Unilateral Ureteral Obstruction Model Research Articles

N1F(Improved-Nephropathy 1 Formula) Ameliorates Renal Interstitial Fibrosis via Inhibiting Extracellular Matrix Deposition and Regulating the FGF23/P38MAPK/Wnt Pathway.

Renal fibrosis is the primary pathway in the progression of chronic kidney disease (CKD) towards end-stage renal failure. The currently used drugs currently are ineffective, and their mechanisms of action remain unclear. This study aims to investigate the nephroprotective effect of Improved-Nephropathy 1 Formula (N1F) in a rat model of unilateral ureteral obstruction (UUO) and explore the potential mechanisms of N1F-containing serum in treating TGF-ß1-induced human renal tubular epithelial cells (HK-2). SD rats received 2-week continuous N1F gavage starting on day 2 after UUO. HK-2 cells were pretreated with a P38MAPK inhibitor for 1 h in vitro, followed by induction of the cells with TGF-ß1 and treatment with N1F 48 h later. The chemical composition of N1F was analyzed using high-performance liquid chromatography-Q-Orbitrap high-resolution liquid mass spectrometry. Renal function was assessed by measuring serum creatinine (Scr), blood urea nitrogen (BUN) and urine protein (Upro) levels. Hematoxylin and eosin (HE) and Masson's trichrome (Masson) staining were used to evaluate the extent of renal tissue damage and fibrosis. Western blotting, immunohistochemistry, and immunofluorescence were used to detect the protein levels of relevant indices. The RNA levels of the relevant indices were detected using real-time fluorescence quantitative PCR (RT-qPCR). We identified 361 chemical components in the water extract of N1F. These chemical components of N1F significantly reduced the area associated with interstitial fibrosis in the kidneys of UUO rats and the levels of serum creatinine, urea nitrogen, and urinary protein. Additionally, N1F decreased the protein levels of FGF23, Wnt1, ß-catenin and p-P38MAPK/P38MAPK, along with the expression of renalfibrosis-associated proteins, α-SMA, FN, Collagen III, and Vimentin in the renal tissues of the UUO rats, while enhancing klotho and DKK1 protein levels. In vitro experiments revealed that inhibition of P38MAPK signaling significantly suppressed the expression of proteins related to the Wnt signaling pathway, with a concomitant decrease in the expression of FGF23 and an increase in the expression of Klotho. Notably, the P38MAPK inhibitor (SB203580) had similar effects to N1F in altering the above-mentioned indices in vitro. N1F may exhibit potential therapeutic efficacy against renal fibrosis by inhibiting the FGF23/P38MAPK/Wnt signaling pathway, consequently inhibiting extracellular matrix deposition due to renal injury.

Up-regulation of long non-coding RNA H19 ameliorates renal tubulointerstitial fibrosis by reducing lipid deposition and inflammatory response through regulation of the microRNA-130a-3p/long-chain acyl-CoA synthetase 1 axis

Long non-coding RNA (lncRNA) H19 is an extensively studied lncRNA that is related to numerous pathological changes. Our previous findings have documented that serum lncRNA H19 levels are decreased in patients with chronic kidney disorder and lncRNA H19 reduction is closely correlated with renal tubulointerstitial fibrosis, an essential step in developing end-stage kidney disease. Nonetheless, the precise function and mechanism of lncRNA H19 in renal tubulointerstitial fibrosis are not fully comprehended. The present work utilized a mouse model of unilateral ureteral obstruction (UUO) and transforming growth factor-β1 (TGF-β1)-stimulated HK-2 cells to investigate the possible role and mechanism of lncRNA H19 in renal tubulointerstitial fibrosis were investigated. Levels of lncRNA H19 decreased in kidneys of mice with UUO and HK-2 cells stimulated with TGF-β1. Up-regulation of lncRNA H19 in mouse kidneys remarkably relieved kidney injury, fibrosis and inflammation triggered by UUO. Moreover, the increase of lncRNA H19 in HK-2 cells reduced epithelial-to-mesenchymal transition (EMT) induced by TGF-β1. Notably, up-regulation of lncRNA H19 reduced lipid accumulation and triacylglycerol content in kidneys of mice with UUO and TGF-β1-stimulated HK-2 cells, accompanied by the up-regulation of long-chain acyl-CoA synthetase 1 (ACSL1). lncRNA H19 was identified as a sponge of microRNA-130a-3p, through which lncRNA H19 modulates the expression of ACSL1. The overexpression of microRNA-130a-3p reversed the lncRNA H19-induced increases in the expression of ACSL1. The suppressive effects of lncRNA H19 overexpression on the EMT, inflammation and lipid accumulation in HK-2 cells were diminished by ACSL1 silencing or microRNA-130a-3p overexpression. Overall, the findings showed that lncRNA H19 ameliorated renal tubulointerstitial fibrosis by reducing lipid deposition via modulation of the microRNA-130a-3p/ACSL1 axis.

