Study On Mechanism Of PAX2 Gene Silencing In The Renal Tubular Epithelial Cell Transdifferentiation Process Of Obstructive Nephropathy

IntroductionObstructive nephropathy is a clinical syndrome resulted from structural and (or) functional changes of urinary tract that impede urination, leading to renal pathology and dysfunction. Obstructive nephropathy is not a independent primary disease but a kind of mutual clinical manifestation originated from different etiological factors. Obstructive nephropathy is a frequent reason to cause children chronic renal failure. According to the statistics in north America, obstructive nephropathy for children occupys 0.3% in children end-stage renal diseases and 16.1% in children kidney transplantation and it brings grave economic burden to families and the society. Accordingly, to delay, arrest and even reverse the advancement of obstructive nephropathy has momentous economic and social sense.Many scholars did empirical studies in vivo and vitro and have indicated that renal interstitial fibrosis is the extreme common pathway of obstructive nephropathy. Up to now, though people have done considerable studies about renal interstitial fibrosis, its mechanism is still not completely elucidated. It is commonly accepted that epithelial-mesenchymal transition (EMT) is an important pathogenesis of renal interstitial fibrosis at present. Tissular reparative process after kidney damage is analogous to the playback of embryonic development of definite kidney. It is that some factors and proteins (prepro-EGF, Tamm-Horsfall protein, PAX-2 protein etc.) highly expressed in the process of kidney embryonic development when mature nephrons is in the pathologic state start again to express highly. Besides, transdifferentiation display embryonic mesenchymal phaenotype and loss mature cell signal, which is analogous to the reversion of metanephridium mesenchymal epithelial transition (MET) in the physiological development process of embryonic kidney.PAX2 (Paired Box2) gene encodes a nuclear transcription factor, a key gene to induce the renal tubular epithelial cells transition during embryonic development, expressing in the developmental complete process of pronephron, mesonephron and metanephron, which is involved in the regulation of embryonic development of kidney at various stages. Its expression is turned off in mature nephrons. It was found in recent years that, PAX2 gene is re-expressed in renal interstitial fibrosis model, but there are divergences in the aspect of expressing location of PAX2 gene and the influence of PAX2 re-expression injuring kidney. Unilateral ureteral obstruction (UUO) model resulting in obstructive nephropathy is a classic model inducing renal interstitial fibrosis and it is a most extensively applied experimental animal model in the study of the development mechanism of renal interstitial fibrosis and transdifferentiation of renal cells and estimating the effect of renal fibrotic therapy. Our study is to identify the exact location of PAX2 expression and the relationship between PAX2 re-expression and renal interstitial fibrosis in obstructive nephropathy via establishing UUO rat model. Moreover, this research is to approach the condition that PAX2 gene silencing influences renal interstitial fibrotic extent and renal function, utilizing RNA interfering skill to silence PAX2 gene of renal tubular epithelial cells in rats with obstructive nephropathy in order to elucidate the effect of PAX2 gene silencing in the transdifferentiation of renal tubular epithelial cells.Materials and Methods1. Animal model and group168 Wistar rats (120-150g,4-6w), male, were obtained from the Laboratory Animal Center of Shengjing Hospital, China Medical University. Animals were randomly distributed into model group (UUO group) 32 rats and sham operated group 32 rats. Then rats were distributed into 4 groups by 3,5,7,14 days after surgery and 8 rats each group; Rats were distributed into 5 groups by engineered siRNA sites:siRNA1, siRNA2, siRNA3, siRNA4 and negative control group,8 rats each group; Others were distributed into silencing group (RNAi) 32 rats and negative control group (NC) 32 rats and they were further divided into 4 groups by 3d,5d,7d and14d after transfection,8 rats each group.Model group (UUO group):to establish obstructive nephropathy model by unilateral