Effects Of Rosiglitazone On Nephrotoxicity And Hepatotoxicity In Rats Induced By CsA And Its Molecular Mechanism

Cyclosporine A (CsA) has already become the cornerstone of clinical immunosuppressant. In fact, it predominantly increases the short-term survival rate of organ transplantation patients. Although lots of new ones come to emerge, CsA is still the first-line drug for the treatment of rejection after transplantation. However CsA has its own shortcomings in the period of clinical practice, especially its nephrotoxity and hapatotoxity. Fibrosis of liver and kidney induced by CsA which is the main problem has been an urgent problem-in-discussing of the development of transplantation. Studies show that the nephtotoxicity and hepatotoxicity induced by CsA in the acute phase is mainly inflammatory cell infiltration, reversible, and irreversible fibrosis in the chronic phase. Rosiglizone (RGZ), a member of thiazolidinediones (TZDs), has universal effects by activating peroxisome proliferator-activated receptor Y (PPAR Y). PPAR Y, a member of typeⅡnuclear receptor superfamily, not only is believed involving in the cellular metabolism, but also plays an important role on ameliorating inflammation and fibrosis. Based on these evidences, we assumed that PPAR Y may be a new target for the management of conditions induced by CsA. Until now there is no such report. We established the hepatical and renal fibrosis model resulted from CsA at the basis of low salt diet (LSD), observed the protective effects of RGZ on the nephrotoxity and hapotoxity induced by CsA, and then explored the mechanism of the effects in normal rat kidney (NRK) and hepatic stellate cells (HSCs)Therefore, the study was further divided into two parts.PartⅠThe Protective Effects Of Rosiglizone On Nephrotoxicity in Rats Induced by CsA and Its MolecularMechanismExperiment 1 Research In VivoMethods1. Experimental group:Healthy male SD rats (BW 220-250g) fed with LSD (natrium mass fraction 0.038%) one week later were randomly divided into four groups:(1) control group (n=12):LSD+olive oil (1.5mg·kg-1·d-1) subcutaneous injection+NS (5ml·kg-1·d-1) intragastric administration; (2) RGZ group(n=12) LSD++olive oil(1.5ml·kg-1·d-1)subcutaneous injection+RGZ(5ml·kg-1·d-1) intragastric administration; (3) CsA group (n=15):LSD+CsA(15ml·kg-1·d-1) subcutaneous injection+NS(5ml·kg-1·d-1)intragastric administration; (4) RGZ+ CsA group (n=15):LSD+CsA(15ml·kg-1·d-1)subcutaneous injection+RGZ (5ml·kg-1·d-1)intragastric administration.6 ones of each group were sacrificed randomly 14 days later, the rest were sacrificed at the end of the experiment (35 days later).2. Body weight and biochemical indicator investigation (Ccr, ALB):Body weight was measured at the beginning and the end of the experiment. Serum creatine, urine creatine, and albumin were investigated by Hitachi 7600-automatic biochemical analyzer. 3. Histology:Interstitial mononuclear cells were counted with HE staining, and interstitial fibrosis was calculated with Masson's staining.4. Immunohistochemistry (IHC):CD68 and CollagenⅣ(ColⅣ) were stained with SP kit.5. The mRNA expressions of osteopontin (OPN) and regulated upon activation normal T-cell expressed and secreted (RANTES) were detected with Real-time polymerase chain reaction (RT-RCR).6. The protein expressions of OPN, RANTES and Fibronectin (FN) were detected with Western blotting.7. The mRNA expressions of MMP-9 and TIMP-1 were detected with Reverse transcription-polymerase chain reaction (RT-RCR).Results1. Body weight:There was significant difference of body weights of rats in each group both at the 14th day and the 35th day (P<0.05). RGZ markedly reduced the decrease of body weight compared with that in CsA-treated rats at the 35th day (P<0.05)..2. Biochemical indicator:Ccr of CsA-treated rats decreased significantly (P<0.05). and RGZ ameliorated the decrease of Ccr markedly at the 35th day(P<0.05). The level of serum ALB decreased markedly in CsA-treated rats (P<0.05). RGZ failed