| Objective:To study the protective effects of PNS on cisplatin-and aristolochic acid (AA)-induced kidney damage, and explore its possible mechanism from the perspective of proteomics, to provide theoretical basis for treatment to cisplatin- and AA -induced kidney damage with PNS.Methods:Established the model of cisplatin-and AA-induced kidney damage in rats and the vitro model of cisplatin-induced renal tubular epithelial cell damage by culturing the HK-2, which was intervened by PNS. And then detected the Urine beta N-ethyl amide base glycosidase enzymes (NAG),24 hours urinary protein, serum creatinine (Scr),urea nitrogen (BUN) and the proliferation of renal tubular epithelial cell,screened the differential expression protein in renal tissue and HK-2 cells of rats with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS), isolated and identified some of these differential expression proteins with matrix-assisted laser desorp-tion/ionization time-of-flight mass spectrometry/ Mass spectrometry(MALDI-TOF-MS/MS). 1. The protective effects of PNS on cisplatin-induced kidney damage in rats: Sprague-Dawley rats were divided randomly into sit groups:control group(received the same volume of saline with PNS for ten days),cisplatin model group(first day was given 5mg/kg cisplatin, and injected with saline the next day until the tenth day.),positive control group(received cisplatin after administration of amifostine 30 minutes, the same volume of saline with amifostine was given in the next nine days.), high,middle and low dose of PNS groups(was given cisplatin and High,middle and low dose of PNS respectively on the first day,only PNS was injected in the next nine days.).Drugs and saline were given into rats by intraperitoneal injection once daily.Ten days later,Urine,blood and renal tissue were collected for biochemical detection, electron microscopic examination,screening and identification of the differential expression protein.2. The protective effects of PNS on AA-induced kidney damage in rats: Wistar rats were divided randomly into blank control group,AA model group, positive control group and three PNS groups(high dose, middle dose and low dose).The rats were treated with extract of Aristolochia manshuriensis Kom by gastric gavage,and 4 hours later, PNS groups were gavaged with different doses of PNS;positive control group(prednisone treated group) received 3.15 mg·kg-1.d-lprednisone,while AA model group was only given the same volume of saline. Then rats were killed at 12th,16th, and 20th weeks after experiencing different treatment.Blood, urine and renal tissue were collected; and 24 hours urinary protein, urinary NAG,serum creatinine (Scr) and urea nitrogen (BUN) were detected.Renal tissues were provided for light and electron microscopy examination and also for screening and identification of the differential expression protein. 3. Effects of PNS on HK-2 cells with cisplatin-induced damage:(1) Effects of cisplatin and PNS on proliferation of HK-2 cells. Three groups were established in the experiment:①Cisplatin cell group:Cells were cultivated in culture fluid containing different concentrations of cisplatin;②PNS cell group:Different concentrations of PNS were added to culture medium;③blank cell group,which were cultivated in culture fluid containing 10% newborn calf serum. All group were cultivated for 48 hours.(2) Effects of PNS on proliferation of HK-2 cells inhibited by cisplatin.Three groups were established in the experiment:①Blank cell group:only containing 10% newborn calf serum;②Cisplatin cell group: containing 12.5ug/L Cisplatin and 10% newborn calf serum;③Cisplatin with PNS group,which was grown in culture fluid containing 12.5ug/L cisplatin and 10% newborn calf serum for 12 hours and then was cultivated for another 48 hours after adding different concentrations of PNS. Blank cell group,Cisplatin cell group(12.5μg/L Cisplatin) and Cisplatin with PNS group(50μg/L PNS after 12.5μg/L Cisplatin for 12h) were taken to screen and identify the differential expression protein.Results:1. The protective effects of PNS on cisplatin-induced kidney damage in rats:①SerumBUN,serum creatinine, and urinary NAG enzyme were significantly increased(p<0.05 or p<0.01) in cisplatin model group compared to that in controls,while these indexes were significantly decreased in PNS group and amifostine group, especially in middle dose of PNS group.