Unilateral Ureteral Obstruction Group Research Articles

Microvesicles from Mesenchymal Stem CellsOverexpressing MiR-34a Ameliorate Renal Fibrosis In Vivo.

We recently discovered that microvesicles (MVs) derived from mesenchymal stem cells (MSCs) overexpressing miRNA-34a can alleviate experimental kidney injury in mice. In this study, we further explored the effects of miR34a-MV on renal fibrosis in the unilateral ureteral obstruction (UUO) models. Methods. Bone marrow MSCs were modified by lentiviruses overexpressing miR-34a, and MVs were collected from the supernatants of MSCs. C57BL6/J mice were divided into control, unilateral ureteral obstruction (UUO), UUO + MV, UUO + miR-34aMV and UUO + miR-34a-inhibitor-MV groups. MVs were injected to mice after surgery. The mice were then euthanized on day 7 and 14 of modeling, and renal tissues were collected for further analyses by Hematoxylin and eosin, Masson's trichrome, and Immunohistochemical (IHC) staining. Results. The UUO + MV group exhibited a significantly reduced degree of renal interstitial fibrosis with inflammatory cell infiltration, tubular epithelial cell atrophy, and vacuole degeneration compared with the UUO group. Surprisingly, overexpressing miR-34a enhanced these effects of MSC-MV on the UUO mice. Conclusion. Our study demonstrates that miR34a further enhances the effects of MSC-MV on renal fibrosis in mice through the regulation of epithelial-to-mesenchymal transition (EMT) and Notch pathway. miR-34a may be a candidate molecular therapeutic target for the treatment of renal fibrosis. DOI: 10.52547/ijkd.7673.

Identification of Cell-Cell Communications by Single-Cell RNA Sequencing in End Stage Renal Disease Provides New Insights into Immune Cell Heterogeneity.

Impaired immune system characterized by low-grade inflammation is closely associated with kidney chronic kidney disease (CKD) progression. To reveal the alterations of the function, component, and intercellular communication of immune cells during the progression of CKD. We conducted a case-control study enrolling regular hemodialysis patients and healthy controls. Clinical data, serum and peripheral blood mononuclear cell (PBMC) samples were collected. Flow cytometry and single-cell RNA sequencing were performed to quantitatively analyze the immune cell subsets and T-cell subsets of PBMCs. scRNA data of GSE140023 containing mouse unilateral ureteral obstruction (UUO) models were analyzed the heterogeneity of immune cells. Overall reduction in peripheral blood lymphocyte subsets in patients with end-stage renal disease (ESRD) was observed. A higher ratio of Th17/Treg, Th1/Treg, and b-cell/Treg in the ESRD group was associated with a decrease in eGFR, PTH, and ferritin. Among T cell subsets identified by scRNA analysis, Th17 cells were significantly increased in the ESRD and UU0 group. TFH, Th1, and Th2 cells are located at the final stage in the developmental tree, while Treg and memory CD8+ T cells are at the beginning site. Early developmental differentiation of Th17, Th1, and Tfh cells was observed in the ESRD and UUO group. Analysis of intercellular communication between t-cell subpopulations identified two major input and output signaling pathways: the CD40 and macrophage inhibitory factor (MIF) pathways. The MIF signaling pathway primarily mediates intercellular communication among th17 effects, CD8+ t-cell, and Th17-Treg in the ESRD group, the serum level of MIF showed significant upregulation, which was closely related to Th17/Treg cells. A global immune imbalance was closely associated with the deterioration in renal function and complication development. The MIF signaling pathway mediates Th17/Treg communication and promotes the trans-differentiation of Treg cells to Th17 cells in CKD progression.

Guben Xiezhuo Decoction inhibits M1 polarization through the Raf1/p-Elk1 signaling axis to attenuate renal interstitial fibrosis

Ethnopharmacological relevanceGuben Xiezhuo Decoction (GBXZD) is an herbal compound used to treat chronic kidney disease (CKD) under the guidance of traditional Chinese medicine (TCM). Its main components are Astragalus membranaceus (Fisch.) Bunge, Codonopsis pilosula (Franch.) Nannf., Centella asiatica (L.) Urb., Salvia miltiorrhiza Bunge, Cuscuta chinensis Lam., and Rheum palmatum L.. Clinical studies have shown that it can relieve fatigue, nausea and other symptoms and improve kidney function in patients; however, its specific mechanism of action requires further study. Aim of the studyRenal interstitial fibrosis (RIF) is the ultimate characteristic manifestation of various CKD, that cannot be cured, and appropriate treatments to delay its progression require further exploration. GBXZD, widely used in clinical practice for RIF treatment, can effectively relieve the syndrome in patients with CKD. However, the specific mechanism of action of GBXZD in RIF is unknown and requires further study. This study aimed to explore the specific effects of GBXZD on RIF through the regulation of M1 macrophages. Materials and methodsAn in vivo RIF model was obtained through unilateral ureteral obstruction (UUO), and the Sprague-Dawley (SD) rats were randomly divided into sham operation, UUO, UUO + GBXZD-low dose (GBXZD-L) and UUO + GBXZD-high dose (GBXZD-H) groups. Pathological changes in rat kidney specimens were observed using hematoxylin and eosin (HE) and Masson staining. The expression of collagen I (COL I), fibronectin (FN), α-smooth muscle actin (α-SMA), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α) was detected using immunohistochemistry, and immunofluorescence was used to detect the expression of CD86 and inducible nitric-oxide synthase (iNOS) in kidney tissue. An in vitro experiment was performed usingM1 polarization model in RAW264.7 macrophages induced by lipopolysaccharide (LPS). Cells were divided into control, LPS, LPS + GBXZD-low dose (GBXZD-L) and LPS + GBXZD-high dose (GBXZD-H) groups. The changes in expression of CD86, iNOS, IL-1β, IL-6, and TNF-α were measured using western blotting, flow cytometry, immunofluorescence and enzyme-linked immunosorbent assay (ELISA). We analyzed the action pathway of GBXZD in regulating M1 polarization of macrophages using antibody microarray and verified the results using western blotting. ResultsHistopathological results showed that the UUO group exhibited significant fibrotic injury compared to the sham group. After GBXZD treatment, the degree of kidney injury, RIF, and inflammatory factor expression were lower than those in the UUO group. Compared with LPS-treated cells, the expression of the M1 markers CD86, iNOS, and pathway proteins Raf1 and p-Elk1 was down-regulated in RAW 264.7 cells treated with LPS and GBXZD. The secretion of the inflammatory factors IL-1β, IL-6, and TNF-α in the LPS group was more than that in the control group. However, the levels of these factors were significantly reduced in the GBXZD-H group compared to those in the LPS group. ConclusionsThis study indicates that GBXZD ameliorates RIF and inhibits the inflammatory response and macrophage M1 polarization by a potential mechanism related to the downregulation of Raf1 and p-Elk1. GBXZD therefore has therapeutic potential value for patients with CKD.