ureteral deligation, rats in UUO group were anaesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg). A left flank incision was made supra left pubic bone. Left ureter was identified along the inferior pole of left kidney, which was then ligated at both proximal and distal points using 4-0 silk suture. Ureter was severed between these two ligation to prevent it from retrograde infection. Every layer was sewn up and the abdominal cavity was closed.Sham operated group (sham group):Ureters were separated without deligation and the abdominal cavity was closed immediately.siRNA1, siRNA2, siRNA3,siRNA4 and negative control group:After modeling, 200μl PAX2-siRNA-invivo jetPEI composite was injected into the renal capsule immediately. Every layer suturation and the abdominal cavity closure were operated.Silencing group (RNAi):After modeling,200μl PAX2-siRNA3- invivo jetPEI composite was injected into the renal capsule immediately. Every layer suturation and the abdominal cavity closure were operated.Negative control group (NC):After modeling,200μl NC-siRNA- invivo jetPEI composite was injected into the renal capsule immediately. Every layer suturation and the abdominal cavity closure were operated.2. Specimen collection and processing2.1 Nephridial tissueRats in every group were anaesthetized by intraperitoneal injection of 10% chloral hydrate (0.3ml/kg) at 3,5,7 and 14 days after surgery or transfection and then sacrificed. The belly was cleanse by conventional degermation. Under the axenic condition, the left renal tissue was taken and douched with stroke-physiological saline solution. Surface fatty tissue was detached and superfic water was blotted with filter paper. Moiety was in 4% paraformaldehyde and operated by paraffin imbedding for pathomorphology and immunohistochemical assays. Renal cortex was separated from medulla after surface fatty tissue was detached. Then the renal tissues were wrapped in sterile aluminum foil, flash-frozen by liquid nitrogen and stored in -80℃for real-time fluorescence quantitative PCR and western blot assay.2.2 UrineRats were put in the metabolic cage after transfection at 1d,2d,3d,5d,7d and 14d to collect the twenty-four-hour urine. Samples of fresh urine were centrifuged for 15 minutes at 3000rpm and ablated sediment. Then the samples were conserved in -80℃chest freezer for urineβ2-microglobulin (β2-MG) content assay.2.3 Blood Under the shallow anaesthesia with aether, rats were operated haemospasia with glass capillary for 1 ml venous blood of angulus oculi medialis at the end of Id,2d after transfection. Rats were operated haemospasia for 4 ml abdominal aorta and 3000 rpm centrifugalization at the end of 3d,5d,7d,14d after transfection.Blood serum was conserved in -80℃chest freezer for serum creatinine content assay.3. Experimental methods3.1 Macroscopic observation of rats in every group at different time points includes colour of kidney, smooth and glossy superficies or not, intact renal capsule or not, hydronephrosis or not, thinner renal parenchyma, compressed pelvis and calices renales, etc.3.2 Degree of interstitial injury in renal tubules was observed under light microscope after HE staining.3.3 Degree of renal interstitial fibrosis was observed under light microscope after Masson staining.3.4 Expression of PAX2, E-cadherin andα-SMA protein in nephridial tissue was detected by immunohistochemistry.3.5 Expression of PAX2, E-cadherin andα-SMA protein in renal cortex was detected by Western Blotting.3.6 Expression of PAX2 mRNA, E-cadherin mRNA andα-SMA mRNA in renal cortex was detected by real-time fluorescence quantitative PCR.3.7 Examination of renal function:the content of urineβ2-MG and serum creatinine was detected by automatic biochemical analyzer belonged to the Docimastic Department of Shengjing Hospital, China Medical University.3.8 Transfection of PAX2-FAM-siRNA-PEI in nephridial tissue was observed by fluorescence microscope.4. Statistical AnalysisExperimental data were shown as mean±standard deviation. SPSS 13.0 software was used for statistical analysis. Inter-group comparison was done by t test, intra-group comparison was done by one-way ANOVA and correlation analysis between two variables was done by Pearson correlation analysis. P<0.05 was considered statistical significance.Results1. Morphological changes of renal tissue1.1 Macroscopic observationBulk