Renal Tubulointerstitial Fibrosis Research Articles

MON-226 FSH Promotes Renal Tubulointerstitial Fibrosis Via AKT/GSK-3beta/beta-catenin Pathway

Background: Estrogen withdraw in postmenopausal women undoubtedly contributes to the progression of chronic kidney disease (CKD). However, the effect of high circulating follicle-stimulating hormone (FSH) level on renal dysfunction remains unknown. We aimed to investigate whether FSH participates in renal development during postmenopausal period. Methods: 3055 postmenopausal blood samples were collected and tested. The effects of FSH on renal function were evaluated in cell and mice model of high FSH level. Results: Based on clinical data analyses, we found that there was a strong negative correlation between eGFR and FSH levels (P<0.001), especially independent of LH, testosterone and estradiol. Functional FSHR was detected in renal tubular epithelial cell. In vivo, high FSH promoted a phenotype of tubulointerstitial fibrosis, characterized by increase in 24h uric protein, serum Cr, serum BUN, as well as ECM deposition. Similar results obtained from cultured HK-2 cells, FSH increased the transcriptional and protein expressions of profibrotic mediators (Collagen IV, Fibronectin and PAI-1). Furthermore, this fibrosis promotion by FSH was through activation of AKT/GSK-3β/β-catenin pathway that could be attenuated by silence of FSHR by siRNA or LY294002. Besides, FSH-stimulated HK-2 secreted chemokines, such as IL-8, promoted macrophages migration to exacerbate tubulointerstitial fibrosis. Conclusion: Functional FSHR was expressed in renal tubular epithelial cells and FSH aggravated renal dysfunction and tubulointerstitial fibrosis via FSHR-mediated AKT/GSK-3β/β-catenin pathway. Our results revealed a previously unknown contribution of FSH to postmenopausal kidney injury.

The antifibrotic and anti-inflammatory effects of icariin on the kidney in a unilateral ureteral obstruction mouse model

BackgroundThe pathology change of renal tubulointerstitial fibrosis is a critical feature of chronic kidney disease (CKD), regardless of the primary insults. The infiltration of inflammatory cells and the consecutive secretion of profibrotic factors are frequently and conspicuously observed during the development of renal fibrosis. Icariin, an active polyphenol of the Epimedium genus, has been found to alleviate the symptoms of chronic diseases like diabetes, neurodegeneration, and heart and renal diseases. The effect and mechanism of icariin on the prevention of CKD-associated renal fibrosis still needed clarification. PurposeThe aims of this study were to investigate whether icariin treatment improves the development of CKD-associated renal fibrosis and its possible mechanism. MethodsAn experimental unilateral ureteral obstruction (UUO)-induced chronic renal fibrosis mouse model was used. Mice were orally administered with icariin (20 mg/kg/day) for 3 consecutive days before and 14 consecutive days after UUO surgery. ResultsThe pathological changes, collagen deposition, and protein expressions of profibrotic factors (transforming growth factor-β and connective tissue growth factor) and fibrotic markers (α-smooth muscle actin and fibronectin), which were significantly elevated in the kidneys of UUO mice, could be significantly reversed by icariin treatment. Icariin treatment also significantly inhibited the increased Smad2/3 and decreased E-cadherin protein expressions in the kidneys of UUO mice. Icariin treatment prominently mitigated the protein expression of proinflammatory factors like nuclear factor-κB, cyclooxygenase-2, interleukin 1-β and prooxidative enzyme (NADPH oxidase-4), and it increased the protein expression of antioxidative enzymes (superoxide dismutase and catalase). ConclusionIcariin treatment protects against CKD-associated renal fibrosis via its antifibrotic and anti-inflammatory properties. Icariin may serve as a therapeutic agent in the prevention of CKD-associated renal fibrosis.

Inhibition of p300/CBP-Associated Factor Attenuates Renal Tubulointerstitial Fibrosis through Modulation of NF-kB and Nrf2.

p300/CBP-associated factor (PCAF), a histone acetyltransferase, is involved in many cellular processes such as differentiation, proliferation, apoptosis, and reaction to cell damage by modulating the activities of several genes and proteins through the acetylation of either the histones or transcription factors. Here, we examined a pathogenic role of PCAF and its potential as a novel therapeutic target in the progression of renal tubulointerstitial fibrosis induced by non-diabetic unilateral ureteral obstruction (UUO) in male C57BL/6 mice. Administration of garcinol, a PCAF inhibitor, reversed a UUO-induced increase in the renal expression of total PCAF and histone 3 lysine 9 acetylation and reduced positive areas of trichrome and α-smooth muscle actin and collagen content. Treatment with garcinol also decreased mRNA levels of transforming growth factor-β, matrix metalloproteinase (MMP)-2, MMP-9, and fibronectin. Furthermore, garcinol suppressed nuclear factor-κB (NF-κB) and pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6, whereas it preserved the nuclear expression of nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and levels of Nrf2-dependent antioxidants including heme oxygense-1, catalase, superoxide dismutase 1, and NAD(P)H:quinone oxidoreductase 1. These results suggest that the inhibition of inordinately enhanced PCAF could mitigate renal fibrosis by redressing aberrant balance between inflammatory signaling and antioxidant response through the modulation of NF-κB and Nrf2.

