Mouse Model Of Ureteral Obstruction Research Articles

MO402: Symmetric Dimethylarginine Inhibits Renal Fibrosis in Obstructive Kidneys

Abstract BACKGROUND AND AIMS Symmetric dimethylarginine (SDMA) is regarded as an independent cardiovascular risk factor in patients with chronic kidney diseases. Renal interstitial fibrosis is a common pathway of all kinds of chronic kidney diseases progressing to the end-stage of renal diseases. In this study, we investigated the role of SDMA in renal fibrosis and its underlining mechanisms. METHOD Normal saline (NS) and SDMA (2.50 µmol/kg) were administered to the kidney through the left ureter in a mouse model of unilateral ureteral obstruction (UUO). UUO kidneys were harvested on day 7. Western blotting and Masson's trichrome staining were performed to evaluate renal fibrosis. Moreover, human kidney 2 (HK2) cells were treated with various concentrations of SDMA (0.01 µM–10 µM) in the presence of 2.5 ng/mL TGF-β. Protein samples were collected from cells to measure the expression of fibrotic markers. RESULTS We observed that intrarenal administration of SDMA attenuated renal fibrosis as shown by Masson staining and Western blotting analysis of the expression of fibronectin, collagen-I and α smooth muscle actin (αSMA). In parallel, SDMA dose-dependently reduced the expression of pro-fibrotic proteins in TGF-β stimulated HK2 cells. Phosphorylation of Smad3 protein was analyzed in vivo and in vitro, which showed that SDMA inhibited phosphorylation of Smad3 in UUO kidneys and TGF-β stimulated HK2 cells. CONCLUSION Thus, our data suggest that renal SDMA exerts direct anti-fibrotic effects in fibrotic kidneys probably through inhibition of the Smad3 signalling pathway.

Abstract 07: Collecting Duct (Pro) Renin Receptor(PRR) Promotes Kidney Fibrosis Via Macrophage Alternative Activation Mediated By Soluble PRR (sPRR)-Dependent Activation Of Yap/Taz Axis

Renal collecting duct (CD), an important site for fine-tuning urinary Na + and water excretion, is not traditionally thought to play a role in pathogenesis of chronic kidney disease. In the present study, we examined a profibrotic role of CD PRR in a mouse model of unilateral ureteral obstruction (UUO) and further defined the underlying mechanism involving soluble PRR (sPRR)-dependent activation of alternative macrophages activation. We subjected mice with CD-specific deletion of PRR (CD PRR KO) and their floxed controls to UUO or sham surgery. After 7 days of UUO, CD PRR KO decreased the fibronectin (28.6±6.8%) and α-SMA (39.2±7.8%) protein expression in obstructed kidneys. The Masson’s trichrome staining data also showed that CD PRR KO significantly attenuated UUO-induced collagen deposition and histological damage in the kidney. In parallel, CD PRR KO reduced TGF-β1 (56.7±3.2%) and TGF-β2 (53±1.2%) mRNA expression in obstructed kidneys. Moreover, in macrophages sorted from obstructed kidneys of CD PRR KO mice showed a significantly reduced of M2 macrophage markers such as mannose receptor ( MR ) (58.7±2.1%), arginase-1 ( Arg-1 ) (50±1.5%), chitinase-like lectins ( YM-1 ) (59.3±1.9%), inflammatory zone-1 ( Fizz1 ) (39.1±4.1%) mRNA expression and Yes-associated protein ( Yap ) (77±6.8%)/ transcriptional coactivator with PDZ-binding motif ( Taz ) (54.5±1.8%) mRNA expression compared with macrophages sorted from obstructed kidneys of floxed mice. Meanwhile, plasma sPRR was elevated in floxed mice by UUO and this elevation was blunted by CD PRR KO (23.7±0.4%). Administration of site-1 protease (S1P) inhibitor PF-429242 to C57BL/6 mice with UUO almost completely recapitulated the antifibrotic action as well as the inhibitory effect on M2 activation of CD PRR KO. In bone marrow-derived macrophages, sPRR-His treatment promoted macrophage M2 polarization, fibrosis and Yap/Taz expression. Overall, these results suggest that activation of CD PRR releases sPRR that activates M2 polarization via Yap/Taz axis, leading to renal fibrosis during UUO.