②It had found that patchy necrosis in rat renal tubular epithelial cells,obvious swelling of mitochondria under the electron microscopy and mitochondria cristae were obscured, even vanished completely; PNS group and amifostine group can reduce pathological damage of renal tubular epithelial cells,however,there was no obvious lesion in control group.③There were kinds of differential expression protein in renal tissue among cisplatin model group, control group and PNS treated group.④Protein at m/z 10815.42 was identified as heat shock protein of mitochondria and it showed high expression in rat renal tissue in PNS treated group and more than two times in cisplatin model. Besides, protein at m/z 16021.67 was identified as hemoglobinβ1,β2-chain,which showed higher expression in cisplatin model group than in control group.2. The protective effects of PNS on AA-induced kidney damage in rats:①Compared with blank control group, serum creatinine (Scr), urea nitrogen (BUN),4 hours urinary protein and urinary NAG enzyme showed a obvious increasing(p<0.05 or p<0.01) in AA model group,and the increased degree had been gradually serious along with the prolonged time of administration of treatment.These indexes were significantly decreased in PNS groups and prednisone treated group when compared to AA model group.②Light microscopy showed that model rats developed tubular swelling, atrophy and interstitial fibrosis gradually. Renal tubular lesion and interstitial fibrosis were improved in PNS groups and prednisone treated group. On the other hand,Electron microscopy showed that there were renal tubular epithelial cell mitochondrial swelling and increased lysosome and phagocytic vacuole in renal tubular cells in AA model group. PNS group and prednisone treated group can reduce pathological changes③Renal tissue in AA model group, blank control group, PNS 20w group and prednisone treated group were found a number of differentially expressed proteins.④Protein at m/z 10082.07 was identified as Acyl-CoA-binding protein that showed two times higher expression in prednisone treated group than in AA model group.⑤There are five common differential expression proteins in the experiment of cisplatin-and AA-induced kidney damage. These indicated that the occurrence or development of cisplatin-and AA-induced kidney damage may involve the changes of common proteins. In addition, PNS can decrease expression of protein at m/z 1428 in cisplatin model group, but raised in AA model group. The specific reasons need further research.3. Effects of PNS on HK-2 cells with cisplatin-induced damage:①cisplatin can inhibit the appreciation rate of HK-2 cells while PNS can promote it in a concentration-dependent manner.②PNS can reduced the inhibition of cisplatin-induced proliferation of HK-2 Cell.③The differential expression protein was failed to screen out for the time being.Conclusions:1. PNS may play a protective role in rats respectively with cisplatin-induced kidney damage, AA-induced kidney damage and HK-2 cells with cisplatin-induced damage2. There were kinds of differential expression protein in renal tissue among cisplatin model group, control group and PNS treated group. Protein at m/z 10815.42 was identified as heat shock protein of mitochondria and it showed high expression in rat renal tissue in PNS treated group and more than two times in cisplatin model. Besides, protein at m/z 16021.67 was identified as hemoglobinβ1,β2-chain.It need further studies to realize the relationship among the two proteins and cisplatin kidney damage, protection of PNS.3. There are a number of differentially expressed proteins in renal tissue between AA model group and blank control group, PNS20w group, prednisone group respectively. The protein at m/z 10082.07 was identified as Acyl-CoA-binding protein whose effects on AA-induced kidney damage need further studies.4. There are five common differential expression proteins in the experiment of cisplatin- and AA- induced kidney damage. These indicated that the occurrence or development of cisplatin-and AA-induced kidney damage may involve the changes of proteins5. The differential expression protein that had been screened need to be the next step of isolation and identification to confirm their character and function. Meanwhile,it is important to realize effects of differential expression protein on cisplatin-,AA-induced kidney damage and PNS treated.6. Though the study on this topic had only obtained preliminary results at present,it can provide new ideas and methods for follow-up research work, and theoretical basis for discussing mechanism of cisplatin-,AA-induced kidney damage and protective action of PNS.
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