangenes(46.24±6.58)% vs wild-type (36.33±5.12)%,P < 0.05], the number of positive F4/80 cells[trangenes(68.9±15.6) vs wild-type (52.4±13.3)cell/field,P < 0.05], and the protein expression of ICAM-1 were increased after UUO, which were more significant in transgenic group than in wild-type group at day 14 after UUO(P < 0.05). The protein expression and activity of TIMP-1 were increased significantly after UUO and with a maximum at day 14 after UUO, and were higher in transgenic group than in wild-type group(P < 0.05). The protein expression and activities of MMP-2 and MMP-9 were decreased after UUO, and were lower in transgenic group than in wild-type group at day 14 after UUO (P < 0.05). Conclusions Overexpression of TIMP-1 exacerbates renal tubulointerstitial injury by enhancing inflammation.

Objective To study the plasma level of vasopressin (AVP or ADH) and the localization, expression as well as trafficking status of aquaporin-2 on protein kinase C (PKC)-alpha knockout mice and to elucidate the potential mechanism of PKC-alpha in mediating urine concentration ability. Methods PKC alpha knockout and wild type (SV129)mice(n=6, each group) were placed in metabolic cages to assess 24 hours urine output, urinary osmolality by freezing-point depression, urinary urea excretion by ELISA, plasma AVP level by radioimmunoassay (RIA). The localization and expression of aquaporin-2(AQP-2) in inner medulla were determined by immunohistochemical and semi-quantitative Western blotting techniques. After i.p. injected with vasopressin V2-receptor antagonist SR121463 and different concentrations of DdAVP separately, the urinary osmolality and urinary output of 3 hours were checked to observe trafficking status of AQP-2. Results As compared to wild type mice, urinary urea excretion and plasma AVP level [(5.03±0.44) pmol/L vs (4.64±0.43) pmol/L,P = 0.55] were not different between tow groups under basal condition consisting with high urinary volume [(2.85±0.35) ml vs (1.80±0.18) ml, P = 0.02] and lower urinary osmolality in PKC-alpha knockout mice [(2.41±0.04) mmol/L vs (3.13±0.11) mmol/L, P = 0.00]. Immunohistochemical and semi-quantitative Western blotting studies revealed that the localization and expression of AQP-2 (P = 0.48) in inner medulla were not different between these two groups. The urinary output presented no difference under DdAVP experiment as well as changes of urinary osmolality [(0.20±0.02) ml vs. (0.20±0.04) ml,P = 0.97] under V2 receptor experiment. Conclusions PKC alpha mediates mouse urine concentration ability independent of inner medulla AQP-2 statues and plasma AVP level.

Objective To evaluate the role of low salt (LS) medium and the involvement of p38 mitogen-activated protein kinase (p38 MAPK) in the expression of cyclooxygenase-2 (COX-2) and production of PGE2 in a mouse macula densa derived cell line (MMDD1 cells). Methods Confluent MMDD1 cells were exposed to serum free DMEM/F12 in a 1∶1 mixture with normal saline (NS), 300 mmol/L mannitol (to reduce NaCl to half, LS solution). Semi-quantitive reverse transcription and polymerase chain reaction amplication (RT-PCR) and Western blotting were used to detect the expression of COX-2 mRNA and protein with LS solution. The content of PGE2 in the supernatant was examined by enzyme linked immunosorbent assay(ELISA). Expression of total and phosphorylated p38 MAPK kinsae was examined by Western blotting in LS solution. Results The COX-2 mRNA abundance and protein expression of MMDD1 cells were up-regulated in LS group as compared to NS group (16 h mRNA 0.94±0.12 vs 0.26±0.09, 28 h protein 0.59±0.02 vs 0.25±0.07, rspectively, all P < 0.01), and PGE2 release was elevated as well [(644.33±26.54) vs (224.0±18.33)ng/L, P < 0.01]. Phosphorylated p38 MAPK increased significantly in LS group( 0.28±0.01 vs 0.17±0.01, P < 0.01). The up-regulatyion of COX-2 protein expression in LS group was reduced by 20 μmol/L p38 inhibitior SB 203580(from 0.58±0.01 to 0.19±0.02, P < 0.01). Conclusions Low salt induces the expression of COX-2 and the secretion of PGE2 in a time-dependent manner in MMDD1 cells through the activation of p38 MAPK.