P0859ACTIVATE ENDOGENOUS HYDROXYMETHYLATION TO RESTORE ERYTHROPOIETIN PRODUCTION IN FIBROTIC KIDNEYS

Abstract Background and Aims Our previous studies have shown that DNA methyltransferases (Dnmt) and Epo hypermethylation are upregulated in myofibroblasts in fibrotic murine kidneys. Demethylation leads to redifferentiation of myofibroblasts into pericytes. Furthermore, nontoxic doses of 5-azacytidine can restore EPO production and ameliorate anemia in mice with kidney fibrosis. Physiologic hydroxymethylation in zygotes is mediated by the 10 − 11 translocation enzymes (Tet1, Tet2, and Tet3). Method The mouse model of unilateral ureteral obstruction (UUO)-induced kidney fibrosis has been found to display enhanced Tet1 and Tet3 mRNA expression at day 7 and marked increase with the disease progression. Results According to the results, Tet expression significantly increased in fibrotic kidney. To study the effect of myofibroblast-specific Tet3 knockout in murine models of renal fibrosis. Tet3F/F mice were generated by a targeting vector with floxed region contains exons 8-9 of Tet3, which included the coding region for the conserved Fe2+-binding motif of dioxygenase. Gli1CreERT2/+;Tet3F/F mice were used to induce knockout of Tet3 in pericytes and myofibroblasts after tamoxifen administration. Tet3F/F mice were used as the control. Oral gavage of tamoxifen 8 mg/day for 3 days was used to activate Cre recombinase in Gli1+ pericytes and myofibroblasts in adult mice (6-8 week). After two-week washout period, mice were subjected to UUO for 7 or 14 days. The body weight, plasma creatinine and BUN levels showed no significant differences between groups in mice after induction and also UUO surgery. Plasma levels of BUN and creatinine were maintained within the normal range by normally functioning contralateral kidneys after UUO surgery. In UUO kidneys, knockout of Tet3 decreased renal α-SMA and Col1a1 expression. But plasma hematocrit and renal Epo mRNA level were not different between groups. In addition to Gli1CreERT2/+ mice we also used Pdgfrb-rtTATg;LC1Tg mice to breed with Tet3F/F mice for inducible knockout of Tet3. The plasma creatinine, BUN, and hematocrit were no different between groups after UUO surgery again. Conclusion Overall, these findings suggested increased Tet3 in myofibroblasts may play an important role in myofibroblast activation and fibrosis. Further studies need to explore the mechanisms.

Anti-Apoptotic Effect of G-Protein-Coupled Receptor 40 Activation on Tumor Necrosis Factor-α-Induced Injury of Rat Proximal Tubular Cells

G-protein-coupled receptor 40 (GPR40) has an anti-apoptotic effect in pancreatic β-cells. However, its role in renal tubular cell apoptosis remains unclear. To explore the role of GPR40 in renal tubular apoptosis, a two-week unilateral ureteral obstruction (UUO) mouse model was used. The protein expression of GPR40 was decreased, while the Bax/Bcl-2 protein expression ratio, the expression of tumor necrosis factor (TNF)-α mRNA, and angiotensin II type 1 receptor (AT1R) protein were increased in mice with UUO. In vitro, pretreatment of rat proximal tubular (NRK52E) cells with GW9508, a GPR40 agonist, attenuated the decreased cell viability, increased the Bax/Bcl-2 protein expression ratio, increased protein expression of cleaved caspase-3 and activated the nuclear translocation of nuclear factor-κB (NF-κB) p65 subunit induced by TNF-α treatment. TNF-α treatment significantly increased the expression of AT1R protein and the generation of reactive oxygen species (ROS), whereas GW9508 treatment markedly reversed these effects. Pretreatment with GW1100, a GPR40 antagonist, or silencing of GPR40 in NRK52E cells promoted the increased expression of the cleaved caspase-3 protein by TNF-α treatment. Our results demonstrate that decreased expression of GPR40 is associated with apoptosis via TNF-α and AT1R in the ureteral obstructed kidney. The activation of GPR40 attenuates TNF-α-induced apoptosis by inhibiting AT1R expression and ROS generation through regulation of the NF-κB signaling pathway.

