| Objectives:To investigate the protective effects of Rhiizoma Dioscoreae pretreatment in easing renal ischemia-reperfusion injury and promoting the tubular cell regeneration in rat.Methods:1. Two weeks before experimentation, 60 Spargue-Dawley rats (3-4 week-old) were injected with BrdU intraperitoneally in a daily dose of 100mg/kg body weight for three days and then randomly assigned into 4 group: (1) normal control group (N, n=6); (2) sham operated control group (SO, n=6); (3) the ischemia-reperfusion group with pretreatment of Rhiizoma Dioscoreae by intragastric administration (I/R Rhiizoma Dioscoreae group, n=24): the rats were pretreated with Rhiizoma Dioscoreae lavage in a daily dose of 10g/kg body weight for five days before being subjected to bilateral renal vascular occlusion for 45 minutes and then at 2h, 12h, 24h and 48h after reperfusion six rats each were randomly euthanized for blood sampling and kidney harvesting; (4) the ischemia-reperfusion group with aseptic normal saline lavage (I/R aseptic saline group, n=24): the rats were pretreated with placebo aseptic normal saline for five days before being subjected to 45 minutes bilateral renal ischemia and reperfusion same as the third group.2. The blood urea nitrogen (BUN) and serum creatinine (Scr) were measured with automatic biochemistry analyzer. The serum content of Malondialdehyde (MDA) was determined with TBA methods. Renal morphologic changes were scored with Paller's criterion on hematoxylin and eosin (H&E) stained sections. Renal tubular cell apoptosis were detected with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling procedure (TUNEL). PCNA positive cells of renal tubular epithelia were detected immunohistochemically as proliferation index. Confocal microscopy was used to identified the distribution of BrdU and Pax-2 positive tubular cells. The renal expression of HGF, C-met, BMP-7 and Pax-2 were also investigated with semiquantitive reverse transcription polymerase chain reaction(RT-PCR) method.Results:1. Histological examination: 12h, 24h and 48h after renal ischemia-reperfusion injury, the renal morphologic score in I/R Rhiizoma Dioscoreae group were 7.50±0.55, 6.17±0.75, 4.17±0.75 respectively, significantly lower than that in I/R aseptic saline group (8.50±0.55, 7.00±0.89, 5.17±0.98 respectively, P < 0.05). The morphological scores in both I/R Rhiizoma Dioscoreae group and I/R aseptic saline group are higher than that in SO group (0.67±0.52, P < 0.01)and the histological damage was most obvious at 12h and 24h after renal ischemia–reperfusion injury.2. The change of serum creatinine and urea nitrogen level: the Scr and BUN value in N group were 23.95±3.20μmol/L, 5.79±0.54mmol/L respectively, not significantly different from those in SO group (25.12±3.95μmol/L, 6.15±0.40mmol/L respectively, P >0.05).In I/R Rhiizoma Dioscoreae group and I/R aseptic saline group Scr and BUN value at 12h and 24h after renal ischemia-reperfusion injury were singnificantly higher than that in SO group (P < 0.01). At 2h,12h, 24h and 48h after renal ischemia-reperfusion injury, the Scr value of I/R Rhiizoma Dioscoreae group were 34.23±5.08μmol/L,56.38±8.70μmol/L, 81.53±10.17μmol/L,55.01±9.71μmol/L respectively, significantly lower than those in I/R aseptic saline group (46.8±3.69μmol/L, 98.70±12.37μmol/L, 128.5±17.30μmol/L, 80.72±11.13μmol/L respectively, P < 0.05); At 2h,12h, 24h and 48h after renal ischemia-reperfusion injury, the BUN value of I/R Rhiizoma Dioscoreae group were 15.3±2.60mmol/L,19.56±3.89mmol/L, 14.72±2.99 mmol/L respectively, significantly lower than I/R aseptic saline group (22.07±4.57mmol/L,34.75±6.32mmol/L, 20.16±2.40mmol/L respectively, P < 0.05 ).3. The contents of MDA in plasma and tubular cell apoptosis: the MDA value in SO group was 3.65±0.98nmol/ml, not significantly different from that in N group(3.70±0.70nmol/ml, P > 0.05);At 2h, 12h, 24h and 48h after renal ischemia-reperfusion injury, the MDA value in I/R Rhiizoma Dioscoreae group were 4.74±0.50nmol/ml, 6.19±0.68nmol/ml, 5.16±0.58nmol/ml respectively, significantly lower than those in I/R aseptic saline group(7.45±1.03nmol/ml,10.52±1.57nmol/ml, 6.93±1.32nmol/ml, P < 0.05 ); 48h after renal ischemia-reperfusion