P38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients.

Activation of p38 mitogen-activated protein kinase (MAPK) is associated with tissue fibrosis, and inhibition of p38 MAPK can attenuate the progression of fibrosis. We aimed to investigate whether p38 MAPK activity in kidney tissue confirmed by immunohistochemical staining is associated with renal tubulointerstitial fibrosis in chronic kidney disease patients with IgA nephropathy. We collected kidney biopsy specimens from 341 IgA nephropathy patients and 15 control patients to identify the clinical and histopathological factors associated with kidney tubulointerstitial fibrosis and to find an association between kidney phosphorylated p38 immunoactivity and pathological grading. In addition, we aimed to investigate whether the anti-fibrotic effect of p38 MAPK inhibition can be identified by assessing the immunostaining intensity of phosphorylated p38 in kidney tissue. A renal tubulointerstitial fibrosis model was introduced using 7-week-old C57BL/6 mice subjected to unilateral ureteral obstruction (UUO). The p38 MAPK inhibitor SB-731445 was injected intraperitoneally every day for 7 days, and changes in renal fibrosis-associated markers were investigated. Assessment of kidney biopsy specimens from IgA nephropathy patients revealed that the degree of interstitial fibrosis was significantly associated with the tissue immunoactivity of phosphorylated p38. High-grade interstitial fibrosis was associated with a low glomerular filtration rate, high proteinuria, and high-grade histopathological changes, including tubular atrophy, interstitial inflammation, and glomerular sclerosis. In a mouse UUO model, renal protein expression of COL1 and phosphorylated p38 were significantly increased, and the protein expression of COL1 and phosphorylated p38 decreased in mice administered 10 mg/kg/day p38 MAPK inhibitor. We found that kidney interstitial fibrosis is associated with increased immunoactivity of phosphorylated p38 in a UUO mouse model and in human IgA nephropathy patients and that the anti-fibrotic effect of p38 MAPK inhibition can be confirmed using immunohistochemical staining for phosphorylated p38 in kidney tissue.

The C5a/C5aR1 axis promotes progression of renal tubulointerstitial fibrosis in a mouse model of renal ischemia/reperfusion injury

C5a is a potent proinflammatory agonist that mediates renal ischemia reperfusion (IR) injury, but the potential for modulating chronic post-ischemic fibrosis and use of therapeutic antagonist are undefined. Here we determine whether C5a receptor 1 (C5aR1) signaling is essential to the development of post-ischemic fibrosis and if it is a valid target for therapeutic blockade with soluble receptor antagonist. C5aR1 is required for the development of renal tubulointerstitial fibrosis in a murine model of renal ischemia/reperfusion injury. Deficiency of C5aR1 protected mice from the development of the fibrosis. This protection was associated with attenuated deposition of extracellular matrix components (fibronectin, collagen I), reduced cellular infiltrates (CD45, F4/80), and gene expression of proinflammatory and profibrogenic mediators in the kidney. In an invitro model of hypoxia/reoxygenation, C5a stimulation caused renal fibroblast proliferation and activation, and upregulated gene expression of interleukin-1α (IL-1α), IL-6 and transforming growth factor-α (TGF-α) in renal tubular epithelial cells and monocytes/macrophages. Administration of a C5aR1 antagonist (PMX53) significantly reduced renal injury and tubulointerstitial fibrosis. Thus, our results demonstrate a pathogenic role for C5aR1 in the progression of tubulointerstitial fibrosis following renal IR injury and support that C5aR1-mediated local inflammatory responses to hypoxic renal injury contribute to tubulointerstitial fibrosis through several cellular pathways, namely, promoting tubule injury, interstitial fibroblast proliferation and epithelial-to-mesenchymal transition of renal tubular epithelial cells. Our results also suggest the C5a-C5aR1 interaction is a therapeutic target for chronic post-ischemic fibrosis.