The attenuation of renal fibrosis by histone deacetylase inhibitors is associated with the plasticity of FOXP3+IL-17+ T cells

BackgroundThe histone deacetylase (HDAC) inhibitor, which has potential effects on epigenetic modifications, had been reported to attenuate renal fibrosis. CD4+ forkhead box P3 (FOXP3)+ T regulatory (Treg) cells may be converted to inflammation-associated T helper 17 cells (Th17) with tissue fibrosis properties. The association between FOXP3+IL-17+ T cells and the attenuation of renal fibrosis by the HDAC inhibitor is not clear.MethodsThis study evaluated the roles of the HDAC inhibitor, Treg cells and their differentiation into Th17 cells, which aggravate chronic inflammation and renal fibrosis in a unilateral ureteral obstruction (UUO) mouse model. The study groups included control and UUO mice that were monitored for 7, 14 or 21 days.ResultsJuxtaglomerular (JG) hyperplasia, angiotensin II type 1 receptor (AT1R) expression and lymphocyte infiltration were observed in renal tissues after UUO but were decreased after trichostatin A (TSA) treatment, a HDAC inhibitor. The number of CD4+FOXP3+ T cells increased progressively, along with the number of FOXP3+interleukin (IL)-17+ T cells, after 14 days, and their numbers then progressively decreased with increasing CD4+IL-17+ T cell numbers, as demonstrated by double immunohistochemistry. Progressive renal fibrosis was associated with the loss of CD4+FOXP3+IL-17+ T cells in splenic single-cell suspensions. FOXP3+IL-17+ T cells expressed TGF-β1 both in vitro and in vivo, and TGF-β1 expression was significantly knockdown by IL-17 siRNA in vitro. These cells were found to play a role in converting Tregs into IL-17- and TGF-β1-producing cells.ConclusionsTSA treatment decreased JG hyperplasia, the percentage of FOXP3+IL-17+ cells and the degree of fibrosis, suggesting that therapeutic benefits may result from epigenetic modifications.

15-Deoxy-Δ12,14-prostaglandin J2 Exerts Antioxidant Effects While Exacerbating Inflammation in Mice Subjected to Ureteral Obstruction.

Urinary obstruction is associated with inflammation and oxidative stress, leading to renal dysfunction. Previous studies have shown that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has both antioxidant and anti-inflammatory effects. Using a unilateral ureteral obstruction (UUO) mouse model, we examined the effects of 15d-PGJ2 on oxidative stress and inflammation in the kidney. Mice were subjected to UUO for 3 days and treated with 15d-PGJ2. Protein and RNA expression were examined using immunoblotting and qPCR. 15d-PGJ2 increased NF-E2-related nuclear factor erythroid-2 (Nrf2) protein expression in response to UUO, and heme oxygenase 1 (HO-1), a downstream target of Nrf2, was induced by 15d-PGJ2. Additionally, 15d-PGJ2 prevented protein carbonylation, a UUO-induced oxidative stress marker. Inflammation, measured by nuclear NF-κB, F4/80, and MCP-1, was increased in response to UUO and further increased by 15d-PGJ2. Renal injury was aggravated by 15d-PGJ2 treatment as measured by kidney injury molecule-1 (KIM-1) and cortical caspase 3 content. No effect of 15d-PGJ2 was observed on renal function in mice subjected to UUO. This study illustrates differentiated functioning of 15d-PGJ2 on inflammation and oxidative stress in response to obstructive nephropathy. High concentrations of 15d-PGJ2 protects against oxidative stress during 3-day UUO in mice; however, it aggravates the associated inflammation.

