| Objective Peroxisome proliferator-activated receptor y (PPAR-y) which is a ligand-dependent nuclear factor receives increasing attention recently not only it plays an important role in regulating many important physiological processes including glucose and lipid metabolism, but also its anti-inflammatory effects. This study aimed to observe the effects of PPAR-y agonist and the synergistic effect of telmisartan to PPAR-y agonist and the relationship between PPAR-y and TLR4 in animal model of IgA nephropathy which was induced by bovine gamma-globulin (BGG).Methods 77 male Lewis rats(40-50g) were maintained with free access to food in standard cages at 25℃ with a 12-h light-dark cycle. The animals were provided standard rodent chow and water to drink ad libitum for 1 week before the initiation of the studies. Then the rats were divided into 5 groups randomly. Groups:1) Control group(n=18):Rats from the control group were maintained with normal drinking water containing 6mM HCl; 2) IgAN group(IgAN, n=28): continuous oral immunization with BGG in the drinking water for 9 weeks, followed by intravenous injection of 1 mg BGG daily for 3 successive days; 3) Pioglitazone group(Pio, n=9):Pioglitazone tablets were grinded into powder, 1g was dissolved in 90ml normal saline to make suspension every day and shake well before use. Rats were administrated the suspension intragastricly at 3ml every day for 4 weeks; 4) Pioglitazone+telmisartan group(Pio+ARB, n=10):Pioglitazone and telmisartan tablets were grinded into powder. 1g pioglitazone and 0.3g telmisartan were dissolved in 120ml normal saline to make suspension every day and shake well before use. Rats were administrated the suspension intragastricly at 3ml every day for 4 weeks; 5) TLR4 inhibitor group(TAK242, n=12):Prepard lipid emulsion containing TAK242 was injected into the tail vein at 7.5ml/kg for 8 successive days. At the end of week 4, 6 and 9, urine was gathered for the quantitative test of hematuria and urinary albumin-creatinine ratio (ACR). Blood was taken through abdominal aorta for the test of serum creatinine(Scr) and blood urea nitrogen(BUN). Changes of mesangial cells and matrix in the kidney were observed by light microscopy. The gene expression of PPAR-γ and TLR4 were examined by real-time RT-PCR while the protein expression of PPAR-γ TLR4 and IL-1β were detected by western blot. IgA deposition was examined by immunofluorescence.Results ①At the end of week 9, ACR of model group was significantly higher than that in control group(4.45±1.33mg/mmol vs 2.89±0.96mg/mmol, P=0.05). There was no significant difference in urinary red blood cells between two groups. Compared with control group, Scr, BUN, ALT and AST in model group had no obvious changes(P>0.05) and the proliferation of mesangial cells and matrix was found to be markedly higher under light microscope. The numbers of the cells in cross section of glomeruli were markedly increased(51±4 vs 41±2, P<0.01) and the volumn of the glomeruli increased obviously. Few renal tubular epithelial cells showed swelling and mild renal interstitial inflammatory cell infiltration was observed. Immunofluorescence showed no IgA deposition in control group but bright green conglomerate or flocculent IgA deposition could be observed in model group.② Compared with control group, ACR in IgAN group increased significantly(1.72± 0.41mg/mmol vs 1.27±0.15mg/mmol, P=0.013). ACR in pioglitazone group had no obvious differences with that in control group(1.13±0.44mg/mmol vs 1.27± 0.15mg/mmol, P=0.41), but much lower than that in IgAN group(1.13± 0.44mg/mmol vs 1.72±0.41mg/mmol, P=0.015). There was no significant differences in ACR between pioglitazone+telmisartan group and control group(1.01± 0.45mg/mmol vs 1.27±0.15mg/mmol, P=0.41). ACR in pioglitazone+telmisartan group was much lower than that in IgAN group(1.01±0.45mg/mmol vs 1.72± 0.41mg/mmol, P=0.00). The differences of ACR between pioglitazone+telmisartan group and pioglitazone group didn’t reach the statistical significant(1.01mg/mmol± 0.45 vs 1.13±0.44mg/mmol, P=0.18). Compared with control group, there was a notable increase of glomerular cross section cells in IgAN group(50±8 vs 40±6, P=0.03). The glomeruli and few renal tubular epithelial cells showed swelling. The glomerular volumn became bigger and mild renal interstitial inflammatory cell infiltration was observed. There were no significant differences of