of rat kidney of UUO group was accrescent with prolonged obstruction. The kidney appeared as grayish-white in color, lacklustre, rough surface, obvious grainy, visible hydronephrosis, thinner renal parenchyma, dilated renal pelvis, compressed calyceal papilla, flat and blunt fornix papilla, some of which became concave shape; rat kidney of sham group was dark-red color with smooth surface and intact renal capsule that was not attached to renal parenchyma. Clear boundary between cortex and medulla could be observed in rat kidney of sham group.1.2 Pathological changes of kidney under light microscopeHE and Masson staining showed appearance of renal fibrosis after urinary obstruction. Macrophage and lymphocyte diffusely infiltrate to renal interstitium. Tubular ectasia, analosis and collapse of lumens appeared, renal interstitium widened and collagen fibers were deposited. It was proved that the UUO model was successfully built in this experiment.2. Expression of PAX2 protein and mRNA in kidney in UUO ratsImmunohistochemistry showed that PAX2 protein had no expression in renal tubular epithelial cells in sham group, while relatively abundant expression was identified in nuclei of collecting duct epithelial cells. In UUO group, PAX2 protein expression appeared in renal tubular epithelial cells at 3d after the operation and became more apparent at 14d after the operation (P<0.05). Expression level of PAX2 was significantly higher than that of sham group (P<0.05).Western blotting results showed that the level of PAX2 protein of UUO group was obviously higher than that of sham group at 3d after the operation (P<0.05) and expression of PAX2 protein was obviously increased with prolonged obstruction (P<0.05). Real time PCR showed that expressed tendency of PAX2 mRNA and that of PAX2 protein were same.3. Correlation analysis of PAX2 protein and interstitial fibrosisWe performed Pearson correlation analysis on PAX2 protein expression level, renal tubular damage extent and relative area of renal interstitium. Results showed that: PAX2 protein level was positively correlated with the severity of renal tubular damage (r= 0.937 and P<0.05) and the level of renal interstitial fibrosis (r= 0.987 and P<0.05).4. Screening and identification of PAX2-siRNA-PEI composite4.1 Transfection of PAX2-siRNA-PEI in nephridial tissueFAM-siRNA-PEI and NC-siRNA-PEI were injected into vivo and kidneys were taken out 3 days later. Freezing cut sheets were made with nephridial tissue immediately and observed under fluorescence microscope. Green fluorescence was present in surround nephric tubule of nephridial tissue because FAM-siRNA-PEI was injected, while there is no green fluorescence in nephridial tissue with NC-siRNA-PEI.4.2 Inhibition of PAX2-siRNA-PEI for PAX2 mRNA and proteinExpression of PAX2 mRNA and protein in nephridial tissue was detected by real-time fluorescence quantitative PCR and Western blotting. The four pairs interfering chi sequence chi indeed inhibited the expression of PAX2 mRNA and protein in renal cortex to different extent. Interference effect of siRNA3 site for PAX2 was greatest and 55% PAX2 mRNA and 81% PAX2 protein were inhibited.5. Identification of PAX2 gene silencing at different time points after transfection with PAX2-siRNA3-PEI compositeChemical synthetic PAX2-siRNA3 and siRNA regarded as negative control and in vivo jetPEI composed PAX2-siRNA3-PEI composite, which was transfected into UUO rats vivo. Fluorescent quantitation PCR and Western blotting assay showed that expression of PAX2 mRNA and protein in the silencing group after transfection obviously degraded (P<0.05) compared with concurrent control group at different time points. 6. Effect of PAX2 gene silencing to renal interstitial fibrosis extent and renal function6.1 Renal interstitial fibrosis extentHE and Masson staining showed that in control group with prolonged obstruction, nephric tubular affection aggravated gradually, renal interstitium widened obviously, considerable collagen fibers proliferated and collagen deposition became conspicuous. There was no obvious changes between silencing group and concurrent control group at 3d,5d and 7d(P>0.05), while compared with control group, in silencing group degrees of distension in nephric tubule lessened, pathological changes in interstitium degraded, collagen deposition reduced obviously and