Atorvastatin reduces renal interstitial fibrosis caused by unilateral ureteral obstruction through inhibiting the transcriptional activity of YAP

Renal interstitial fibrosis is the primary pathological basis for the progression and development of various chronic kidney diseases, ultimately leading to renal failure. Obstructive kidney disease caused by conditions such as kidney stones, is a common cause of renal fibrosis. The Hippo pathway is a crucial signaling pathway that senses mechanical forces and is involved in the pathophysiology of fibrosis. In this study, we established a mouse model of obstructive kidney disease induced by unilateral ureteral obstruction (UUO). The UUO procedure significantly upregulated YAP and fibrosis-related gene expression in a time-dependent manner. Morphologically, the renal fibrotic lesions associated with hydronephrosis progressively worsened over time in the UUO group. Atorvastatin, which is widely used to lower blood cholesterol levels, has recently been shown to inhibit Yes1 associated protein (YAP). We treated UUO mice with atorvastatin for 3 and 10 days and observed a decrease in the expression of YAP and fibrosis-related genes at the mRNA and protein levels, along with a reduction in the renal fibrosis analyzed by Masson's staining. These findings suggest that atorvastatin may serve as a preventive agent for fibrosis associated with obstructive kidney disease.

Nicotinamide N-Methyl Transferase as a Predictive Marker of Tubular Fibrosis in CKD.

Chronic kidney disease (CKD) progression is complex. There are not standardized methods for predicting the prognosis of CKD. Nicotinamide N-methyltransferase (NNMT) has been shown to be associated with renal fibrosis. This study aimed to validate NNMT as a prognostic biomarker of progressive CKD. We explored the relationship between NNMT expression and CKD-related outcome variables using the NephroseqV5 and GEO databases. Additionally, a validation set of 37 CKD patients was enrolled to measure the correlation between NNMT expression levels and CKD outcomes. Furthermore, single-cell RNA sequencing data and the Human Protein Atlas were reanalyzed to investigate the expression specificity of NNMT in the kidney. Finally, to detect the status of NNMT expression with tubular fibrosis in vivo, we constructed a unilateral ureteral obstruction (UUO) mouse treated with an NNMT inhibitor. Analyzing the datasets showed that NNMT was expressed mainly in proximal tubule compartments. And patients with high NNMT expression levels had a significantly lower overall survival rate compared to those with low NNMT expression levels (P = 0.013). NNMT was independent of prognosis factors in the multivariate Cox regression model, and the AUCs for CKD progression at 1, 3, and 5 years were 0.849, 0.775, and 0.877, respectively. Pathway enrichment analysis indicated that NNMT regulates the biological processes of tubulointerstitial fibrosis (TIF). In the validation group, NNMT levels were significantly higher in the CKD group combined with interstitial fibrosis. In vivo, NNMT was a high expression in the UUO group, peaking at postoperative day 21. Treatment with an NNMT inhibitor improved renal tubular interstitial fibrosis, and expression levels of FN, α-SMA, VIM, and TGF-β1 were decreased compared with UUO (P < 0.05). NNMT was expressed mainly in tubular renal compartments, and associated with CKD prognosis. It holds potential as a diagnostic biomarker for tubular fibrosis in CKD.