Iguratimod prevents renal fibrosis in unilateral ureteral obstruction model mice by suppressing M2 macrophage infiltration and macrophage–myofibroblast transition

Iguratimod is a novel synthetic, small-molecule immunosuppressive agent used to treat rheumatoid arthritis. Through ongoing exploration of its role and mechanisms of action, iguratimod has been observed to have antifibrotic effects in the lung and skin; however, its effect on renal fibrosis remains unknown. This study aimed to investigate whether iguratimod could affect renal fibrosis progression. Three different concentrations of iguratimod (30 mg/kg/day, 10 mg/kg/day, and 3 mg/kg/day) were used to intervene in unilateral ureteral obstruction (UUO) model mice. Iguratimod at 10 mg/kg/day was observed to be effective in slowing UUO-mediated renal fibrosis. In addition, stimulating bone marrow-derived macrophages with IL-4 and/or iguratimod, or with TGF-β and iguratimod or SRC inhibitors in vitro, suggested that iguratimod mitigates the progression of renal fibrosis in UUO mice, at least in part, by inhibiting the IL-4/STAT6 signaling pathway to attenuate renal M2 macrophage infiltration, as well as by impeding SRC activation to reduce macrophage–myofibroblast transition. These findings reveal the potential of iguratimod as a treatment for renal disease.

CD226 promotes renal fibrosis by regulating macrophage activation and migration.

It has been found that CD226 plays an important role in regulating macrophage function, but its expression and function in macrophages during renal fibrogenesis have not been studied. Our data demonstrated that CD226 expression in macrophages was obviously upregulated in the unilateral ureteral obstruction model, while CD226 deficiency attenuated collagen deposition in renal interstitium along with fewer M1 within renal cortex and renal medulla and a lower level of proinflammatory factors compared to that of control littermates. Further studies demonstrated that Cd226-/- bone marrow-derived macrophages transferring could significantly reduce the tubular injury, collagen deposition, and proinflammatory cytokine secretion compared with that of Cd226+/+ bone marrow-derived macrophages transferring in the unilateral ureteral obstruction model. Mechanistic investigations revealed that CD226 promoted proinflammatory M1 macrophage accumulation in the kidney via suppressing KLF4 expression in macrophages. Therefore, our results uncovered a pathogenic role of CD226 during the development of chronic kidney disease by promoting monocyte infiltration from peripheral blood into the kidney and enhancing macrophage activation toward the inflammatory phenotype by suppressing KLF4 expression.

Exosomes From Human Umbilical Cord Stem Cells Suppress Macrophage-to-myofibroblast Transition, Alleviating Renal Fibrosis.

Renal fibrosis, a progressive scarring of the kidney, lacks effective treatment. Human umbilical cord mesenchymal stem cell-derived exosomes (HucMSC-Exos) hold promise for treating kidney diseases due to their anti-inflammatory properties. This study investigates their potential to lessen renal fibrosis by targeting macrophage-to-myofibroblast transformation (MMT), a key driver of fibrosis.We employed a mouse model of unilateral ureteral obstruction (UUO) and cultured cells exposed to transforming growth factor-β (TGF-β) to mimic MMT.HucMSC-Exos were administered to UUO mice, and their effects on kidney function and fibrosis were assessed. Additionally, RNA sequencing and cellular analysis were performed to elucidate the mechanisms by which HucMSC-Exos inhibit MMT. HucMSC-Exos treatment significantly reduced kidney damage and fibrosis in UUO mice. They downregulated markers of fibrosis (Collagen I, vimentin, alpha-smooth muscle actin) and suppressed MMT (α-SMA + F4/80 + cells). Furthermore, ARNTL, a specific molecule, emerged as a potential target of HucMSC-Exos in hindering MMT and consequently preventing fibrosis.HucMSC-Exos effectively lessen renal fibrosis by suppressing MMT, suggesting a novel therapeutic strategy for managing kidney damage and fibrosis.