Autophagy attenuates tubulointerstital fibrosis through regulating transforming growth factor-\u03b2 and NLRP3 inflammasome signaling pathway

Renal fibrosis is the final common pathway of various renal injuries and it leads to chronic kidney disease. Autophagy is a cellular process of degradation of damaged cytoplasmic components and regulates cell death and proliferation. Cellular response during unilateral ureteral obstruction (UUO) is tubular segment specific. Thus the role of autophagy on renal tubulointerstitial fibrosis (TIF) after UUO may be different according to segment of nephron. The role of autophagy during UUO remains unclear especially in distal tubules. In this study, we investigated the role of autophagy in distal tubules on renal TIF using conditional knockout mice in which Atg7 was genetically ablated specifically in distal tubular epithelial cell (TEC). In green fluorescent protein (GFP)-LC3 transgenic mice, GFP-LC3 puncta was highly expressed in distal tubular cells of the obstructed kidneys after UUO. Genetic deletion of Atg7 specifically in distal TEC increased renal tubulointerstial fibrosis and epithelial-mesenchymal transition-like phenotype change after UUO through Smad4-dependent transforming growth factor (TGF)-β pathway. Distal tubule-specific autophagy-deficient mice increased the accumulation of damaged mitochondria and SQSTM1/p62-positive aggregates in the obstructed kidney and resulted in increased expression of NLRP3 inflammasome, interleukin (IL) 1-β and caspase-1. Distal TEC-specific Atg7 deletion enhanced apoptosis of TECs after UUO. In summary, our data showed that autophagy in distal TEC plays a protective role in development of renal tubulointerstial fibrosis through regulating the expression of TGF-β an IL1-β after UUO.

Febuxostat inhibits TGF‑β1‑induced epithelial‑mesenchymal transition via downregulation of USAG‑1 expression in Madin‑Darby canine kidney cells invitro.

Our previous study demonstrated that febuxostat, a xanthine oxidase inhibitor, can alleviate kidney dysfunction and ameliorate renal tubulointerstitial fibrosis in a rat unilateral ureteral obstruction (UUO) model; however, the underlying mechanisms remain unknown. Increasing evidence has revealed that epithelial-mesenchymal transition (EMT) is one of the key mechanisms mediating the progression of renal tubulointerstitial fibrosis in chronic kidney disease (CKD). Uterine sensitization-associated gene-1 (USAG-1), a kidney-specific bone morphogenetic protein antagonist, is involved in the development of numerous types of CKDs. The present study aimed to investigate the role of febuxostat in the process of EMT in Madin-Darby canine kidney (MDCK) cells in vitro. Western blotting, reverse transcription-semiquantitative polymerase chain reaction analysis and immunofluorescence staining were used to evaluate the expression levels of bone morphogenetic protein 7, USAG-1, α-smooth muscle actin (α-SMA) and E-cadherin, respectively. The results demonstrated that the expression of USAG-1 and α-SMA increased, and that of E-cadherin decreased significantly in MDCK cells following treatment with transforming growth factor-β1 (TGF-β1). The application of small interfering RNA-USAG-1 potently inhibited TGF-β1-induced EMT. Subsequently, the effects of febuxostat on TGF-β1-induced EMT was investigated. The results demonstrated that febuxostat downregulated the expression of USAG-1, and reversed TGF-β1-induced EMT in MDCK cells. Furthermore, pretreatment with febuxostat significantly restored the decreased expression levels of phosphorylated Smad1/5/8 induced by TGF-β1 in MDCK cells. The results of the present study suggested that USAG-1 may be involved in the EMT process of MDCK cells induced by TGF-β1, and febuxostat inhibited EMT by activating the Smad1/5/8 signaling pathway via downregulating the expression of USAG-1 in MDCK cells.

Ursolic acid inhibits epithelial-mesenchymal transition in vitro and in vivo

Context: Ursolic acid (UA; 3β-hydroxy-urs-12-en-28-oic acid), one of the pentacyclic triterpenoids found in various plants and herbs, possesses some beneficial effects under pathological conditions, including combating hepatic fibrosis.Objective: This study investigates the effects of UA on renal tubulointerstitial fibrosis in vivo and in vitro.Materials and methods: In vivo, 24 male C57BL6 mice were divided into four groups. Eighteen mice were subjected to unilateral ureteral obstruction (UUO) and the remaining six sham-operated mice served as control. UUO mice received either vehicle or UA (50 or 100 mg/kg) by gastric gavage for 6 days. In vitro, HK-2 cells were treated with 10 or 50 μM UA and 10 ng/mL recombinant human transforming growth factor-β1 (TGF-β1). The molecular mechanisms of fibrosis were investigated.Results: UUO induced marked interstitial collagen I and fibronectin deposition and epithelial-mesenchymal transition (EMT), as evidenced by increased α-smooth muscle actin (α-SMA) and decreased E-cadherin. However, UA treatment significantly reduced collagen I and fibronectin accumulation in the fibrotic kidney. UA treatment also decreased α-SMA and preserved E-cadherin in vivo. In vitro, TGF-β1-treated HK-2 cells demonstrated elevated α-SMA, snail1, slug, TGF-β1, and p-smad3, as well as diminished E-cadherin. UA pretreatment prevented E-cadherin loss and diminished α-SMA expression in HK-2 cells. UA downregulated mRNA expression of snail1 and slug. UA also lowered TGF-β1 protein expression and p-Smad3 in HK-2 cells.Conclusions: UA attenuated renal tubulointerstitial fibrosis by inhibiting EMT, and such inhibition may be achieved by decreasing profibrotic factors. UA may be a novel therapeutic agent for renal fibrosis.