#2685 SLC34A2 EXACERBATES TUBULAR INTERSTITIAL FIBROSIS VIA INDUCTION OF APOPTOSIS AND CELL CYCLE ARREST IN S PHASE

Abstract Background and Aims Chronic kidney disease (CKD) is an urgent public health issue in the world. However, effective treatments for intervention of CKD remain undefined. Phosphate (Pi) is an essential element for synthesis of DNA, RNA, ATP, phospholipid membranes, and for regulation of phosphorylation/dephosphorylation, cellular signaling and metabolic pathways. Solute carrier family 34 (SLC34) is responsible for Pi absorption in the small intestine. However, the role of SLC34 in progression of CKD is still unknown. Method A membrane protein, solute carrier family 34 member 2 (SLC34A2), was discovered by using big data mining from the National Center for Biotechnology Information (NCBI). SLC34A2 was overexpressed in human proximal tubular cells, HK-2, by Lipofectamine 3000. RNA sequencing was carried out to uncover SLC34A2-mediated signaling pathways by using Illumina platform. Apoptosis was evaluated by Annexin V-PI staining and TEUNEL assay in cells and mouse kidneys, respectively. Cell cycle was measured by flow cytometry. Unilateral ureteral obstruction (UUO) and bilateral renal ischemia-reperfusion injury (bIRI) were employed to generate CKD mouse models. Results By using big data mining from the NCBI, we identified a membrane protein, SLC34A2, was upregulated in various types of nephropathies including diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, ANCA-associated vasculitis. Overexpression of SLC34A2 suppressed viability of HK-2 cells. RNA sequencing revealed that apoptosis and cell cycle are high scored pathways modulated by SLC34A2. The results were further verified in HK-2 cells. Ectopic SLC34A2 promoted apoptosis via downregulation of BCL-2. Moreover, SLC34A2 induced cell cycle arrest in S phase via downregulation of CDK2. CKD mouse models of UUO and bIRI were carried out to confirm in vitro findings. In mice kidneys, the expression of Slc34a2 in UUO and bIRI groups was superior to that in sham groups. Enriched Bax and TUNEL-positive cells were observed in the fibrotic kidneys of mice. Moreover, increased expression of p21 and downregulated Cyclin A2 in the fibrotic kidneys further confirmed that Slc34a2 induced cell cycle arrest in S phase. Conclusion Slc34a2 induced apoptosis and halted cell cycle in S phase, resulting in subsequent tubulointerstitial fibrosis in mice. Our results suggest that targeting SLC34A2 might be a promising strategy for intervention of CKD.

The effect of tannic acid on renal renin-angiotensin signaling pathway in a model of unilateral ureteral obstruction in male Wistar rats.

The purpose of current study was to elucidate polyphenol tannic acid effect on renal function and activity of the renin-angiotensin system after unilateral ureteral obstruction (UUO). Male Wistar rats were divided into three groups of six randomly: 1) Sham, 2) UUO, and 3) UUO + Tannic acid. Rats in the UUO and UUO + Tannic acid groups experienced unilateral ureteral obstruction. In the Sham group, the abdominal cavity was exposed without UUO induction. In the UUO + Tannic acid group, animals received tannic acid (20mg/kg) intraperitoneally, 6 and 12h after clamping the left ureter and 6 and 12h after the right nephrectomy. Blood samples were taken to measure blood urea nitrogen (BUN) and creatinine levels. Kidney tissue samples were obtained for assessment of oxidative stress, inflammatory indices and the levels of renin-angiotensin system components. Tannic acid administration significantly improved UUO-induced kidney dysfunction (serum BUN: 66.42 ± 14.414mg/dl, p < 0.05; serum creatinine: 1.67 ± 0.258mg/dl, p < 0.05), oxidative stress (MDA level: 95.29 ± 37.35µmol/g tissue, p < 0.05; SOD activity: 59.82 ± 13.41 U/g protein, p < 0.01) and inflammation (renal TNF-α: 57.05 ± 15.653pg/g tissue, p < 0.05; renal IL-6: 117.015 ± 24.076pg/g tissue, p < 0.001). The treatment caused a reduction in the amount of renal angiotensinogen, renin and ACE genes expression compared to the UUO group (Angiotensinogen: 8.9 ± onefold, p < 0.05, Renin: 6.5 ± 1.14 fold, p < 0.05, ACE: 4.9 ± 0.64 fold, p < 0.05). Angiotensin II type 1 receptor protein levels decreased in the tannic acid-treated rats in comparison with the UUO group (0.61 ± 0.136, p < 0.05). According to the result of the current study, tannic acid considerably attenuated the complications of unilateral ureteral obstruction through renin-angiotensin system modulation. Trial registration: IR.TUMS.MEDICINE.REC.1400.802.

Podocarpusflavone alleviated renal fibrosis in obstructive nephropathy by inhibiting Fyn/Stat3 signaling pathway.

Tubulointerstitial fibrosis is a common pathological change in end-stage renal disease. However, limited treatment methods are developed, and unexplained potential mechanisms of renal diseases are urgent problems to be solved. In the present research, we first elucidated the role of podocarpusflavone (POD), a biflavone compound, in unilateral ureteral obstruction (UUO) in rodent model which is characterized by inflammation and fibrosis. The changes in histology and immunohistochemistry were observed that POD exerted renoprotective effects by retarding the infiltration of macrophage and aberrant deposition of ɑ-SMA, Col1a1, and fibronectin. Consistent with in vivo assay, POD treatment also ameliorated the process of fibrosis in TGF-β1-stimulated renal tubular epithelial cells and inflammation in LPS-induced RAW264.7 cells in vitro. In terms of mechanism, our results showed that treatment with POD inhibited the aggravated activation of Fyn in the UUO group, and weakened the level of phosphorylation of Stat3 which indicated that POD may alleviate the process of fibrosis by the Fyn/Stat3 signaling pathway. Furthermore, the gain of function assay by lentivirus-mediated exogenous forced expression of Fyn abrogated the therapeutic effect of the POD on renal fibrosis and inflammation. Collectively, it can be concluded that POD exerted a protective effect on renal fibrosis by mediating Fyn/Stat3 signaling pathway.

Relative comparison of chronic kidney disease-mineral and bone disorder rat models.