Renoprotective effect of diacerein in rats with partial unilateral ureteral obstruction model

Abstract Objectives We aimed to analyze the effects of diacerein in a rat model of partial unilateral ureteral obstruction (PUUO). Methods We randomly divided 24 female rats into three groups. Control group, PUUO group and PUUO + diacerein group. The PUUO group was subjected to the PUUO model for seven days. The PUUO + diacerein group received oral diacerein (80 mg/kg) for seven days. Spectrophotometric methods were employed to measure oxidative stress parameters, including malondialdehyde (MDA), protein carbonyl (PC) and antioxidant enzyme levels, including glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD), while indicators of renal function, such as kidney injury molecule-1 (KIM-1) and neutrophil gelatinous-associated lipocalin (NGAL), along with inflammatory parameters interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6), were assessed using the ELISA method. Inflammatory parameters were measured in blood samples, and other parameters were analyzed in kidney tissue. Hematoxylin-eosin method examinations were used for histological analyses. Results IL-1beta, TNF-alpha, and IL-6 levels were found to be significantly decreased in the PUUO + diacerein group compared to the PUUO group (p=0.006, p=0.002 and p=0.001, respectively). In the PUUO + diacerein group, GSH-Px and SOD activities increased compared with the PUUO group (p=0.031 and p=0.037, respectively). We also observed a significant improvement in renal function parameters, such as KIM-1 and NGAL levels in the PUUO + diacerein group compared to PUUO (p=0.002 and p=0.012, respectively). The PC and MDA levels were highest in the PUUO group (p<0.001). Similarly, the histopathologic tissue damage was the most prominent PUUO group (p<0.001). Conclusions Our study found that diacerein is a highly effective pharmacologic agent in alleviating oxidative damage in PUUO model rats.

Modulation of Stem Cell Factor by Forskolin Inhibits the Progression of Tubule Interstitial Fibrosis

Purpose: Forskolin is primarily found in the roots of the Coleus forskohlii plant, historically employed in Southeast Asian and Indian Ayurvedic medicine. Mast cells play a crucial role in fibrosis progression, yet their activation and inhibition in animal models are understudied; thus, we explored forskolin’s impact on kidney fibrosis. Materials and Methods: Forskolin was evaluated in a mouse model for stem cell factor-induced histamine release, and plasma histamine levels were measured using the enzyme-linked immunosorbent assay. Kidney fibrosis was developed by unilateral ureteral obstruction (UUO). Renal function was assessed by spectrophotometric measurements of serum blood urea nitrogen (BUN) and creatinine. The gene expression of collagen, transforming growth factor-beta (TGF-β), α-smooth muscle actin (α-SMA), interleukin-1 beta (IL-1β), and mast cell protease-5 (MCPT-5) in the kidney was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Histopathological changes in the renal tissues were examined by hematoxylin and eosin (H&E) and Masson’s trichome stain. Results: Our results showed that 3 mg/kg forskolin inhibited SCF-induced plasma histamine release in a mouse model. In the 7-day UUO model, forskolin significantly showed inhibition of serum creatinine and blood urea nitrogen compared with the disease group. Forskolin significantly inhibited elevated expression of collagen, TGF-β, α-SMA, IL-1β, and MCPT-5 in the kidneys. Histopathological observation of H&E and Masson trichome-stained kidney forskolin demonstrated a reduction in inflammatory cells, pelvic and tubular dilation, and fibrosis. Conclusion: Forskolin showed an anti-fibrotic effect in UUO-induced renal fibrotic mice. Most significantly, forskolin administration showed a decrease in the expression of the mast cell protease MCPT5 in the kidneys. These results imply that forskolin, through modifying SCF activity, may be a viable potential treatment for the attenuation of tubule-interstitial fibrosis.