injury the MDA value of I/R Rhiizoma Dioscoreae group was not significantly different from that in I/R aseptic saline group. The tubular cell apoptosis was detected in the kidney at 2h after ischemia-reperfusion injury and reached the highest level at 12h. Renal apoptosis score in I/R Rhiizoma Dioscoreae group were 10.17±2.14, much less than that in I/R aseptic saline group (13.67±2.94, P < 0.05).4. Tubular cell proliferation and distriburion of the BrdU-positive cells in the kidney: the proliferating cell nuclear antigen (PCNA) positive cells and BrdU-positive cells were examined by light microscopy. Most of PCNA-positive cells were located in the proximal and distal tubule, while most glomeruli and interstitial tissue had no PCNA-positive cells; At 2h, 2h, 24h and 48h after renal ischemia-reperfusion injury, the PCNA-positive cells in I/R Rhiizoma Dioscoreae group were 2.67±1.21, 9.50±1.87, 14.83±2.71, 10.83±2.04 respectively, signifcantly more than those in I/R aseptic saline group (1.83±0.75, 7.00±1.41, 11.33±1.75, 8.50±1.05 respectively, P < 0.05). A large number of BrdU-retaining cells were found in the renal papilla regions, while rarely found in cortical tubules in SO group; At 48h after renal ischemia-reperfusion injury, there was a marked reduction in the number of BrdU-positive cells in the papilla region in I/R Rhiizoma Dioscoreae group while there were more BrdU-positive cells in cortical tubules. At 24h after renal ischemia-reperfusion injury, the BrdU-positive cells in I/R Rhiizoma Dioscoreae group were 13.17±3.19, significantly more than that I/R aseptic saline group (8.33±2.16, P < 0.05) and SO group(1.17±0.75, P < 0.05).5. The gene expression of HGF, C-met, BMP-7 and Pax-2 in renal tissue:(1) At 12h after renal ischemia-reperfusion injury, renal HGF mRNA expression in I/R Rhiizoma Dioscoreae group were 0.51±0.10,0.46±0.09 respectively, significantly higher than those in I/R aseptic saline group (10.36±0.02, 0.31±0.15 respectively, P < 0.05) and SO group (0.28±0.05, P < 0.05), and the C-met mRNA expression in I/R Rhiizoma Dioscoreae group were 1.22±0.28,0.46±0.09 respectively, significantly higher than those in I/R aseptic saline group (0.86±0.08, 0.31±0.15, P < 0.05).(2) The expression of BMP-7 mRNA was downregulated at 2h, and then upregulated, and peaked at 24h after renal ischemia-reperfusion injury; The expression of BMP-7 mRNA in I/R Rhiizoma Dioscoreae group were 0.6±0.09, significantly higher than that in I/R aseptic saline group (0.39±0.08, P < 0.05) and SO group at 12h (0.42±0.07, P < 0.05).(3) No expression of Pax-2 in proximal tubules was observed in the N group or the SO group. At 12h after renal ischemia-reperfusion injury, the Pax-2 mRNA was re-expressed in tubular cells in the I/R Rhiizoma Dioscoreae group and I/R aseptic saline group, reached peak at 24h after renal ischemia-reperfusion injury. Similarly the Pax-2 positive cells were detected in renal tubules in I/R Rhiizoma Dioscoreae group, and the BrdU and Pax-2 double positive staining cells were localized in the renal tubules by confocal microscopy.Conclusions:1. Renal ischemia/reperfusion injury may result in significant histological damage and renal dysfunction. The pretreatment of Rhiizoma Dioscoreae lavage in rats results in lessening the oxidative damage and apoptosis, lowering the level of Scr,BUN and MDA, thereby protecting kidney from ischemia reperfusion injury.2. The pretreatment of Rhiizoma Dioscoreae lavage in rats regulates and improves renal microenvironment by upregulating the expression of HGF, C-met and BMP-7, and thereby promotes the tubular cell regeneration and remodeling of the injured tubular architecture.3. After renal ischemia-reperfusion injury, intrarenal BrdU-label-retaining cells participated in the tubular cell regeneration and remodeling of the tubular architecture. Some BrdU-positive tubular cells co-expressed with Pax-2, which may present in the metanephric mesenchyme, but not in the mature nephron. The BrdU and Pax-2 double positive staining cells may have been the renal progenitor cells, and the renal papilla may be a niche for adult renal stem cells or progenitor cells.
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