Objective: The aim of this study is to establish a suitable animal model of chronic kidney disease-mineral and bone disorder (CKD-MBD) by comparing CKD-MBD rat models induced by 5/6 Nx, AN, and UUO, accompanied by a low-calcium and high-phosphorus diet. Methods: Sprague‒Dawley rats were randomly divided into four groups: control group, 5/6 nephrectomy (5/6 Nx) group, Adriamycin nephropathy (AN) group, and unilateral ureteral obstruction (UUO) group. Serum biochemical indices were measured to evaluate renal function, mineral and bone metabolism, the severity of CKD-MBD, and the status of bone transformation. Hematoxylin-eosin staining (HE) and Masson's trichrome (Masson) staining were used for histopathological analysis of the kidney. Goldner's trichrome (Goldner) and tartrate-resistant acid phosphatase (TRAP) staining were utilized to observe bone mineralization and osteoclasts in the femur, respectively. Micro-CT images were applied to study the structure of the femur. The expression levels of osterix and cathepsin K in the femur were measured by immunohistochemistry (IHC) to confirm the status of bone transformation. Results: The levels of serum creatinine (Scr) and blood urea nitrogen (BUN) in the 5/6 Nx and AN group rats were significantly higher than those in the control rats, and this change was accompanied by marked changes in the levels of calcium (Ca), phosphate (Pi), intact parathyroid hormone (i-PTH), fibroblast growth factor 23 (FGF23), osteocalcin (OC), and cross-linked C-telopeptide of type 1 collagen (CTX-1); UUO group rats exhibited slight and inconsistent variations in the levels of Scr, BUN, Ca, Pi, i-PTH, FGF23, OC, and CTX-1 in serum. Histopathological analysis of the kidney showed that the UUO group rats suffered serious fibrosis and 5/6 Nx group rats exhibited severe focal calcification. Histopathological analysis of the femur showed that the AN group rats had minimal bone mineralization and that the 5/6 Nx group rats had overactive osteoclasts. Micro-CT revealed that the AN model had the most severe bone destruction and that the 5/6 Nx model had the least severe bone loss among the three models. The expression of cathepsin K in the femur was significantly increased in all models, while the expression of osterix in the femur was only significantly increased in the 5/6 Nx model. Conclusion: 5/6 Nx, AN, and UUO accompanied by a low-calcium and high-phosphorus diet successfully induced CKD-MBD in rats. The 5/6 NX model presented the progression of high-turnover bone disease, with consistency between biochemical indices in serum and histomorphometric analysis of the femur, and the AN and UUO models developed a severe deterioration in bone quantity and severe bone resorption; however, the changes in biochemical indices were subtle in the UUO model, and liver injury was obvious in the AN model.

Acteoside alleviates UUO-induced inflammation and fibrosis by regulating the HMGN1/TLR4/TREM1 signaling pathway.

Acteoside (Act), a phenylethanoid compound that was first isolated from mullein, has been widely used for the investigation of anti-inflammatory and anti-fibrotic effect. However, the mechanism of Act against unilateral ureteral obstruction (UUO)-mediated renal injury is largely unknown. Therefore, this study aimed to explore the effects of Act on UUO rats and possible mechanisms. A total of 20 Sprague-Dawley (SD) rats were divided randomly into three groups (n≥6): (i) sham-operated group (Sham); (ii) UUO group (UUO+Saline); and (iii) UUO + Act 40 mg/kg/day, (UUO+Act); Continuous gavage administration for 2 weeks postoperatively, while the rats in Sham and UUO+saline groups were given equal amounts of saline. All rats were sacrificed after 14 days, the urine and blood samples were collected for biochemical analysis, the renal tissues were collected for pathological staining and immunohistochemistry. Correlations between individual proteins were analyzed by Pearson correlation analysis. The results of renal function indexes and histopathological staining showed that Act could improve renal function by reducing serum creatinine, blood urea nitrogen and urine protein at the same time, Act could alleviate renal inflammation and fibrosis. In addition, the results of immunohistochemistry showed that Act could reduce the expression of inflammation and kidney injury-related proteins F4/80, Mcp-1, KIM-1 proteins, as well as the expression of fibrosis-related protein α-SMA and β-catenin. More importantly, Act can also reduce the expression of HMGN1, TLR4 and TREM-1 proteins. These data demonstrate that Act can ameliorate UUO-induced renal inflammation and fibrosis in rats probably through triggering HMGN1/TLR4/TREM-1 pathway.

PS-BPB10-5: POSSIBLE INVOLVEMENT OF RENAL TUBULAR NFAT5 IN AGING-ASSOCIATED RENAL DYSFUNCTION AND SALT-SENSITIVE HYPERTENSION

Objective: Japan is currently facing a super-aging society, where an incidence of lifestyle-related diseases has become a greater social burden. The number of patients with chronic kidney disease (CKD) and hypertension is being increased with age; however, it is not clearly defined why and how aging causes renal dysfunction and hypertension. Nuclear factor of activated T-cells 5 (NFAT5) is a transcription factor that is activated upon hypertonic conditions as observed in the renal medulla. We have shown that the renal tubular cell-specific NFAT5 conditional knockout (KO) mice exhibit salt-sensitive hypertension, while the mice exhibit impaired urine concentrating ability and are susceptible to renal fibrosis. These phenotypes resemble aging-associated renal dysfunction, i.e., urine concentrating disorder, salt-sensitive hypertension, and renal fibrosis. We therefore investigated the possible involvement of NFAT5 in aging-related changes of the kidney. Methods: Male wild-type (WT) and KO mice (12 weeks old) were subjected to unilateral ureteral obstruction (UUO) or sham operation. After 7 days, mice were sacrificed and kidneys were removed for analysis. Renal fibrosis was evaluated by AZAN staining. Senescence-associated beta-galactosidase (SA-β;-Gal) activity was examined by fluorescence study in the whole kidney slices. mRNA expression for senescence-associated secretory phenotype (SASP)-related factors (TGF-β;1, COL1A1, ICAM1, PAI-1, p21, p16, and p53) in the whole kidney tissue was examined by real-time PCR. Results: AZAN staining showed that UUO induced renal fibrosis both in WT and KO mice. The UUO-induced fibrotic area was significantly larger in KO mice than in WT mice in the medulla. In sham-operated mice, overall SA-β;-Gal activity was larger in KO mice, in which the activity was dominant in the corticomedullary region. In UUO groups, overall SA-β;-Gal activity was low compared to sham-operated groups. The mRNA expression of SASP-related factors was not different between WT and KO mice in sham-operated groups. UUO upregulated the mRNA expression of all SASP-related factors both in WT and KO mice. In UUO groups, the expression of TGF-β;1, COL1A1, ICAM1, and PAI-1 was significantly larger in KO mice than in WT mice. The expression of p21, p16, and p53 tended to be larger in KO mice than in WT mice. Conclusions: Renal tubular NFAT5 deficiency showed senescence-related phenotypes at baseline. Deficit of renal tubular NFAT5 may further induce cell senescence upon insult, which might accelerate aging-associated renal inflammation and dysfunction in CKD.