Atg5 deficiency in macrophages protects against kidney fibrosis via the CCR6-CCL20 axis

BackgroundAutophagy is a lysosome-dependent degradation pathway that regulates macrophage activation, differentiation, and polarization. Autophagy related 5 (Atg5) is a key protein involved in phagocytic membrane elongation in autophagic vesicles that forms a complex with Atg12 and Atg16L1. Alterations in Atg5 are related to both acute and chronic kidney diseases in experimental models. However, the role of macrophage-expressed Atg5 in acute kidney injury remains unclear.MethodsUsing a myeloid cell-specific Atg5 knockout (MΦ atg5−/−) mouse, we established renal ischemia/reperfusion and unilateral ureteral obstruction models to evaluate the role of macrophage Atg5 in renal macrophage migration and fibrosis.ResultsBased on changes in the serum urea nitrogen and creatinine levels, Atg5 deletion had a minimal effect on renal function in the early stages after mild injury; however, MΦ atg5−/− mice had reduced renal fibrosis and reduced macrophage recruitment after 4 weeks of ischemia/reperfusion injury and 2 weeks of unilateral ureteral obstruction injury. Atg5 deficiency impaired the CCL20-CCR6 axis after severe ischemic kidneys. Chemotactic responses of bone marrow-derived monocytes (BMDMs) from MΦ atg5−/− mice to CCL20 were significantly attenuated compared with those of wild-type BMDMs, and this might be caused by the inhibition of PI3K, AKT, and ERK1/2 activation.ConclusionsOur data indicate that Atg5 deficiency decreased macrophage migration by impairing the CCL20-CCR6 axis and inhibited M2 polarization, thereby improving kidney fibrosis.

Secreted frizzled-related protein 5 protects against renal fibrosis by inhibiting Wnt/β-catenin pathway.

Renal fibrosis (RF) is an important pathogenesis for renal function deterioration in chronic kidney disease. Secreted frizzled-related protein 5 (SFRP5) is an anti-fibrotic adipokine but its direct role on RF remains unknown. It was aimed to study the protective effect of SFRP5 against RF and interference with Wnt/β-catenin signaling pathway for the first time. First, the therapeutic efficacy of SFRP5 was evaluated by adenovirus overexpression in rats with unilateral ureteral obstruction (UUO) in vivo. Thirty-six rats were randomly divided into the sham, UUO, and SFRP5 (UUO + Ad-SFRP5) groups. Half rats in each group were selected at random for euthanasia at 7 days and the others until 14 days. Then, the transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) was established in HK-2 cells in vitro. The cells were divided into four groups: the control group, the TGF-β1 group, the TGF-β1 + SFRP5 group, and the TGF-β1 + SFRP5 + anti-SFRP5 group. The makers of EMT and Wnt/β-catenin pathway proteins were investigated. In the UUO model, expression of SFRP5 showed compensatory upregulation, and adenoviral-mediated SFRP5 over-expression remarkably attenuated RF, as demonstrated by maintenance of E-cadherin and suppression of α-smooth muscle actin (SMA). In vitro, SFRP5 was shown to inhibit TGF-β1-mediated positive regulation of α-SMA, fibronectin, collagen I but negative regulation of E-cadherin. Furthermore, SFRP5 abrogated activation of Wnt/β-catenin, which was the essential pathway in EMT and RF pathogenesis. The changes after a neutralizing antibody to SFRP5 confirmed the specificity of SFRP5 for inhibition. These findings suggest that SFRP5 can directly ameliorate EMT and protect against RF by inhibiting Wnt/β-catenin pathway.

Pyxinol Fatty Acid Ester Derivatives J16 against AKI by Selectively Promoting M1 Transition to M2c Macrophages.

Acute kidney injury (AKI) is a common, multicause clinical condition that, if ignored, often progresses to chronic kidney disease (CKD) and end-stage kidney disease, with a mortality rate of 40-50%. However, there is a lack of universal treatment for AKI. Inflammation is the basic pathological change of early kidney injury, and inflammation can exacerbate AKI. Macrophages are the primary immune cells involved in the inflammatory microenvironment of kidney disease. Therefore, regulating the function of macrophages is a crucial breakthrough for the AKI intervention. Our team chemically modified pyxinol, an ocotillol-type ginsenoside, to prepare PJ16 with higher solubility and bioavailability. In vitro, using a model of macrophages stimulated by LPS, it was found that PJ16 could regulate macrophage function, including inhibiting the secretion of inflammatory factors, promoting phagocytosis, inhibiting M1 macrophages, and promoting M1 transition to the M2c macrophage. Further investigation revealed that PJ16 may shield renal tubular epithelial cells (HK-2) damaged by LPS in vitro. Based on this, PJ16 was validated in the animal model of unilateral ureteral obstruction, which showed that it improves renal function and inhibits renal tissue fibrosis by decreasing inflammatory responses, reducing macrophage inflammatory infiltration, and preferentially upregulating M2c macrophages. In conclusion, our study is the first to show that PJ16 resists AKI and fibrosis by mechanistically regulating macrophage function by modulating the phenotypic transition from M1 to M2 macrophages, mainly M2c macrophages.