Connexin 43 Prevents the Progression of Diabetic Renal Tubulointerstitial Fibrosis by Regulating the SIRT1-HIF-1α&amp;nbsp;Signaling&amp;nbsp;Pathway

Background: Diabetic renal fibrosis is a combination of glomerular sclerosis and renal tubulointerstitial fibrosis (RIF). Our previous studies have shown that connexin 43 (Cx43) activation could inhibit the development of diabetic renal glomerular sclerosis. Renal epithelial to mesenchymal transition (EMT) is a key pathological factor in diabetic RIF. Whether Cx43 affect the EMT and the underlying mechanism remains to be elucidated. Methods: The effects of Cx43 on EMT were assessed by transfections of plasmids and short hairpin RNAs respectively in NRK-52E cells and caudal vein injection of Cx43 adenovirus in the db/db spontaneous diabetic model mice. The downstream factors were screened through western blot, Immunofluorescence, co-immunoprecipitation, EMSA assay and dual luciferase reporter assay. Findings: Cx43 protein expression was decreased in the kidney tissues of db/db mice and in NRK-52E cells cultured in HG. Cx43 overexpression inhibited the HG-induced EMT process and reduced the expression of the extracellular matrix (ECM) components. Depletion of Cx43 further promoted the EMT process and increased those ECM components. Cx43, in a carboxyl terminal signal transduction-dependent manner, could up-regulate SIRT1 expression and enhance SIRT1-dependent deacetylation of HIF-1α to reduce HIF-1α activity, which eventually ameliorated diabetic RIF. Caudal vein injection of Cx43 adenovirus alleviated renal injury in db/db spontaneous diabetic model mice, accompanied by increased SIRT1 levels, decreased HIF-1α expression, and reduced production of EMT markers and ECM components. Interpretation: Cx43 ameliorates renal EMT and diabetic RIF by regulating the SIRT1-HIF-1α signaling pathway, which provides an experimental basis for Cx43 as a potential target of diabetic nephropathy. Funding Statement: This work was supported by research grants from the National Natural Foundation of China (No. 81603168, No. 81573477, No. 81770816, No.81973375), the Key Projects of Guangdong Natural Science Foundation (No. 2017A030311036), Guangdong Provincial Key Laboratory of Construction Foundation (No. 2017B030314030), and the Natural Science Foundation of Guangdong Province, China (No. 2017A030313678). Declaration of Interests: The authors have declared that no conflicts of interest exist. Ethics Approval Statement: All animal care and experimental procedures complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996), in line with the China Animal Welfare Legislation, and were approved by the Ethics Committee on the Care and Use of Laboratory Animals of Sun Yat-sen University, Guangzhou, Guangdong, China.

Gemigliptin Attenuates Renal Fibrosis Through Down-Regulation of the NLRP3 Inflammasome.

BackgroundThe hypoglycemic drugs dipeptidyl peptidase-4 (DPP-4) inhibitors have proven protective effects on diabetic kidney disease, including renal fibrosis. Although NOD-like receptor protein 3 (NLRP3) inflammasome activation is known to play an important role in the progression of renal fibrosis, the impact of DPP-4 inhibition on NLRP3-mediated inflammation while ameliorating renal fibrosis has not been fully elucidated. Here, we report that the renoprotective effect of gemigliptin is associated with a reduction in NLRP3-mediated inflammation in a murine model of renal fibrosis.MethodsWe examined the effects of gemigliptin on renal tubulointerstitial fibrosis induced in mice by unilateral ureteral obstruction (UUO). Using immunohistochemical and Western blot analysis, we quantitated components of the NLRP3 inflammasome in kidneys with and without gemigliptin treatment, and in vitro in human kidney tubular epithelial human renal proximal tubule cells (HK-2) cells, we further analyzed the effect of gemigliptin on transforming growth factor-β (TGF-β)-stimulated production of profibrotic proteins.ResultsImmunohistological examination revealed that gemigliptin ameliorated UUO-induced tubular atrophy and renal fibrosis. Gemigliptin-treated kidneys showed a reduction in levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin-1β, which had all been markedly increased by UUO. In line with the in vivo results, TGF-β markedly increased NLRP3 inflammasome markers, which were attenuated by gemigliptin treatment. Furthermore, gemigliptin treatment attenuated phosphorylated nuclear factor-κB levels, which had been increased in the UUO kidney as well as in TGF-β-treated cultured renal cells.ConclusionThe present study shows that activation of the NLRP3 inflammasome contributes to UUO-induced renal fibrosis and the renoprotective effect of gemigliptin is associated with attenuation of NLRP3 inflammasome activation.