Yiqi Huoxue Tongluo recipe regulates NR4A1 to improve renal mitochondrial function in unilateral ureteral obstruction (UUO) rats

Context Yiqi Huoxue Tongluo recipe (YHTR) is a traditional Chinese medicine for the treatment of chronic kidney disease, but its exact mechanism is not clear. Objectives To monitor the potential improvement of renal mitochondrial function in unilateral ureteral obstruction (UUO) rats by regulating NR4A1 using the YHTR. Materials and methods Wistar rats were randomly divided into four groups: sham, UUO (left ureteral ligation for 14 days), eplerenone (EPL) (UUO + EPL), and YHTR (UUO + YHTR). UUO rats were established and intragastrically administered EPL (100 mg/day/kg) or YHTR (11.7 g/day/kg) for 14 days. The expression of related proteins in kidneys was detected by immunohistochemistry, western blot, RT-PCR, and chemical colorimetric assay, respectively. Results In vivo, YHTR treatment reduced the levels of BUN and Scr (by 17.9% and 23.5%) in UUO rats. Moreover, YHTR improved the renal mitochondrial function via increasing key enzymes of the tricarboxylic acid (TCA) cycle (p < 0.05) and activity of the mitochondrial complex (I–V) (by 30.8%, 29.1%, 19.7%, 35.9%, and 22.4%) in UUO rats. Compared with the UUO group, the expression of NR4A1 and Bcl-2 were significantly increased (p < 0.05), the expression of caspase-3 and caspase-9 were significantly decreased (p < 0.05) in the YHTR group. YHTR could upregulate key enzymes of the TCA cycle via promoting NR4A1 expression in HK2 cells, leading to inhibition of TGF-β1 induced cell apoptosis. Conclusions YHTR significantly improved the development of CKD; this study may provide new ideas for the pathogenesis of CKD and new strategies for the development of new drugs against CKD.

Acteoside Alleviates Renal Fibrosis by Inhibiting β-Catenin/CTGF Signaling Pathway in UUO Rats

Objective: Acteoside (ACT) has been reported to regulate the inflammation and immune response. The study aims to explore the effect of ACT on renal fibrosis in unilateral ureteral obstruction (UUO) rats. Methods: Eighteen Sprague-Dawley rats were randomly divided into 3 groups: sham group, opened the abdominal cavity and sutured abdominal; UUO group, performed UUO surgery; and ACT + UUO group, ACT (40 mg/kg) was given by gavage every day after UUO surgery. After 2 weeks of rat model construction, urine and blood samples were collected for biochemical analysis, while kidney tissues were harvested for hematoxylin and eosin (H&amp;E), Masson's trichrome, and immunohistochemistry staining. The expression of connective tissue growth factor (CTGF), alpha smooth muscle actin (α-SMA), collagen III, heat shock protein 47 (HSP47), and β-catenin in the renal tissue was detected and the correlation between these proteins was analyzed. Results: ACT improved the parameters of renal function in UUO rats, including decreased creatinine and urea nitrogen, and declined urinary protein. Pathological analysis suggested that ACT improved the conditions of renal tubule lesion (including structure destruction, atrophy and lumen obstruction), renal interstitial fibrosis and inflammatory cell infiltration in UUO rats. It also down-regulated the expressions of fibrin-related proteins β-catenin, CTGF, α-SMA, collagen III, and HSP47. Correlation analysis found that β-catenin and CTGF were correlated with the expressions of α-SMA, collagen III, and HSP47. Conclusions: ACT could alleviate renal fibrosis in UUO rats probably via inhibiting β-catenin/CTGF signaling pathway.

Toll-like receptor 4 signaling pathway mediates both liver and kidney injuries in mice with hepatorenal syndrome.

