Significance Of PP2Ac Y127 Nitration On EndMT In Peritubular Capillary:a Peptide-based Preventive Strategy

Objective Peritubular capillary endothelial to mesenchymal transition (EndMT) has recently proven to be an important source of renal interstitial myofibroblasts, and the mechanism by which EndMT is initiated is involved in the function loss of tight junction in the endothelium. Evidence has demonstrated that tyrosine nitration can protect the catalytic subunit of protein serine/threonine phosphatase 2a (PP2Ac) and activate PP2A, then down-regulated serine/threonine residues on occluding, subsequently disrupt the function of tight junction in endothelial cells. On the basis of the preliminary study, we aim to find out the effect of critical nitrated tyrosine residue of PP2Ac on the function of tight junction and EndMT both in vivo and in vitro in order to explore the new therapeutic targets and preventive strategies of progressive renal fibrosis.Methods Tandem mass spectrometry was used to map the nitration sites of PP2Ac treated with Peroxinitrite (a nitric oxide with superoxide-product) in vitro. To specifically determine the critical nitration site on PP2Ac, the surface exposure of the nitrated residues were displayed by modeling the known crystal structure of PP2Ac. A tyrosine 127 residue domain mimic peptide of PP2Ac with fused TAT (TAT+Y127WT) and its randomly scrambled variant peptide(TAT+Y127Scr) were constructed and detected for antifibroic effects of the peptide in EndMT cell model. In vivo experiments, whole body fluorescence imaging was used to detect the in vivo distribution and degeneration of the peptides and determine whether TAT-127WT exhibited the same efficiency in vivo by administration TAT-127WT into an experimental animal model of renal fibrosis (i.e., UUO) in mice.Results Six peptides containing nitrotyrosine residues (Tyr-127,130,218,265,267 and 284) were found in PP2Ac. The surface exposure and local electrostatic environment of the tyrosine127 appear to constitute structural requirements, serving as the preferred nitration site followed by Tyr265,130,284,267 and Tyr218. Pretreatment of TAT-127WT, protects against TGF-?1-induced EndoMT by competitive-binding with peroxyintrite to alleviate PP2Ac pro-fibrotic effects. In vivo experiments, fluorescence was strongly detected in liver and kidney. Compared with TAT-Y127Scr, UUO mice treated with TAT-127WT significantly inhibited the de-phosphorylation of serine and threonine residues on occludin and maintain PTC numbers by inhibiting EndMT, resulting in decreased renal interstitial fibrosis.Conclusion PP2Ac Y127 mimic peptide can effectively intervening renal interstitial fibrosis, which is the potential biological targets against renal interstitial fibrosis.Part1 Study the relation between EndMT and renal interstitial fibrosisObjective To explore the evidence of EndMT in the development of renal interstitial fibrosis in patients with primary and secondary nephropathy and to observe the effect of TGF-?1 on promoting the transformation of endothelial cells into mesenchymal cells and confirm the existence of endothelial-originated myofibroblasts in renal fibrosis.Methods Using double labeling of tissue for endothelial marker (CD31) and fibroblast marker (a-SMA) on sequential sections to demonstrate the appearance of EndMT in patients with primary and secondary nephropathy and in UUO-14d mice.3D reconstructed technology was used to analyze the exact construction of a-SMA+/CD31+ areas. In vitro, human umbilical vein endothelial cells (HUVECs) were stimulated with TGF-?1(10ng/ml) for 72h. Light microscopy was performed to observe the morphological change in HUVECs after stimulation; cellular immunofluorescence and western blot were used to investigate the expression of a-SMA and VE-cadherin.Results Co-expression of a-SMA and CD31 were substantially increased on serial kidney sections in renal tissues from patients and UUO mice but rarely observed in control group (P<0.05).3D reconstructed images illustrated microvascular endothelial cells underwent endothelial-to-mesenchymal transition. Compared with basal conditions, certain subsets of endothelial cells exhibited a fibroblast-like morphological phenotype upon stimulation with TGF-?1 for 72 h; in cells stained by immunofluorescence with endothelial (VE-cadherin) and mesenchymal marker (a-SMA), we observed loss of VE-cadherin expression and gain of a-SMA in response to TGF-?1 treatment, which was confirmed by analyzing protein expression using western blot (P<0.05).Conclusions Endothelial cells can undergo endothelial-to-mesenchymal transition both in vivo and in vitro, suggesting the contribution of endothelial-origin myofibroblasts to interstitial fibrosis in kidney disease.Part 2 The role of PP2A activation and PP2Ac nitration in EndMTObjective To investigate the role of PP2A activation and PP2Ac nitration in the process of EndMT.Methods PP2A activity was evaluated both in tissue lysates from UUO mice and cell lysates from TGF-?1-treated HUVECs using the Serine/Threonine Phosphatase Assay System. After co-cultured with PP2A specific inhibitor-okadaic acid (OA) and TGF-?1, HUVECs were harvested. Western blot were used to investigate the expression of a-SMA and VE-cadherin and immunoprecipitation assay was performed to evaluate the phosphorylation status of PP2A physiological substrate-occludin. PP2Ac distribution and expression were examined to determine whether there was a correlation between PP2Ac and endothelial cells using immunohistochemical staining and western blot, respectively. To clarify