Urinary excretion profile of microRNAs related to renal fibrosis in Fabry disease patients. A pilot study

Irreversible renal histological lesions, including glomerulosclerosis and tubulo-interstitial fibrosis, have been described in asymptomatic Fabry disease (FD) patients with normal estimated glomerular filtration rate (eGFR) and mild or absent proteinuria. However, it is currently recommended to start FD treatment in presence of clinical manifestations. During renal fibrosis miR-21, miR-192, and miR-433 that promote fibrosis are activated by TGF-β, and miR-29 and miR-200 that suppress fibrosis, are inhibited by TGF-β. The aim of this work is to compare the urinary excretion of microRNAs related to renal fibrosis in FD patients with healthy subjects. In addition, the microRNAs urinary excretion was related to study population clinical variables. Results: 24 FD patients and 10 healthy subjects of similar characteristics were compared. Asymptomatic FD women with normal α-galactosidase-A (α-gal-A) enzyme activity and symptomatic FD women with normal α-gal-A enzyme activity, who were receiving enzyme replacement therapy (ERT) presented a microRNAs urinary profile excretion similar to controls. Asymptomatic FD men or women patients with decreased α-gal-A enzyme activity presented significant differences in miR-29 (p = 0.0478) and miR-200 (p = 0.0426) compared with controls. Symptomatic FD men with decreased α-gal-A enzymatic activity, who were receiving ERT presented significant differences in miR-200 (p = 0.0238) compared with controls. Conclusion: In asymptomatic FD patients with decreased α-gal-A enzymatic activity a microRNA profile indicative of renal fibrosis was found. These patients, at present, are not treated because they do not present clinical criteria to start ERT. In this population, the microRNAs urinary profile excretion could be useful to detect renal fibrosis in early stages.

Autophagy in FOXD1 stroma-derived cells regulates renal fibrosis through TGF-β and NLRP3 inflammasome pathway

Renal fibrosis is the final common pathway of various renal injuries and it leads to chronic kidney disease. Recent studies reported that FOXD1-lineage pericyte plays a critical role in tubulointerstitial fibrosis (TIF). However the regulatory mechanisms remain unclear. Autophagy is a cellular process of degradation of damaged cytoplasmic components that regulates cell death and proliferation. To investigate the role of autophagy in FOXD1-lineage pericytes on renal TIF, we generated the FOXD1-lineage stromal cell-specific Atg7 deletion (Atg7△FOXD1) mice. FOXD1-lineage stromal cell-specific Atg7 deletion enhanced renal TIF through Smad-dependent transforming growth factor (TGF)-β signaling after unilateral ureteral obstruction (UUO). FOXD1-lineage stromal cell-specific Atg7 deletion increased the accumulation of interstitial myofibroblasts and enhanced the differentiation of pericytes into myofibroblasts after UUO. Peritubular capillary rarefaction was accelerated in Atg7△FOXD1 mice after UUO. Atg7△FOXD1 mice increased the accumulation of SQSTM1/p62-positive aggregates in the obstructed kidney and resulted in increased expression of NLRP3 inflammasome, interleukin (IL) 1-β and caspase-1 signaling pathway, which enhanced apoptosis of interstitial cells after UUO. In summary, our data showed that autophagy in FOXD1-lineage stromal cells plays a protective role in renal TIF through regulating the Smad4 dependent TGF-β an NLRP3 inflammasome signaling pathway.

Bone marrow-derived mesenchymal stem cells attenuate tubulointerstitial injury through multiple mechanisms in UUO model.

Current evidence supports the use of bone marrow-derived mesenchymal stem cells (MSCs) for a diverse range of clinical applications, and many studies have shown that MSCs have renal-protective effects, but the mechanism is not well understood. Therefore, in this study, we aim to further identify whether MSCs can attenuate renal fibrosis by decreasing tubulointerstitial injury in a unilateral ureteral obstruction (UUO) model. In this study, we cultured MSCs and then transplanted them into a UUO model through the tail vein. Histology, cell proliferation, peritubular capillary (PTC) loss and myofibroblast markers were examined on days 3, 7 and 14 after surgery. We demonstrated that renal interstitial fibrosis in the MSC group was significantly attenuated compared with the UUO and DMEM groups. Moreover, MSC treatment inhibited the loss of PTCs and increased parenchymal cell proliferation. In addition, UUO-induced activation and proliferation of myofibroblasts were suppressed by MSC infusion. Furthermore, MSCs attenuated tubulointerstitial infiltration of macrophages in UUO mice. Tubulointerstitial damage plays a very important role in the progression of chronic kidney disease (CKD). PTC loss, macrophage recruitment, and myofibroblast activation are directly correlated with the development of renal tubulointerstitial fibrosis. Our results suggest that MSC infusion in the UUO model is a promising therapeutic strategy for promoting kidney repair.