Hepatorenal syndrome (HRS) is a complication of cirrhosis with high morbidity and mortality. Nevertheless, the underlying mechanism involving how kidney injury aggravates the progression of cirrhosis remains unclear. This study aims to explore the role of the Toll-like receptor 4 (TLR4) signaling pathway in mediating liver and kidney injuries in HRS mice induced by unilateral ureteral obstruction (UUO) and/or bile duct ligation (BDL). Two weeks after UUO, there were no obvious pathological changes in mouse liver and the unligated side of kidney. Nevertheless, impaired liver and kidney functions, inflammatory response, and fibrosis were examined in mice after 2 wk of BDL. Compared with those of other groups, mice in the BDL + UUO group presented severer liver and kidney injuries, higher levels of inflammatory factors, and faster deposition of collagens, suggesting that kidney injuries accelerated the aggravation of HRS. Correlation analysis identified a positive correlation between expression levels of inflammatory factors and fibrotic levels. Meanwhile, TLR4 and its ligand MyD88 were upregulated during the process of liver and kidney injuries in HRS mice. Further animal experiments in transgenic TLR4-/- mice or in those treated with TAK242, a small molecule inhibitor of TLR4, showed that blocking the TLR4 signaling pathway significantly improved survival quality and survival rate in HRS mice by alleviating liver fibrosis and kidney injury. It is concluded that kidney dysfunction plays an important role in the aggravation of cirrhosis, which may be attributed to the TLR4 signaling pathway. Targeting TLR4 could be a promising therapeutic strategy for protecting both liver and kidneys in patients with HRS.NEW & NOTEWORTHY Our study established BDL, UUO, and BDL + UUO models, providing a novel idea for analyzing liver and kidney diseases. It is highlighted that the kidney injury accelerated the aggravation of HRS via inflammatory response, which could be protected by inhibiting the TLR4 signaling pathway. We believed that targeting TLR4 was a promising therapeutic strategy for protecting both liver and kidney functions in patients with HRS.

ShenKang Injection Attenuates Renal Fibrosis by Inhibiting EMT and Regulating the Wnt/β-Catenin Pathway

Shenkang Injection (SKI) is a traditional Chinese medicine injection commonly used in the clinical treatment of chronic kidney disease. Although it has been confirmed that SKI has anti-kidney fibrosis effects, the underlying mechanism remains unclear. To investigate the effects of SKI on epithelial-mesenchymal transition (EMT) and Wnt/β-catenin pathway and explore its potential anti-fibrosis mechanism. A unilateral ureteral obstruction (UUO) model was induced by ligating the left ureter of male SD rats. A total of 24 rats were randomly divided into the following four groups: sham group, model group, SKI group, and benazepril group. The rats in each group were treated for 28 days, and renal function was evaluated by blood urea nitrogen (BUN) and serum creatinine (Scr). The degree of renal fibrosis was assessed by hematoxylin and eosin (HE) and Masson staining. Extracellular matrix (ECM) deposition was evaluated by immunohistochemistry. Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting were used to detect the expression of genes and proteins in the Wnt/β-catenin signaling pathway. Further studies were performed in vitro using HK-2 cells treated with TGF-β1. At 28 days postoperation, the levels of BUN and Scr expression were significantly increased in the UUO group. SKI and benazepril reduced the levels of BUN and Scr, which displayed protective renal effects. Pathological staining showed that compared with the sham operation group, the renal parenchymal structure was severely damaged, the number of glomeruli was reduced, and a large amount of collagen was deposited in the kidney tissue of the UUO group. SKI treatment reduced morphological changes. Immunohistochemistry showed that compared with the sham operation group, the content of collagen I and FN in the kidney tissue of the UUO group were significantly increased, whereas the SKI content was decreased. In addition, compared with the UUO group, the levels of Wnt1, active β-catenin, Snail1, and PAI-1 expression were reduced in the SKI group, suggesting that SKI may reduce renal fibrosis by mediating the Wnt/β-catenin pathway. Further in vitro studies showed that collagen I, FN, and α-SMA levels in HK-2 cells were significantly increased following stimulation with TGF-β1. SKI could significantly reduce the expression of collagen I, FN, and α-SMA. A scratch test showed that SKI could reduce HK-2 migration. In addition, by stimulating TGF-β1, the levels of Wnt1, active β-catenin, snail1, and PAI-1 were significantly upregulated. SKI treatment could inhibit the activity of the Wnt/β-catenin signaling pathway in HK-2 cells. SKI improves kidney function by inhibiting renal fibrosis. The anti-fibrotic effects may be mediated by regulation of the Wnt/β-catenin pathway and EMT inhibition.

Polymethoxyflavone-rich Fraction from Citrus sunki Leaves Alleviates Renal Dysfunction in Mice with Unilateral Ureteral Obstruction

Polymethoxyflavones (PMFs) are flavonoid compounds present in citrus plants that are proposed to be advantageous to human health. However, the advantageous effects of PMFs in the context of renal dysfunction are unclear. In this study, we made a PMF-rich fraction (PRF) from the leaves of Citrus sunki Hort ex. Tanaka and identified its components using liquid chromatography and mass spectrometry. We then investigated the effect of PRF—comprising 9 types of PMF—on renal dysfunction induced by unilateral ureteral obstruction (UUO) in mice. Animals were divided into four experimental groups ( n = 7 per group): I) sham-operated group (Sham); II) UUO group (UUO); III) UUO + Enalapril 0.1 mg/1 mL (UUO + Enap); IV) UUO + PRF 100 mg/kg/day (UUO + PRF). All mice were orally administered with the drugs once a day from 7 days before UUO to 1 week after UUO. After the experiments were over, serum and tissues were taken for biochemical and histological analysis. PRF promoted the recovery of body weight in the background of UUO. Biochemical and histological analysis revealed that PRF ameliorated UUO-induced renal dysfunction and moderately reversed inflammation and tubulointerstitial fibrosis. Further, PRF inhibited the expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), transforming growth factor-β (TGF-β), collagen I (Col-I), and collagen IV (Col-IV). These results suggest that PRF improves UUO-induced renal dysfunction by regulating the expression of inflammatory and fibrotic response-related genes.

FuZhengHuaYuJiangZhuTongLuoFang Prescription Modulates Gut Microbiota and Gut-Derived Metabolites in UUO Rats.