Oxymatrine inhibits renal fibrosis of obstructive nephropathy by downregulating the TGF-β1-Smad3 pathway

This study investigated whether oxymatrine (OMT) treatment can ameliorate renal interstitial fibrosis in unilateral ureteral obstruction (UUO) mice model. Moreover, the potential mechanisms of such treatment were analyzed. Twenty-four C57/BL6 mice were randomly divided into three groups, namely sham group, vehicle plus unilateral ureteral obstruction (UUO)-treated group, and 100 mg/kg/d OMT plus UUO-treated group. All mice were euthanized seven days after surgery, and their kidneys were harvested. Renal injury, fibrosis, expression of proinflammatory cytokines, and the transforming growth factor-β1/Smads (TGF-β/Smads) and nuclear factor-kappa B (NF-κB)-signaling pathways were assessed. The results showed OMT significantly prevented kidney injury and fibrosis, as evidenced by decreased expression of collagen-1 and fibronectin. Furthermore, OMT administration inhibited the release of inflammatory factors including tumor necrosis factor-α, (TNF-α) interleukin-1β (IL-1β), and interleukin-6 (IL-6), as well as phosphorylated NF-κB p65. In addition, OMT blocked the activation of myofibroblasts by inhibiting the TGF-β/Smad3-signaling pathway. The findings indicate that OMT-attenuated renal fibrosis and inflammation, and this renoprotective effect may be ascribed to the inactivation of the TGF-β/Smad3 and NF-κB p65 pathways.

Dipeptidyl peptidase IV inhibitor protects against renal interstitial fibrosis in a mouse model of ureteral obstruction

Dipeptidyl peptidase IV (DPPIV) is an exopeptidase that modulates the function of several substrates, among which insulin-releasing incretin hormones are the most well known. DPPIV also modulate substrates involved in inflammation, cell migration, and cell differentiation. Although DPPIV is highly expressed in proximal renal tubular cells, the role of DPPIV inhibition in renal disease is not fully understood. For this reason, we investigated the effects of LC15-0444, a DPPIV inhibitor, on renal function in a mouse model of renal fibrosis. Eight-week-old C57/BL6 mice were subjected to unilateral ureteral obstruction (UUO) and were treated with LC15-0444 (a DPPIV inhibitor) at a dose of 150 mg/kg per day in food or vehicle for 14 days. DPPIV activity was significantly increased in obstructed kidneys, and reduced after treatment with LC15-0444. Administration of LC15-0444 resulted in a significant decrease in albuminuria, urinary excretion of 8-isoprostane, and renal fibrosis. DPPIV inhibition also substantially decreased the synthesis of several proinflammatory and profibrotic molecules, as well as the infiltration of macrophages. UUO significantly increased, and LC15-0444 markedly suppressed, levels of phosphorylated Smad2/3, TGFβ1, toll-like receptor 4, high-mobility group box-1, NADPH oxidase 4, and NF-κB. These results suggest that activation of DPPIV in the kidney has a role in the progression of renal disease and that targeted therapy inhibiting DPPIV may prove to be a useful new approach in the management of progressive renal disease, independent of mechanisms mediated by glucagon-like peptide-1.

Vascular endothelial growth factor-C and -D are involved in lymphangiogenesis in mouse unilateral ureteral obstruction

Lymphatic remodeling in inflammation has been found in tracheal mycoplasma infection, human kidney transplant, skin inflammation, peritonitis, and corneal inflammation. Here we investigated lymphangiogenesis in fibrotic area in unilateral ureteral obstruction, a model of progressive renal fibrosis, and evaluated the roles of vascular endothelial growth factor (VEGF)-C and -D in the obstructed kidney. Compared to sham-operated mice, the number of LYVE-1-positive lymphatic vessels, the proliferation of LYVE-1-positive lymphatic endothelial cells, along with VEGF-C and -D mRNA expression were all significantly increased following ureteral obstruction. Depletion of macrophages with clodronate decreased lymphangiogenesis in the obstructed kidney. VEGF-C expression was higher in M2- than in M1-polarized macrophages from bone marrow-derived macrophages, and also increased in Raw 264.7 or renal proximal tubule cells by stimulation with TGF-β1 or TNF-α. VEGF-D reversed the inhibitory effect of TGF-β1 on VEGF-C-induced migration, capillary-like tube formation, and proliferation of human lymphatic endothelial cells. Additionally, the blockade of VEGF-C and VEGF-D signaling decreased obstruction-induced lymphangiogenesis. Thus, VEGF-C and VEGF-D are associated with lymphangiogenesis in the fibrotic kidney in a mouse model of ureteral obstruction.