glomerular cross section cells between pioglitazone group and control group(46±6 vs 40±6,P>0.05), pioglitazone group and IgAN group(46±6 vs 50±8,P=0.23). The renal tubules were normal in pioglitazone group and no obvious inflammatory cell infiltration was observed. The number of glomerular cross section cells in pioglitazone+telmisartan group had no significant differences with control group(41±4 vs 40±6, P>0.05), but much less than that in IgAN group(41±4 vs 50±8, P=0.03). There was no obvious changes in the number of glomerular cross section cells in pioglitazone+telmisartan group and pioglitazone group(41±4 vs 46±6, P=0.08). The renal tubules were normal in pioglitazone+telmisartan group and no obvious inflammatory cell infiltration was observed. Compared with control group, serum IL-1β and IL-1β protein of renal tissues in IgAN group were increased obviously(47.45±12.91pg/ml vs 34.49±12.09pg/ml, P=0.01; 0.46±0.21 vs 0.27±0.10,P=0.04). Serum IL-1β and IL-1β protein of renal tissues in pioglitazone group had no significant differences with control group (39.06±17.92pg/ml vs 34.49±12.09pg/ml, P>0.05; 0.32±0.15 vs 0.27 ±0.10, P=0.45) and IgAN group(39.06±17.92pg/ml vs 47.45±12.91pg/ml, P=0.30; 0.37±0.20 vs 0.46±0.21, P=0.44). Compared with control group, serum IL-1β and IL-1β protein of renal tissues in pioglitazone+telmisartan group were almost not changed(34.49±14.55pg/ml vs 34.49±12.09pg/ml, P>0.05; 0.28±0.09 vs 0.27± 0.10, P=0.94), but much lower than those in IgAN group(34.49±14.55pg/ml vs 47.45 ±12.91pg/ml, P=0.04; 0.28±0.09 vs 0.46±0.21, P=0.04). There was no marked differences of serum IL-1β and IL-1β protein of renal tissues between pioglitazone+telmisartan group and pioglitazone group(34.49±14.55pg/ml vs 39.06 ±17.92pg/ml, P=0.59; 0.28±0.09 vs 0.32±0.15, P=0.46). Compared with control group, the expression of PPAR-y protein was much more than that in IgAN group(0.64±0.14 vs 0.42±0.04, P=0.03), but the expression of PPAR-y mRNA was almost not changed(1.20±0.42 vs 0.98±0.03, P=0.39). The expression of PPAR-γ protein and mRNA in pioglitazone group were much more than control group(0.71± 0.19 vs 0.42±0.04, P=0.03; 1.58±0.20 vs 0.98±0.03, P=0.001). The differences of PPAR-y protein and mRNA between pioglitazone group and IgAN group didn’t reach the statistical significance(0.71±0.19 vs 0.64±0.14, P=0.44; 1.58±0.20 vs 1.20± 0.42, P=0.15). There was also no obvious change in PPAR-y protein and mRNA between pioglitazone+telmisartan group and control group, pioglitazone+telmisartan group and IgAN group(all P>0.05). Compared with pioglitazone group, the expression of PPAR-y protein and mRNA in pioglitazone+telmisartan group were much lower(0.56±0.08 vs 0.71±0.19, P=0.047; 1.02±0.17 vs 1.58±0.20, P=0.005). ③Compared with IgAN group, ACR in TLR4 inhibitor group was decreased significangtly(1.13±0.44mg/mmol vs 1.72±0.41mg/mmol, P=0.015). Mesangial cells and matrix were obviously improved and the cross section cells in glomeruli were markedly reduced (35±3 vs 45±3, P<0.01). There was no significant difference of serum IL-1β and IL-1β protein in renal tissues between the two groups(30.20±4.93pg/ml vs 32.99±5.64pg/ml,0.56±0.22 vs 0.63±0.17, both P>0.05) and so was the expression of PPAR-y mRNA (1.00±0.54 vs 0.87±0.35, P=0.63). The expression of PPAR-y protein was elevated siginificantly(0.86±0.20 vs 0.65± 0.13, P=0.03). Compared with IgAN group, the expression of TLR4 protein in pioglitazone group was reduced markedly(0.12±0.03 vs 0.21±0.05, P=0.001) and TLR4 mRNA had a tendency to decrease(0.78±0.21 vs 0.95±0.09, P=0.13).Conclusion ①Mild-to-moderate IgA nephropathy could be established by continuous oral immunization with BGG in the drinking water for 9 weeks and intravenous injection of 1 mg BGG daily for 3 successive days in rats. ②In the animal models of IgA nephropathy, PPAR-y agonist (pioglitazone) and angiotensin receptor blocker(telmisartan) could decrease the levels of inflammatory factor, ACR and restrain the proliferation of mesangial cells and matrix to improve IgA nephropathy. The synergistic effect of telmisartan and pioglitazone was not obvious. ③TLR4 inhibitor-TAK242 could improve IgA nephropathy, decrease the levels of ACR and restrain the proliferation of mesangial cells and matrix. In the animal models of IgA nephropathy, TLR4 and PPAR-y could interact with each other and inhibit each other. |
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