fibrotic proceeding delayed greatly at 14d (P<0.05).6.2 Renal functionBlood and urine were reserved after transfection for the content of serum creatinine and urineβ2-MG of rats in silencing group and control group at the end of 1d,2d,3d,5d,7d and 14d in this research. Results show that the level of serum creatinine appeared fluctuant after transfection at different time points in both silencing group and control group, but there are no statistical difference (P>0.05); the content of urineβ2-MG started to step up from the fifth day after transfection in both groups. Moreover, there were not distinctions in the content ofβ2-MG between silencing group and concurrent control group at Id,2d,3d,5d and 7d, but the content ofβ2-MG in silencing group was obviously less than that in control group at 14d.7. Epithelial-mesenchymal transition (EMT) in UUO ratsExpression of E-cadherin and that of a-SMA mRNA and protein were detected by Immunohistochemistry, Western blotting and real time fluorescent quantitation PCR. Relative expression of E-cadherin mRNA and protein in UUO Group decreased at 3d postop, while relative expression of a-SMA mRNA and protein was increased; with prolonged obstruction, expression of E-cadherin degraded gradually and expression of a-SMA increased gradually; E-cadherin in renal cortex appeared micro expression but a-SMA expressed markedly in UUO Group at 14d postop and there were distinctions in statistics between the UUO group and the sham group (P<0.05). Moreover, there were statistical distinctions in the relative expression of E-cadherin mRNA and protein and expression ofα-SMA mRNA and protein in UUO Group at different time points (P<0.05).8. Effect of PAX2 gene silencing to renal tubular epithelial cell mesenchymal transdifferentiation in UUO ratsWith prolonged obstruction, expression of E-cadherin mRNA and protein decreased gradually but expression ofα-SMA mRNA and protein increased gradually in the control group. There were no distinctions in indices between the silencing group and the concurrent control group at 3d,5d,7d after transfection(P>0.05). However, the relative expression of E-cadherin mRNA and protein in the silencing group was obviously higher than the control group at 14d after transfection P<0.05), while the relative expression ofα-SMA mRNA and protein in the silencing group was less obviously than the control group (P<0.05).Discussion1. Embryonic development gene PAX2 is re-expressed in renal tubular epithelial cells in obstructive nephropathy rats and expression of PAX2 will increase with prolonged obstruction.2. Expression level of PAX2 protein were positively correlated with degrees of renal tubular damage and renal interstitial fibrosis.3. PAX2-siRNA mediated by in vivo jetPEI can be injected under the renal capsule of rats and reach to the renal parenchyma successfully.4. Expression of PAX2 mRNA and protein in nephridial tissue was detected by real-time fluorescence quantitative PCR and Western blotting. The four pairs interfering chi sequence chi indeed inhibited the expression of PAX2 mRNA and protein in renal cortex to different extent. Interference effect of siRNA3 site for PAX2 was greatest and 55% PAX2 mRNA and 81% PAX2 protein were inhibited.5. Effect of PAX2 gene silencing to changes of renal interstitial fibrosis extent in the earlier period of obstruction is not obvious but PAX2 gene silencing can delay the proceeding of renal interstitial fibrosis, lessen nephric tubular injury and it has obvious therapeutical effect to renal interstitial fibrosis in the advanced stage of obstruction.6. The content of urineβ2-MG in UUO rats can be degraded by PAX2 gene silencing in the advanced stage of obstruction.7. With prolonged obstruction, expression of E-cadherin mRNA and protein decreased gradually but expression of a-SMA mRNA and protein increased gradually in UUO model of rats. The result suggests that EMT happens in renal tubular epithelial cells.8. PAX2 gene silencing has no effect to EMT in the earlier period of obstruction and it can inhibit the procedure of nephric tubular EMT in the advanced stage of obstruction. PAX2 gene silencing postpone the proceeding of renal interstitial fibrosis.9. Re-expressed PAX2 has the dual biological significance of impair recovery and aggravating pathological changes in renal tubular epithelial cells in the UUO model of rats.