BackgroundAlteration of intestinal flora and metabolites is closely related to chronic kidney disease (CKD) across early to advanced stages. FuZhengHuaYuJiangZhuTongLuoFang prescription (FZHY) is a Chinese herb that has been proven to effectively treat CKD, but the underlying mechanism is not clear.MethodsRats were subjected to intragastric treatment with FZHY 7, 14, and 21 days after unilateral ureteral obstruction (UUO) surgery, and kidney tissue, colon tissue, serum, and stool samples were collected.ResultsFZHY treatment effectively ameliorated UUO-induced renal function loss, renal injury and renal fibrosis, and colon tissue damage and fibrosis on day 7. The results of 16S flora analysis (day 7) showed that, compared with the UUO group, both the FZHY group and the sham group showed decreased levels of g_Monoglobus, g_Papillibacter, g_Eubacterium_nodatum, and g_Family_XIII_AD3011. Additionally, FZHY obviously induced the reduction of serum citrulline, glycoursodeoxycholic acid, 23-nordeoxycholic acid, 7-ketodeoxycholic acid, kahweol, lipoid B4, 4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-2-methyl-1,3-thiazole, taurolithocholic acid sodium salt, indoline-2-carboxylic acid, 5(S),15(S)-diHETE, and others and the increase of bilirubin, asparagine, and others, which were positively associated with the above four candidate bacteria. Moreover, FZHY increased the levels of ZO-1, occludin, and claudin-1 in the colonic mucosa and reduced the levels of CRP, TNF-α, IL-6, and IL-1 in the serum and LN, FN, Col-I, and Col-III in the tubulointerstitium of UUO rats on day 7.ConclusionOur study revealed that FZHY reduced kidney damage at the early stage of CKD by regulating the above four candidate bacteria biomarkers and gut-derived harmful metabolites, inhibiting the inflammation response and tubulointerstitial fibrosis, providing deep insight into CKD therapeutic strategy.

DNMT3a negatively regulates PTEN to activate the PI3K/AKT pathway to aggravate renal fibrosis

BackgroundRenal fibrosis has become one of the major diseases threatening global public health and harming human life and health. PTEN methylation plays an important role in fibrotic diseases of many organs. However, the relationship between PTEN methylation and renal fibrosis is still elusive. MethodsIn the present study, we established a unilateral ureteral obstruction (UUO) mouse model in vivo and a transforming growth factor β1 (TGF-β1)-stimulated renal tubular epithelial cell (HK−2) model in vitro. The degree of renal interstitial fibrosis was detected by haematoxylin-eosin (HE) staining and Masson's trichrome staining. Western blot (WB), qRT–PCR, immunohistochemistry (IHC) and methylation-specific PCR (MSP) analyses were used to determine the mechanism by which PTEN methylation regulates renal fibrosis. The α-SMA fibrosis marker was detected by immunofluorescence (IF). Additionally, the relationship of PTEN and DNMT3a in UUO was determined by ChIP-qRT–PCR. ResultsOur results showed that the promoter region of PTEN was methylated in UUO. Compared to the sham group, the expression of PTEN was significantly reduced in the UUO group. However, the demethylation reagent significantly inhibited epithelial-mesenchymal transition (EMT), which showed increased expression of E-cadherin and decreased expression of α-SMA and fibronectin. Moreover, treatment of HK-2 cells with 5-aza-dc reversed the activation of the TGF-β1-induced PI3K/AKT signalling pathway, which inhibited renal fibrosis. WB analysis demonstrated that TGF-β1 inhibited the PTEN protein expression level and DNMT3a knockdown reversed the inhibitory effect of TGF-β1 on PTEN expression. Furthermore, ChIP-qRT–PCR showed that DNMT3a interacted with PTEN. Finally, we found that DNMT3a negatively regulated PTEN to activate the PI3K/AKT signalling pathway and aggravate renal fibrosis in vitro and in vivo. ConclusionIn summary, these results indicated that renal fibrosis is related to the downregulation of PTEN. Additionally, DNMT3a negatively regulates PTEN to activate the PI3K/AKT signalling pathway and induce EMT in renal tubular epithelial cells, thereby aggravating renal fibrosis.