to improve the level of serum ALB either at the 14th day or at the 35th day (P<0.05).3. Interstitial mononuclear cells infiltration:At the 14th day, compared with the control group, the amount of interstitial mononuclear cells infiltrated markedly increased in CsA-treated rats (P<0.05). RGZ ameliorated the increase of interstitial mononuclear cells significantly at the 14th day (P<0.05).4. Interstitial fibrosis:Interstitial fibrosis detected with Masson's staining. At the 35th day, in CsA-treated rats the level of renal interstitial fibrosis was remarkably raising (P<0.05). RGZ significantly alleviated the level of renal interstitial fibrosis (P<0.05).5. The IHC staining of CD68:At the 14th day, compared with the control group, the IHC staining of CD68 remarkably increased in CsA-treated rats (P<0.05). RGZ significantly reduced the IHC staining of CD68 (P<0.05). There was no difference between the control group and RGZ group (P>0.05). At the 35th day, compared with the control group, the IHC staining of CD68 remarkably increased, but decreased in CsA-treated rats compared with that of the 14th day CsA-treated rats (P<0.05). The IHC staining of CD68 markedly decreased in CsA+RGZ group(P<0.05). There was no difference between the control group and RGZ group(P>0.05).6. The IHC staining of ColⅣ:At the 14th day, compared with the control group, the IHC staining of Col IV remarkably increased in CsA-treated rats (P<0.05). RGZ reduced the IHC staining of ColⅣ,but there was no significantly difference between the CsA group and RGZ group (P<0.05). There was no difference between the control group and RGZ group (P>0.05). At the 35th day, compared with the control group, the IHC staining of ColⅣremarkably increased. Compared with CsA-treated rats, The IHC staining of ColⅣmarkedly decreased in CsA+RGZ group (P<0.05). There was no difference between the control group and RGZ group (P>0.05).7. OPN:There were mRNA and protein expressions of OPN in earch group. Based on the control group, the mRNA and protein expressions of OPN were significantly up-regulated in the CsA group and CsA+RGZ group (P<0.05). Compared with CsA group, the mRNA and protein expressions of OPN were significantly down-regulated in CsA+RGZ group (P<0.05).8. RANTES:There were mRNA and protein expressions of RANTES in earch group. Based on the control group, the mRNA and protein expressions of RANTES were significantly up-regulated in CsA group and CsA+RGZ group (P<0.05). Compared with CsA group, the mRNA and protein expressions of RANTES were significantly down-regulated in CsA+RGZ group (P<0.05).9. FN:At the 35th day, based on the control group, the expression of FN detected with Western blotting was significantly up-regulated in CsA group and CsA+RGZ group (P<0.05). Compared with CsA group, the protein expression of FN was significantly down-regulated in CsA+RGZ group (P<0.05). There was no difference between the control group and RGZ group (P>0.05).10. MMP-9:Compared with the control group, the mRNA expression of MMP-9 was significantly up-regulated in CsA group and CsA+RGZ group (P<0.05), and at the 14th day the mRNA expression of MMP-9 was higher than that of the 35th day. Compared with CsA group, the mRNA expression of MMP-9 was significantly down-regulated in CsA+RGZ group (P<0.05). There was no difference between the control group and RGZ group (P>0.05).11. TIMP-1:Compared with the control group, the mRNA expression of MMP-9 was significantly up-regulated in CsA group and CsA+RGZ group (P<0.05), and at the 14th day the mRNA expression of MMP-9 was higher than that of the 35th day. Compared with CsA group, the mRNA expression of MMP-9 was significantly down-regulated in CsA+RGZ group (P<0.05). There was no difference between the control group and RGZ group (P>0.05).Research In VitroMethods1. Construction, screening and amplification of the target siRNA vector for PPARy.2. Observe the inhibitory effect of siRNA on the expression of PPARy in normal rat kidney cells.3. Normal Rat