Complement C3 Produced by Macrophages Promotes Renal Fibrosis via IL-17A Secretion

Complement synthesis in cells of origin is strongly linked to the pathogenesis and progression of renal disease. Multiple studies have examined local C3 synthesis in renal disease and elucidated the contribution of local cellular sources, but the contribution of infiltrating inflammatory cells remains unclear. We investigate the relationships among C3, macrophages and Th17 cells, which are involved in interstitial fibrosis. Here, we report that increased local C3 expression, mainly by monocyte/macrophages, was detected in renal biopsy specimens and was correlated with the severity of renal fibrosis (RF) and indexes of renal function. In mouse models of UUO (unilateral ureteral obstruction), we found that local C3 was constitutively expressed throughout the kidney in the interstitium, from which it was released by F4/80+macrophages. After the depletion of macrophages using clodronate, mice lacking macrophages exhibited reductions in C3 expression and renal tubulointerstitial fibrosis. Blocking C3 expression with a C3 and C3aR inhibitor provided similar protection against renal tubulointerstitial fibrosis. These protective effects were associated with reduced pro-inflammatory cytokines, renal recruitment of inflammatory cells, and the Th17 response. in vitro, recombinant C3a significantly enhanced T cell proliferation and IL-17A expression, which was mediated through phosphorylation of ERK, STAT3, and STAT5 and activation of NF-kB in T cells. More importantly, blockade of C3a by a C3aR inhibitor drastically suppressed IL-17A expression in C3a-stimulated T cells. We propose that local C3 secretion by macrophages leads to IL-17A-mediated inflammatory cell infiltration into the kidney, which further drives fibrogenic responses. Our findings suggest that inhibition of the C3a/C3aR pathway is a novel therapeutic approach for obstructive nephropathy.

Dicer deficiency in proximal tubules exacerbates renal injury and tubulointerstitial fibrosis and upregulates Smad2/3.

Renal fibrosis is a common pathological feature in chronic kidney disease (CKD), including diabetic kidney disease (DKD) and obstructive nephropathy. Multiple microRNAs have been implicated in the pathogenesis of both DKD and obstructive nephropathy, although the overall role of microRNAs in tubular injury and renal fibrosis in CKD is unclear. Dicer (a key RNase III enzyme for microRNA biogenesis) was specifically ablated from kidney proximal tubules in mice via the Cre-lox system to deplete micoRNAs. Proximal tubular Dicer knockout (PT- Dicer KO) mice and wild-type (WT) littermates were subjected to streptozotocin (STZ) treatment to induce DKD or unilateral ureteral obstruction (UUO) to induce obstructive nephropathy. Renal hypertrophy, renal tubular apoptosis, kidney inflammation, and tubulointerstitial fibrosis were examined. Compared with WT mice, PT- Dicer KO mice showed more severe tubular injury and renal inflammation following STZ treatment. These mice also developed higher levels of tubolointerstitial fibrosis. Meanwhile, PT- Dicer KO mice had a significantly higher Smad2/3 expression in kidneys than WT mice (at 6 mo of age) in both control and STZ-treated mice. Similarly, UUO induced more severe renal injury, inflammation, and interstitial fibrosis in PT- Dicer KO mice than WT. Although we did not detect obvious Smad2/3 expression in sham-operated mice (2-3 mo old), significantly more Smad2/3 was induced in obstructed PT- Dicer KO kidneys. These results supported a protective role of Dicer-dependent microRNA synthesis in renal injury and fibrosis development in CKD, specifically in DKD and obstructive nephropathy. Depletion of Dicer and microRNAs may upregulate Smad2/3-related signaling pathway to enhance the progression of CKD.