MO289: IL33 Exerts Toxicity in the Heart as Secreted by Renal Inflammation

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is a major cardiovascular risk factor, regardless of the severity or the origin of the AKI. Among this risk, AKI imparts an array of signals and long-term physiologic alterations that can precipitate heart insufficiency and hypertrophy. However, few direct molecular mechanisms have been identified that show how AKI leads to cardiac disease and maladaptation. The aims of this project are to assess the role of IL33 in remote cardiac damage after AKI. METHOD AKI was induced in mice by ischemia–reperfusion injury of both right and left kidneys by 30min of reversible artery occlusion. To examine the role of renal inflammation more specifically, we used the unilateral ureteral obstruction (UUO) model, which does not cause acute injury. For UUO, an incision was made in the lower left quadrant of the abdomen and the left ureter was isolated and ligated permanently. Mice were treated to manipulate select signaling pathways with either Adenovirus-associated virus 9 (AAV9) or recombinant proteins. We generated AAV9 carrying the IL33 gene or an empty vector (EV) under a CMV promoter and injected 1012 viral particles intrathoracically in 6 to 9 days old pups. Recombinant IL33 (rIL33) or vehicle were injected intraperitoneally in IL33 KO adult mice after AKI (at D0, D1 and D2). Echocardiography was performed either at baseline or after 28 days of surgery. Hearts were fixed in 4% paraformaldehyde overnight and paraffin-embedded for fibrosis (picrosirius staining) and cardiomyocyte area measurement (WGA staining and ImageJ software). IL33 citrine reporter mice (IL33cit) in the heterozygous state were used to show IL33 expression. RESULTS Our data showed that UUO and AKI were strong inducers of cardiac dysfunction after 28 days in adult mice, measured with echocardiography (mean ejectional fraction (EF): 58, 46, 47% in Sham, AKI and UUO group respectively, P &amp;lt; .05). Cardiac pathology was also present such as increased fibrosis in hearts from WT mice after AKI compared with Sham (2.3%, 4.5%, 8.3% of fibrosis area in Sham, AKI and UUO respectively, P &amp;lt; 0.05) and an increased mean cardiomyocyte area indicative of hypertrophy (333, 597, 1246 µm2 in Sham, AKI and UUO respectively, P &amp;lt; .05). However, cardiac function and architecture were preserved either after 28 days of AKI or UUO in IL33 KO mice. In parallel, WT mice injected with AAV9-CMV-IL33 showed impaired cardiac function compared with mice injected with a control AAV9-empty vector after 8 weeks (EF 58% versus 42% in AAV-EV and AAV-IL33 respectively, P &amp;lt; .05). After AKI, AAV9-IL33 mice had even more impaired cardiac function compared to AAV-empty control mice (EF 42% versus 35% in AAV-EV and AAV-IL33 respectively). In IL33 KO mice, the injection of 1 µg of rIL33 in the early days of AKI restored cardiac dysfunction and fibrosis after 28 days of AKI (EF 59 versus 43% in Sham and AKI respectively, P &amp;lt; .05), yet vehicle injected mice were still protected. IL33 expression viewed with the Citrine reporter mice showed that it was locally downregulated in the heart after AKI, which was strictly localized in pericytes in the heart (PDGFRß+ cells). ELISA for protein levels of IL33 confirmed the reduction of this cytokine in hearts from WT AKI and UUO mice after 28 days compared with Sham (29.6, 4.2 and 2.7 fg of IL33/µg of protein in Sham, AKI and UUO hearts respectively, P &amp;lt; .05). CONCLUSION IL33 seems to exert toxicity in the heart as secreted by renal inflammation, which is against some previous reports in the literature where it was suggested to be a protective factor to the heart (whether released from the kidney or from within the heart).

MO444: Quercetin, a Novel Exosomes Inhibitor, Alleviates Renal Tubulointerstitial Inflammation and Fibrosis

Abstract BACKGROUND AND AIMS Tubulointerstitial inflammation (TI) and tubulointerstitial fibrosis (TIF) are the common pathological manifestations of all progressive chronic kidney diseases (CKD). Exosomes-mediated cellular communication between tubular epithelial cells (TECs) and macrophages play an important role in the development of TI and TIF. Exosomes from injured kidneys of CKD mice are increased, while inhibition of exosomes secretion prevents renal fibrosis. Hsp90 is known to promote release of exosomes through Skip/Hop. Here, the protective effects against kidney TI and TIF of quercetin, a novel exosomes inhibitor, were studied. METHOD For unilateral ureteral obstruction (UUO), male C57BL/6J mice were subjected to a double-ligating right ureter following a back incision. The UUO mice were randomly classified into four groups: UUO 7d, UUO 7d + Q, UUO 14d, UUO 14d + Q. Quercetin was intra-gastrically administered at a dose of 50 mg/kg daily. For LPS model, mice were administered intraperitoneally with 200μg LPS. The LPS mice were randomly divided into two groups: LPS group, LPS + Q group. The mice within LPS + Q group were intragastrically treated with Quercetin at a dose of 50 mg/kg daily. Mice were euthanized at Day 3 post-LPS injury. In vitro, TECs were treated with LPS or LPS + Quercetin. TECs were co-cultured with Raw264.7 cells in transwell chambers. After 24 h incubation, exosomes from DiI-labeled TECs were released on the other side and were absorbed by macrophages, which were fixed, stained and observed. To prepare TEC-exos, TECs were cultured in 10% FBS in medium for 24 h. And then cells were maintained for 48 h in a medium with 10% exosomes-deprived FBS for exosomes extraction. TEC-exos were used to interfere with macrophages. To investigate the role of Hsp70 and Hsp90 in exosomes generation and secretion, small interfering RNA (siRNAs) and a plasmid containing Hsp70 or Hsp90 expression gene were used to transfect TECs. RESULTS In UUO and LPS mice model, quercetin inhibited the inflammation and fibrosis of the kidney, as illustrated by decreased expression of IL-18, IL-1β and TNF-α in qPCR, reduction of fibrosis area in Masson staining and inhibited expression of collagen Ⅰ and fibronectin in qPCR. The expressions of Hsp70 and Hsp90 were significantly higher in the UUO group and LPS group than in the vehicle group. With the administration of quercetin, the expression of Hsp70 and Hsp90 showed a marked decline. Renal exosomes particles, and expression of exosomes markers (Alix, CD63), were upregulated in UUO and LPS mice models and downregulated in quercetin-treated groups. In vitro, quercetin abolished Hsp70 and Hsp90 upregulation induced by LPS in TECs. Quercetin also reduced LPS-induced increase in exosomes secretion of TECs. Exosomes secretion of TECs was reduced by silencing Hsp70 or Hsp90 by siRNA, while increased by overexpressing Hsp70 or Hsp90 by the plasmid. CONCLUSION We provided evidence that quercetin, an easily available and nontoxic agent, could be applied as a new therapeutic agent for the treatment of TI and TIF via inhibiting the generation and secretion of exosomes.