Inhibitory Effects Of Peroxisome Proliferator-activated Receptor-γ Agonists On Collagen Ⅳ Production In Podocytes

Part 1 The primary culture and indendification of podocytes Objective:Obtain the primary cultured mouse podocytes,which have more stable biological fuction,to piovide basic condition for our research. Methods:Mouse kidneys were removed and the renal capsule stripped using autoclaved surgical instruments. The cortex was isolated and minced with razor dablade, and glomeruli isolated by sieving(sieve pore size 180 l X2, 75 l X1). Glomeruli were then suspended in DMEM/Ham’s F-12(2:1), plated onto collagen type I-coated flask, and incubated. After 5 days, cell colonies began to sprout around the glomeruli. These cells were characterized as podocytes by detection of the podocyte specific markers, synaptopodin and Wilms’ tumor 1(WT-1) by immunofluorescence staining. Results:After the glomeruli were isolated from mouse kidneys and cultured for 5 days,cell colonies began to sprout around the glomeruli. These cells showed an epithelial morphology with a polyhedral shape.Immunofluorescence staining with synaptopodin and WT-1 was performed to confirm those cells as podocytes. there were strongly expressions of synaptopodin and WT-1,in those differentiated podocytes,particularly in the cytoplasm and extending toward cell processes. Conclusion: The results showed that we obtained primary mouse podocytes successfully. Part 2 The expression and location of PPARγ in the mouse podocyte and the effect of PPARγ agonist Objective: To investigate the expression and location of PPARγ podocyte with the experiment in vivo. Methods: Treat podocytes of PPAR-γ agonist, Rosiglitazone(Rosi10μM) and Pioglitazone(Pio1μM), respectively, for 24 h, the expression of PPARγ in mouse podocytes was determined by RT-PCR and Western blotting, and PPARγ expression in podocytes was also confirmed by immunocytochemical staining. To determine the functional activities of PPARγ as a transcription factor in podocytes, the PPARγ luciferase reporter plasmid containing a PPARγ response element was transfected into the mouse podocyte along with TK plasmids as a control of transfection efficiency. Results: PPARγ m RNA expressed in podocytes. PPARγ protein expressed in podocytes treated with Rosi(10μM) and Pio(1μM). Quantitation of PPARγ protein expression in C.E Positive signals of PPARγ were observed in cytoplasm, nuclear, and cell processes of mouse podocytes detected by immunocytochemical staining. The relative level of PPARγ luciferase activity was significantly increased by Rosi or Pio treatment.(P<0.05) Conclusion: PPARγ express in podocyte;PPARγ agonists not only increase the expression of PPARγ m RNA and protein but also enhance it’s fuctional activities.Part 3 The effect of PPARγ agonists on the collagen Ⅳ production ofpodocytes induced by TGFβ Objective:With the investigation of expression changes of collagen Ⅳ production in podocytes stimulated by TGFβ,with or without treatment of PPARγ agonists,we intent to ruveal the effects of PPARγ agonists on collagen Ⅳ production in podocytes. Methods:Immuno-blots were used to assess podocyte expression of collagen IV with or without PPARγ agonist treatments. collagen IV expression was determined by immunofluorescence staining using a rabbit polyclonal antiserum against collagen IV as the primary antibody.To further confirm the regulation of PPARγ on collagen IV production of podocyte specifically, the mouse podocytes were infected with PPARγ adenovirus(Ad-PPARγ) for 24 h or treated with Pio treatment. The specific significance of PPARγ activation on the regulation of collagen IV expression was also evaluated in primary mouse podocytes treated by PPARγ antagonist,GW9662(10μM). Results:TGFβ(10ng/ml) markedly increased collagen extraction compared with untreated cells(P<0.01); but Rosi(10μM) or Pio(1μM) decreased the collagen products in mouse podocytes compared with TGFβ treatment alone(P<0.01). TGFβ(10ng/ml) upregulated the m RNA expressions of collagen IV in podocytes, Rosi and Pio treatment markedly decreased its m RNA levels.Rosi and Pio significantly reduced collagen IV protein expression which was increased by TGFβ stimulation in mouse podocytes(P<0.05). Collagen IV expression determined by immunofluorescence staining was significantly increased in mouse podocytes stimulated by TGFβ, but Pio treatment obviously obliterated collagen IV densities. Pio(1μM) obviously activated PPARγ expression, and Ad-PPARγ infection dramatically enhanced PPARγ expression VS control(P<0.05).Collagen IV expression was significantly decreased in mouse podocytes infected by Ad-PPARγ VS TGFβ stimulation alone. The empty virus vector had no effect on collagen IV expression. was significantly decreased in mouse podocytes infected by Ad-PPARγ VS TGFβ stimulation alone(P<0.05).The increased PPARγ expression responsed to Pio treatment was blunted after exposure to GW9662 in podocytes.Collagen IVexpression was downregulated by adding Pio VS TGFβ treatment alone(P<0.05). GW9662 largely prevented suppression of collagen IV expression by Pio treatment. Conclusion:TGFβ induced the expression of collagenⅣproduction of podocytes; PPARγ agonists blunted that responses by TGFβ.Part 4 The molecular research of PPARγ agonists regulating collagenb Ⅳ production in mouse podocytes Objective:To investigated potential molecular mechanisms through which PPARγ agonist might regulate collagen IV production in mouse podocytes Methods:Mouse podocytes treated with or without Pio were exposed to 10ng/ml of TGFβ for various periods of time(0、15、30 min),the phosphorylation of Smad2/3 was determined by western blot.todetermine the effect of PPARγ agonists on TGFβ-induced transcriptional activation of Smad response elements,SBE luciferase reporters were transfected into podocytes with TK plasmids as a control of transfection efficiency. Results:TGFβ treatment led to Smad2/3 phosphorylation in a time-dependent manner. Pio attenuated TGFβ-driven phosphorylation of Smad2/3 in each single time point,VS TGFβ treatment alone(P<0.05). The promoter analysis indicated that the promoter of Collagen IV contained Smad-binding elements(SBEs). TGFβ induced SBE4-luciferase activity was markedly blocked by cotreatment with either Rosi(10μM) or Pio(1μM), and had statistic significant. Conclusion:TGFβ led to Smad2/3 phosthorylation in a time-dependent manner; PPARγ agonist inhibit the expression of P-Smad2/3 induced by TGFβ; PPARγ agonist inhibit TGFβ-induced SBE luciferase activity.