Progressive glomerular and tubulointerstitial fibrosis develop in 1-year-old rats even after relief (R) of unilateral ureteral obstruction (UUO) at 5 days of age. The present study investigated whether a progressive renal injury model of UUO could be achieved after reversal of UUO (RUUO) in adult instead of neonatal rats. The potential for alpha-tocopherol modulation of mRNA for the fibrogenic cytokine, transforming growth factor-beta1 (TGFbeta1), apoptosis (TUNEL assay), and the presence of the stress protein, heat shock protein-70 (HSP-70), was also studied in this post-obstructive model. Male Sprague-Dawley rats weighing 125-150 g were randomly assigned to groups of 4 animals each for durations of 7 or 14 days of alpha-tocopherol supplementation after RUUO. The groups included: (i) sham, regular chow; (ii) RUUO, regular chow; (iii) RUUO, contralateral nephrectomy (NX); and (iv) RUUO, NX plus alpha-tocopherol supplementation. We found a significant increase in the ratio of kidney weight/body weight in the RUUO+NX group at 14 days compared with the sham and RUUO groups. This rise in the RUUO+NX group was significantly reduced after 14 days of alpha-tocopherol administration. The elevated level of kidney TGFbeta1 mRNA in the RUUO+NX group was only partially reduced at 7 days. But at 14 days this became significantly reduced with the continued alpha-tocopherol treatment. The HSP-70 staining and the apoptosis of the kidney showed results parallel to those of TGFbeta mRNA at 14 days. To separate the effects of hypertrophy after unilateral NX from the RUUO studies, we carried out a second experiment in control animals subjected to NX, with and without alpha-tocopherol supplementation. Fourteen days after NX, the apoptosis and TGFbeta1 mRNA showed no significant differences from the control animals. Our data suggest that a model of progressive renal injury in RUUO can be established in adult rats. After contralateral NX, the progressive injury is evidenced by the increase in the ratio of kidney weight/total body weight, the apoptotic counts, as well as fibrogenic cytokine TGFbeta1 mRNA in the post-obstructed kidney. Finally, our data also support the concept that alpha-tocopherol is renal protective, as judged by TGFbeta1 mRNA, apoptosis, and HSP-70 staining, even in the progressive disease process of the post-obstructed model.
Effective gene therapy requires efficient delivery and expression of the necessary genetic information to the target tissue. We demonstrate here that plasmid DNA, injected as naked, uncomplexed DNA into the cortical region of rat kidney, or intravenously, is localized and expressed in the kidney. The plasmid pRSVZ contained the Rous sarcoma virus promoter and a reporter gene, the beta-galactosidase gene, derived from bacteria. The beta-galactosidase gene hydrolyzes the artificial substrate X-gal to produce an intense blue color in cells that have taken up and expressed the plasmid genes. We have used X-gal staining and Western blotting to study plasmid gene expression 1, 4, and 8 days and 6 months after intrarenal injection of 50 microg of plasmid DNA and at 1 and 4 days after intravenous injection. Expression was apparent in the kidneys and several other tissues 24 h after injection and persisted for at least 8 days; expressed proteins could still be detected in the injected kidney 6 months later. These observations were corroborated by use of a plasmid, pEGFP-Puro, harboring the cytomegalovirus promoter in conjunction with a different reporter gene, the green fluorescent protein (GFP). Histological localization and Western blotting analysis of GFP expression after intrarenal injection of pEGFP-Puro paralleled results obtained with the plasmid pRSVZ. Our findings support the suggestion that intrarenal or intravenous injection of naked plasmid DNA may be an effective means of delivering therapeutic genes to the kidney and several other tissues.
Results.Our results showed that LDL malondialdehyde during UUO was increased 87% over baseline values (P Ͻ 0.005). With R-UUO, the oxidized LDL dropped 23% from the peak values during UUO (P Ͻ 0.005), but was still different from that of the baseline values (P Ͻ 0.025). Rat mesangial cell survival, after 72 h exposure to oLDL, inversely correlated to oLDL cytotoxicity and showed a 14% drop during UUO compared with sham-operated animals (P Ͻ 0.01). Cell survival increased 11% after R-UUO (P Ͻ 0.02) and was not different from control values (P ϭ NS). The apoptotic counts by the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling) technique showed significant increases during UUO and a noticeable reduction after R-UUO. Conclusion. Our data support the proposition that UUO stimulates oxidation of LDL. The cytotoxicity of oLDL plays a significant role in the injury of UUO. A decrease in cytotoxicity was associated with the repair in R-UUO. Our observations that apoptosis follows this same pattern, point to the importance of apoptosis in the injury and repair of obstructive nephropathy. Future studies to interrupt these processes of injury may lead to novel treatment modalities in reversing the injury and hastening the repair of obstructive nephropathy.
Background. We have previously shown that α-tocopherol prevents oxidative stress in experimental IgA nephropathy (IgAN) when administered before or concurrent with the induction of IgAN. We now seek to determine whether α-tocopherol can ameliorate the disease after IgAN is established. Methods. Using the classic IgAN model, 25 male Lewis rats were sorted into five groups of five animals each: 4-week control, 4-week bovine gamma globulin (BGG) treatment, 6-week control, 6-week BGG treatment, and 6-week BGG treatment with α-tocopherol administration started after 4 weeks. Serum α-tocopherol concentrations, kidney and plasma malondialdehyde concentrations, and kidney transforming growth factor beta-1 (TGF 1) mRNA were analyzed. Results. α-Tocopherol modulated IgAN after the disease was established in the 4-week BGG model, as indicated by the reduction in tissue oxidative stress, dampening of fibrogenic cytokine (TGF 1), and abatement of proteinuria in α-tocopherol-treated animals compared with untreated rats. Conclusions. These results substantiate the anti-oxidant role of α-tocopherol in diminishing the indices associated
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