Research on the intervention of Yishen Huoxue prescription to renal fibrosis through the signal regulation by microRNA-126 to VEGF-Notch

To explore the molecular mechanism of Yishen Huoxue prescription in delaying the development of renal fibrosis by regulating the microRNA-126/vascular endothelial growth factor-Notch (miR-126/VEGF-Notch) signaling pathway. Ninety male Sprague-Dawley (SD) rats were randomly divided into sham operation group (sham group), unilateral ureteral obstruction (UUO) model group, losartan group (50 mg×kg-1×d-1) and high, medium and low doses Yishen Huoxue prescription group (25.2, 12.6, 6.3 mL/kg). Each treatment group began to administer drugs by gavage on the day when UUO modeling was finished until the end of the experiment. Left renal tissues of rats were harvested after 7, 14 and 21 days postoperatively. The pathological changes of renal tissue were observed after hematoxylin and eosin (HE) and Masson staining under the microscope, and the renal fibrosis score was calculated. The mRNA expressions of renal tissues miR-126, VEGFA, vascular endothelial growth factor receptor-2 (VEGFR-2), Notch1 were detected by reverse transcription-polymerase chain reaction (RT-PCR). (1) Pathology results: the kidney tissue of sham group was normal. In UUO model group, renal tubules expanded and inflammatory cells in renal interstitium increased; renal tubulointerstitial fibrosis could be seen 7 days after operation, and the degree of fibrosis was gradually increased with time. The renal fibrosis score at each time point after operation in UUO model group was significantly higher than that in sham group. Compared with UUO model group, each treatment group were improved the degree of renal swelling and atrophy of renal parenchyma; the score of renal fibrosis were significantly decreased in the middle and high doses Yishen Huoxue prescription group and losartan group at the 7th day after operation (1.00±1.00, 0.91±0.58, 1.01±0.58 vs. 2.00±0.00, all P < 0.01); but there was no significant difference between low dose Yishen Huoxue prescription group and UUO model group. (2) RT-PCR results: Compared with sham group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissues were significantly increased at each time point after operation in UUO model group. Compared with the UUO model group, the mRNA expressions of miR-126, VEGFA, VEGFR-2 and Notch1 in renal tissue of 7 days postoperatively in the middle and high doses Yishen Huoxue prescription group and the losartan group were significantly increased [miR-126 (2-ΔΔCt): 0.465±0.067, 0.639±0.092, 0.404±0.069 vs. 0.132±0.021; VEGFA (2-ΔΔCt): 0.024±0.005, 0.027±0.007, 0.023±0.006 vs. 0.014±0.006; VEGFR-2 (2-ΔΔCt): 0.021±0.007, 0.023±0.008, 0.019±0.007 vs. 0.012±0.004; Notch1 (2-ΔΔCt): 0.017±0.004, 0.020±0.005, 0.018±0.005 vs. 0.007±0.004; all P < 0.05]; compared with the losartan group, the mRNA expressions of each index in the middle and high doses Yishen Huoxue prescription group were increased at all the time points, but there was no significant difference between them (all P > 0.05). There was no significant difference in mRNA expression of each index between low dose Yishen Huoxue prescription group and UUO model group. The medium and high doses of Yishen Huoxue prescription can effectively antagonize renal fibrosis. Yishen Huoxue prescription may use up-regulation the signaling pathways of miR-126/VEGF-Notch to mediate renal microvascular newly born, eventually to improve renal microvascular damage and delay the development of renal fibrosis progression.

Abstract 030: Stem Cell-Derived Extracellular Vesicles Attenuates Aging-Associated Arterial Stiffness and Hypertension

Objective: Stem cell-derived extracellular vesicles are decreased with aging. Aging is a major risk factor for the development of arterial stiffness and hypertension. This study was designed to assess the effect of inducible pluripotent stem cells (iPSC)-derived extracellular vesicles (EVs) on the aging-related arterial stiffness and hypertension. Methods and Results: Adult (6 months) and aged (20 months) mice were used. Pulse wave velocity (PWV), an index of arterial stiffness and blood pressure were measure twice a week. PWV and blood pressure were increased significantly in aged mice, indicating arterial stiffness and hypertension. One of the exciting findings is that intravenous administration of iPSC-derived EVs significantly decreased aging-related arterial stiffness and hypertension. Treatment with EVs also enhanced endothelium-dependent vascular relaxation and arterial compliance in aged mice. It was noticed that elastin degradation and collagen I deposition (fibrosis) were increased in aortas of aged mice, formulating a structural basis of arterial stiffness. The aging-associated structural remodeling was nearly eliminated by EVs. Mechanistically, EVs abolished downregulation of Sirt1 and eNOS protein expression in aortas of aged mice. In cultured human aortic endothelial cells, we found that EVs promoted the expression and phosphorylation of Sirt1, AMPKα, and eNOS. In addition, EVs attenuated aging-associated renal glomerulosclerosis and tubulointerstitial fibrosis. Conclusion: This study demonstrates for the first time that iPSC-derived EVs improved aging-associated vascular endothelial dysfunction, arterial stiffening, renal remodeling and hypertension likely via regulating the Sirt1-AMPKα-eNOS pathway. A decrease in stem cell-derived EVs plays an important role in aging-associated arterial stiffening and hypertension. This finding sheds light on the therapeutic potential of EVs for arterial stiffness and hypertension associated with aging.

Pentoxifylline protects against loss of function and renal interstitial fibrosis in chronic experimental partial ureteral obstruction

Tubulointerstitial fibrosis (TIF) is a hallmark of chronic kidney disease resulting from diverse etiologies and predicts severity and progression of the kidney disease. To investigate the pathogenesis of TIF, complete unilateral ureteral obstruction (UUO) is the most widely used animal model. However, UUO precludes evaluation of renal function. In the present study, we created a rat model of chronic partial ureteral obstruction (PUO), which allowed assessment of renal function at different intervals after obstruction. We examined the effects of pentoxifylline (PTF), a phosphodiesterase inhibitor used clinically to treat peripheral artery disease, on renal function and TIF. Studies were performed in sham-PUO rats and rats with 14-day PUO or 30-day PUO receiving vehicle in drinking water or PTF (400 mg/liter in drinking water). At day-14 PUO, glomerular filtration rate (GFR) was markedly and similarly depressed in rats receiving vehicle or PTF as compared with sham-operated rats. However, at day-30 PUO, GFR in rats receiving PTF was significantly higher than that in rats receiving vehicle, approaching the level seen in the sham-operated rats. At day-30 PUO, histologic studies also revealed a marked reduction of TIF in rats treated with PTF as compared with the rats receiving vehicle in drinking water. Western blot analysis demonstrated that at day-30 the expression of α-smooth muscle actin (an indicator of renal fibrosis) in the medulla was significantly reduced in PUO rats treated with PTF. In conclusion, PTF treatment ameliorated renal fibrosis and helped preserve renal function in a rodent model of PUO.

Delayed spironolactone administration prevents the transition from acute kidney injury to chronic kidney disease through improving renal inflammation.

Acute kidney injury (AKI) is not as harmless as previously thought since it may lead to chronic kidney disease (CKD). Because most of the time ischemic AKI occurs unexpectedly, it is difficult to prevent its occurrence and there are no specific therapeutic approaches to prevent the AKI to CKD transition. We aimed to determine whether mineralocorticoid receptor blockade (MRB) in the first days after ischemia/reperfusion (IR) can prevent progression to CKD. Four groups of male Wistar rats were included: sham and three groups of bilateral renal ischemia for 45min, one without treatment and the other two receiving spironolactone for 5 or 10 days, starting 24h after IR. The rats were studied at 10 days or 5 months after ischemia induction. After 5months of follow-up, the untreated group exhibited clear evidence of AKI to CKD progression, such as proteinuria, reduced renal blood flow, tubulointerstitial fibrosis, glomerulosclerosis and glomerular hypertrophy. All these alterations were prevented by both spironolactone treatments initiated 24h after IR, the 10-day treatment being more effective. Within the early mechanisms of the MRB protective effect are the reduction of inflammation and increased endothelin-B-receptor expression and endothelial nitric oxide synthase activation in the first 10 days after IR. We propose that MRB, administered 24h after the ischemic injury that leads to AKI, reduces inflammation and promotes efficient tissue repair that avoids the AKI to CKD transition. These data highlight a therapeutic window to preclude CKD development after AKI.

Renal expression of trefoil factor 3 mRNA in association with tubulointerstitial fibrosis in IgA nephropathy.

ABSTRACTAimTrefoil factor 3 (TFF3) is a small peptide that is involved in mucosal protection. TFF3 is widely expressed in multiple tissues including kidney tissue. Previous studies have reported that the levels of urinary TFF3 are significantly increased in patients with chronic kidney disease. The aim of this study is to detect the TFF3 mRNA in kidney and elucidate the relationship between renal TFF3 mRNA and tubulointerstitial fibrosis in IgA nephropathy (IgAN).MethodsWe investigated the renal mRNA expression of TFF3 by real‐time PCR analysis in biopsy specimens from patients with IgAN, other glomerulonephritis (OGN) and minor glomerular abnormalities (MGA). We also determined the renal localization of TFF3 and the levels of urinary TFF3 by immunostaining and ELISA, respectively.ResultsThe renal TFF3 mRNA expression was significantly associated with the urinary TFF3 secretion and the tubulointerstitial fibrosis score in the IgAN group alone. Immunostaining of the renal specimen of IgAN patients revealed that TFF3 is located in the renal tubular epithelial cells. The locations were almost the same as those that showed uromodulin positivity; specifically, the thick ascending limb (TAL) of the loop of Henle and the early portion of the distal tubule. The urinary TFF3 levels were positively correlated with the levels of urinary biomarkers of tubulointerstitial injury in such patients.ConclusionRenal TFF3 mRNA is associated with renal tubulointerstitial fibrosis in IgAN patients. The TFF3 located in the renal tubular epithelial cells may play a role in the progression of tubulointerstitial fibrosis in IgAN patients.