Kidney Cells (NRK) were cultured in the routine way. Experimental groups: The control group (N):single NRK cells without treatment; RGZ group (R):NRK cells with RGZ (final concentration of 10μmol/L); CsA group (M):NRK cells with CsA (final concentration 1.0μg/ml); CsA+RGZ group (Z):NRK cells with CsA (final concentration 1.0μg/ml) and RGZ (final concentration of 10μmol/L); CsA+RGZ+siRNA group (S):236-PP-1 plasmid transfected into NRK cells with CsA (final concentration 1.0μg/ml) and RGZ (final concentration of 10μmol/L). (The choice of time and concentration can refer to the thesis of Professor Liu Zhangsuo)4. The cell proliferation was detected with MTT colorimetry.5. The mRNA expression of PPAR Y was detected with Realtime-RCR.6. The mRNA expressions of MMP-9 and TIMP-1 were detected with reverse transcription-polymerase chain reaction (RT-RCR).7. The protein expression of FN was detected with Western blotting.Results1. Three targeted PPARy siRNA vectors were successfully constructed. The 236-PP-1 plasmid with high transfection efficiency was selected by liposome-mediated plasmid transfected cells.2. Cell proliferations:Cell proliferations of NRK were suppressed by CsA (P<0.05). RGZ accelerated the suppression of NRK resulted from CsA (P<0.05). The application of PPARy siRNA resulted the decreasing of NRK cell proliferation resulted from CsA (P<0.05).3. PPARγ:CsA upregulated the mRNA level of PPARγ(P<0.05), and this tendency was improved by RGZ significantly (P<0.05). The application of PPARy siRNA resulted the decreasing of PPARγmRNA (P<0.05).4. MMP-9:CsA upregulated the mRNA level of MMP-9 (P<0.05), and this tendency was significantly improved by RGZ (P<0.05). The application of PPARy siRNA resulted the increasing of MMP-9 mRNA (P<0.05).5. TIMP-1:CsA upregulated the mRNA level of TIMP-1 (P<0.05), and this tendency was significantly improved by RGZ (P<0.05). The application of PPARy siRNA resulted the increasing of TIMP-1 mRNA (P<0.05).6. FN:CsA upregulated the protein level of FN (P<0.05), and this tendency was significantly improved by RGZ (P<0.05). The application of PPARγsiRNA resulted the increasing of FN protein (P<0.05).Conclusions1. RGZ delayed the progression of renal function induced by CsA through suppressing mononuclear cells infiltration and interstitial fibrosis. 2. The expression of CD68 was downregulated by RGZ. The mRNA and protein expressions of OPN and RANTES were also significantly downregulated by RGZ.in tubular-interstitial.3. RGZ downregulated the protein expressions of COLⅣand FN and inhibited the process of fibrosis through reducing accumulation of extracellular matrix.4. The mRNA expressions of MMP-9 and TIMP-1 were significantly reduced by RGZ. RGZ delayed the occurance of fibrosis through inhibiting the accumulation of extracellular matrix and promoting the degradation of extracellular matrix.5. RGZ suppressed the proliferation of NRK and downregulated the expressions of FN, MMP-9 and TIMP-1. So the accumulation of extracellular matrix could be inhibited.6. Three targeted PPARγsiRNA vectors were successfully constructed. The siRNA with high transfection efficiency on NRK was selected by liposome-mediated plasmid transfected cells.7. The mRNA expression of PPARγin NRK can be effectively blocked by PPARγsiRNA.8. The mRNA expression of PPARγin NRK was blocked by siRNA technology. The protective effect of RGZ on renal interstitial fibrosis induced by CsA was decreased.PartⅡThe Protective Effects Of Rosiglizone On Hepatoprotective in Rats Induced by CsA and Its MolecularMechanismResearch In VivoMethods1. Experimental group:Healthy male SD rats (BW 220～250g) fed with LSD (natrium mass fraction 0.038%) one week later were randomly divided into four groups:(1) control group (n=12):LSD+olive oil (1.5ml·kg-1·d-1) subcutaneous injection+NS (5ml·kg-1·d-1) intragastric administration; (2) RGZ group (n=12) LSD++olive oil (1.5ml·kg-1·d-1) subcutaneous injection+RGZ(5mg·kg-1·d-1) intragastric administration; (3)CsA group (n=15):LSD+CsA(15mg·kg-1·d-1) subcutaneous injection+NS(5mg·kg-1·d-1)intragastric administration; (4) RGZ+ CsA group (n=15):LSD+CsA(15mg·kg-1·d-1)subcutaneous injection+RGZ (5mg·kg-1·d-1)intragastric administration.6 ones of each group were sacrificed randomly 14 days later, the rest were sacrificed at the end of the experiment (35 days later).2. Body weight and biochemical indicator investigation (ALT, ALB):Body weight was measured at the beginning and the end of the experiment. Glutamic pyruvic transaminase and albumin were investigated by Hitachi 7600-automatic biochemical analyzer.3. Histology:Interstitial mononuclear cells were counted with HE staining, and hepatic fibrosis was calculated with Masson's staining.4. The mRNA expressions of OPN and RANTES were detected with RT-RCR.5. The protein expressions of OPN, RANTES and FN were detected with Western blotting.6. The mRNA expressions of TGF-β1, MMP-9 and TIMP-1 were detected with RT-RCR.7. Malondialdehyde (MDA) and reduced Glutathione (GSH) were detected with colorimetry.Results1. Body weight:There was significant difference of body weights of rats in each group both at the 14th day and at the 35th days (P<0.05). RGZ markedly reduced the decrease of body weight compared with that in CsA-treated rats at the 35th day (P<0.05).2. Biochemical indicator (ALB,ALT):The level of serum ALB decreased markedly in CsA-treated rats (P<0.05). RGZ failed to improve the level of serum ALB either at the 14th day or at the 35th day(P>0.05). The serum level of ALT of CsA-treated rats increased significantly (P<0.05), and RGZ ameliorated the increase of ALT markedly (P<0.05).3. Mononuclear cells infiltration:Compared with the control group, the amount of mononuclear cells infiltrated increased markedly (P<0.05). Compared with CsA group, RGZ ameliorated the increase of mononuclear cells significantly in CsA+ RGZ group (P<0.05). There was no difference between the control group and RGZ group (P>0.05).4. Liver fibrosis:The level of Liver fibrosis detected with Masson's staining in CsA group was remarkably raising. (P<0.05). RGZ significantly alleviated the level of liver fibrosis (P<0.05). Similarly, the protein level of FN detected with Western blotting had the similar tendency. At the 35th day, compared with the control group, the expression of FN detected with Western blotting was significantly up-regulated in CsA group and CsA+RGZ group (P<0.05). Compared with CsA group, the protein expression of FN was significantly down-regulated in CsA+RGZ group (P<0.05). There was no difference between the control group and RGZ group (P>0.05).5. OPN:There were mRNA and protein expressions of OPN in earch group. Based on the control group, the mRNA and protein expressions of OPN were significantly up-regulated in CsA group and CsA+RGZ group (P<0.05). Compared with CsA group, the mRNA and protein expressions of OPN were significantly down-regulated in CsA+RGZ group (P<0.05).6. RANTES:There were mRNA and protein expressions of RANTES in earch group. Based on the control group, the mRNA and protein expressions of RANTES were significantly up-regulated in CsA group and CsA+RGZ group (P<0.05). Compared with CsA group, the mRNA and protein expressions of RANTES were significantly down-regulated in CsA+RGZ group (P<0.05).7. TGF-β1:Compared with the control group, the mRNA expression of TGF-β1 was significantly up-regulated in CsA group and CsA+RGZ group (P<0.05) and at the 14th day the mRNA expression of TGF-β1 was higher than that of the 35th day. The mRNA expression of TGF-β1 was ameliorated by RGZ significantly at the two time-points (P<0.05). There was no difference between the control group and RGZ group (P>0.05).8. MMP-9:Compared with the control group, the mRNA expression of MMP-9 was significantly up-regulated in CsA group and CsA+RGZ group (P<0.05), and at the 14th day the mRNA expression of MMP-9 was higher than that of the 35th day. Compared with CsA group, the mRNA expression of MMP-9 was significantly down-regulated in CsA+RGZ group (P<0.05). There was no difference between the control group and RGZ group (P>0.05).9. TIMP-1:Compared with the control group, the mRNA expression of MMP-9 was significantly up-regulated in CsA group and CsA+RGZ group (P<0.05), and at the 14th day the mRNA expression of MMP-9 was higher than that of 35 days. Compared with CsA group, the mRNA expression of MMP-9 was significantly down-regulated in CsA+RGZ group (P<0.05). There was no difference between the control group and RGZ group (P>0.05).10. MDA and GSH:The activity of MDA in liver detected with colorimetry increased markedly in CsA-treated rats (P<0.05), while the activity of GSH decreased significantly (P<0.05) both at the 14th day and at the 35th day. This tendency was significantly improved by RGZ at 14 days (P<0.05).Research In VitroMethods1. Hepatic stellate cells (HSC) were cultured in routine ways.2. Cell proliferations were detected with MTT colorimetry.3. The mRNA expression of PPARγwas detected with Real-time polymerase chain reaction (RT-RCR).4. The mRNA expressions of TGF-β1, MMP-9 and TIMP-1 were detected with reverse transcription-polymerase chain reaction.5. The protein level of FN was detected with Western blotting.6. The protein level of TGF-β1 was detected with ELISA. 7. Malondialdehyde (MDA) and reduced Glutathione (GSH) were detected with colorimetryResults1. Cell proliferations:Cell proliferations of HSC were suppressed by CsA (P<0.05). RGZ accelerated the suppression of HSC resulted from CsA (P<0.05). The application of PPARy siRNA resulted in the decreasing of HSC proliferation resulted from CsA (P<0.05).2. PPAR Y:CsA upregulated the mRNA level of PPARγ(P<0.05), and this tendency was improved by RGZ significantly (P<0.05). The application of PPARy siRNA resulted in the decreasing of PPARγmRNA (P<0.05).3. TGF-β1:CsA upregulated the mRNA and protein level of TGF-β1 (P<0.05), and this tendency was significantly improved by RGZ (P<0.05). The application of PPARy siRNA resulted in the increasing the mRNA and protein level of TGF-β1(P<0.05).4. MMP-9:CsA upregulated the mRNA level of MMP-9 (P<0.05), and this tendency was significantly improved by RGZ (P<0.05). The application of PPARy siRNA resulted in the increasing of MMP-9 mRNA (P<0.05).5. TIMP-1:CsA upregulated the mRNA level of TIMP-1 (P<0.05), and this tendency was significantly improved by RGZ (P<0.05). The application of PPARy siRNA resulted in the increasing of TIMP-1 mRNA (P<0.05).6. FN:CsA upregulated the protein level of FN (P<0.05), and this tendency was significantly improved by RGZ (P<0.05). The application of PPARy siRNA resulted in the increasing of FN protein (P<0.05).7. MDA and GSH:The activity of MDA in HSC detected with colorimetry increased markedly by CsA (P<0.05), while the activity of GSH decreased significantly (P<0.05). This tendency was significantly improved by RGZ (P<0.05). The application of PPARγsiRNA decreased the positive effect of RGZ(P<0.05). Conclusions1. RGZ protected the liver function through suppressing the infiltration of mononuclear cells and interstitial fibrosis.2. RGZ significantly decreased the mRNA and protein expressions of OPN and RANTES on early stage.3. RGZ significantly reduced the mRNA expression of TGF-β1 and the protein expression of FN. RGZ delayed the occurance of liver fibrosis through inhibiting the accumulation of extracellular matrix.4. RGZ downregulated the mRNA expressions of MMP-9 and TIMP-1. It delayed the occurance of fibrosis through inhibiting the accumulation of extracellular matrix and promoting the degradation of extracellular matrix.5. RGZ significantly reduced the accumulation of extracellular matrix by downregulating the protein expressins of FN and the mRNA expression of MMP-9, TIMP-1.6. The mRNA expression of PPARγin HSC can be effectively blocked by the plasmid when it was transfected into HSC.7. The mRNA expression of PPARγin HSC was blocked by siRNA technology. The protective effect of RGZ on liver fibrosis induced by CsA was decreased.