the role of PP2Ac in the regulation of PP2A, PP2Ac post-translational modifications (PTMs) were examined using immunoprecipitation.Results (1) PP2A activity was gradually upregulated, nearly twofold in UUO mice of 2 weeks compared with sham-operated group and significantly decreased in UUO mice of 3 weeks; exposure to TGF-?1 increased PP2A activity in HUVECs, which began at 15 min and peaked at 60 min. (2) Exposure to OA markedly attenuated a-SMA expression induced by TGF-?1 and maintained VE-cadherin expression; compared with TGF-?1 treatment, the abundance of phosphorylated serine and threonine residues in occludin immunoprecipitates was significantly dampened by pretreatment with OA. (3) Bothe immunohistochemical staining and immunofluorescence analysis of double stainings for PP2Ac and CD31 showed that PP2Ac was expressed in the position of endothelium both in glomerular capillaries and interstitial microvasculars of obstructed kidney; no significance was observed in PP2Ac expression between sham-operated mice and UUO mice, which was similar to the result from HUVECs for the indicated periods after TGF-?1 treatment, however, TGF-?1 markedly enhanced the level of 3-nitrotyrosine (a marker of nitration) in PP2Ac. Moreover, compared with other PTMs,3-nitrotyrosine immunoprecipitates isolated from TGF-?1-stimulated endothelial cells also exhibited moderately increased PP2A activity.Conclusions (1) PP2A activates in the process of EndMT and blockade of PP2A signaling can inhibit this process. (2) PP2Ac nitration is a critical mechanism that activates PP2A in EndMT.Part 3 The role of tyrosine 127 nitration of PP2Ac in EndMTObjective Evidence has demonstrated that tyrosine nitration can protect the catalytic subunit of protein serine/threonine phosphatase 2a (PP2Ac) and activates PP2A, then down-regulates serine/threonine residues on occludin, subsequently disrupt the function of tight junction in endothelial cells. On the basis of the preliminary study, we aim to find out the effect of critical nitrated tyrosine residue of PP2Ac on the function of endothelial cells in order to explore the new therapeutic targets and preventive strategies of EndMT.Methods Tandem mass spectrometry was used to map the nitration sites of PP2 Ac treated with peroxinitrite (a nitric oxide with superoxide-product) in vitro. To specifically determine the critical nitration site on PP2Ac, the surface exposure of the nitrated residues were displayed by modeling the known crystal structure of PP2Ac using ZDOCK 3.0.2 software and ZRANK program. Based on the mass spectrometry experiments, mimic peptides of PP2Ac fused with TAT were constructed:TAT+Y127WT, TAT+Y130WT, TAT+Y265WT, TAT+Y267WT and TAT+Y284WT. Their anti-EndMT effects were compared by analyzing the expression of a-SMA and VE-cadherin in HUVECs, prior to TGF-?1 treatment. Results (1) Only Y218 was detected in control group and six peptides containing nitrotyrosine residues (Tyr-284,267,265,218,130 and 127) were found in peroxyintrite-treated group. (2) The surface exposure and local electrostatic environment of the tyrosine 127 appear to constitute structural requirements, serving as the preferred nitration site followed by Tyr265,130,284,267. (3) TAT-Y127WT exhibited higher potency to inhibit TGF-?1-induced VE-cadherin decrease and ?-SMA increase. By analysis their effects on protein expression, an order of inhibitory potency was evaluated: Y127>265>130>284>267.Conclusions PP2Ac Y127 mimic peptide (TAT-Y127WT) exhibited higher inhibitory effects on EndMT than the other four peptides, suggesting that PP2Ac Y127 is critical for PP2Ac nitration and PP2A activation, which is the potential biological target against EndMT.Part 4 The study of TAT-Y127WT anti-EndMT effectsObjective To investigate the anti-EndMT effects of TAT-Y127WT both in vitro and in vivo and to provide experimental evidence and strategies for renal interstitial fibrosis.Methods (1) To further confirm the inhibitory effect of Y127WT on EndMT, Y127WT (ITQVYGFYD) and its randomly scrambled variant peptide Y127Scr (TYGVIYQFD) were synthesized. Their anti-EndMT effects were compared by analyzing the expression of a-SMA, VE-cadherin and the level of occludin phosphorylation in HUVECs, prior to TGF-?1 treatment. (2) Whole body fluorescence imaging was used to detect the in vivo distribution and degeneration of the peptides and determine whether TAT-127WT exhibited anti-EndMT effects in vivo by administration the peptide at a dose of 5 nmol/g per day to UUO mice until the end of the experiments.Results (1) Compared with TAT-Y127Scr, pretreatment withTAT-Y127WT matained the expression of endothelial marker VE-cadherin and inhibited the increase of a-SMA. Meanwhile, HUVECs with TAT-Y127WT significantly increased both serine and threonine phosphorylation of occludin compared with TAT-Y127Scr treatment. (2) The fluorescence signal disappeared within 24h after injection. Organs and tissues were removed at different time points after tail vain injection. Fluorescence was strongly detected in the kidney and liver, with a small signal in the heart and lung but no signal in spleen. (3) Confocal microscopy revealed that the number of CD31+/a-SMA+areas in the interstitium were dramatically decreased after treatment with TAT-Y127WT. TAT-Y127WT administration attenuated excessive extracellular matrix (ECM) production in UUO mice, which was measured by a-SMA and vimentin deposition in immunohistochemical staining.Conclusions TAT-Y127WT blocks EndMT response both in vitro and in vivo, which it is a therapeutic approach in attenuation of renal fibrosis.