RfxCas13d-mediated inhibition of Circ1647 alleviates renal fibrosis via PI3K/AKT signaling pathway

Background Circular RNAs (CircRNAs) have been shown to be involved in the development of chronic kidney disease (CKD). This study aimed to investigate the role of Circ1647 in renal fibrosis, which is a hallmark of CKD. Methods In this study, we established a unilateral ureteral obstruction (UUO) model and delivered Circ1647 RfxCas13d knockdown plasmid into renal parenchymal cells via retrograde injection through the ureter followed by electroporation. After that, the pathological changes were determined by Hematoxylin and Eosin. Meanwhile, Immunohistochemistry, qRT-PCR and Western blot were conducted to assess the degree of fibrosis. In addition, overexpressing of Circ1647 in renal tubular epithelial cells (TCMK1) was performed to investigate the underlying mechanisms of Circ1647. Results Our results displayed that electroporation-mediated knockdown of Circ1647 by RfxCas13d knockdown plasmid significantly inhibited renal fibrosis in UUO mice as evidenced by reduced expression of fibronectin and α-SMA (alpha-smooth muscle actin). Conversely, overexpression of Circ1647 in TCMK1 cells promoted the fibrosis. In terms of mechanism, Circ1647 may mediate the PI3K/AKT Signaling Pathway as demonstrated by the balance of the phosphorylation of PI3K and AKT in vivo and the aggravated phosphorylation of PI3K and AKT in vitro. These observations were corroborated by the effects of the PI3K inhibitor LY294002, which mitigated fibrosis post Circ1647 overexpression. Conclusion Our study suggests that Circ1647 plays a significant role in renal fibrosis by mediating the PI3K/AKT signaling pathway. RfxCas13d-mediated inhibition of Circ1647 may serve as a therapeutic target for renal fibrosis in CKD.

Kaempferitrin attenuates unilateral ureteral obstruction-induced renal inflammation and fibrosis in mice by inhibiting NOX4-mediated tubular ferroptosis.

Tubular ferroptosis significantly contributes to renal inflammation and fibrosis, critical factors in chronic kidney disease (CKD). This study aims to investigate Kaempferitrin, a potent flavonoid glycoside from Bauhinia forficata leaves, renowned for its anti-inflammatory and antitumor effects, and to elucidate its potential mechanisms in mitigating inflammation and fibrosis induced by tubular ferroptosis. The study investigated Kaempferitrin's impact on tubular ferroptosis using a unilateral ureteral obstruction (UUO) model-induced renal inflammation and fibrosis. In vitro, erastin-induced ferroptosis in primary tubular epithelial cells (TECs) was utilized to further explore Kaempferitrin's effects. Additionally, NADPH oxidase 4 (NOX4) transfection in TECs and cellular thermal shift assay (CETSA) were conducted to identify Kaempferitrin's target protein. Kaempferitrin effectively improved renal function, indicated by reduced serum creatinine and blood urea nitrogen levels. In the UUO model, it significantly reduced tubular necrosis, inflammation, and fibrosis. Its renoprotective effects were linked to ferroptosis inhibition, evidenced by decreased iron, 4-hydroxynonenal (4-HNE), and malondialdehyde (MDA) levels, and increased glutathione (GSH). Kaempferitrin also normalized glutathione peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11(SLC7A11) expression, critical ferroptosis mediators. In vitro, it protected TECs from ferroptosis and consistently suppressed NOX4 expression. NOX4 transfection negated Kaempferitrin's antiferroptosis effects, while CETSA confirmed Kaempferitrin-NOX4 interaction. Kaempferitrin shows promise as a nephroprotective agent by inhibiting NOX4-mediated ferroptosis in tubular cells, offering potential therapeutic value for CKD.

FTO-mediated m6A mRNA demethylation aggravates renal fibrosis by targeting RUNX1 and further enhancing PI3K/AKT pathway.

Chronic kidney disease (CKD) is a global health burden, with ineffective therapies leading to increasing morbidity and mortality. Renal interstitial fibrosis is a common pathway in advanced CKD, resulting in kidney function and structure deterioration. In this study, we investigate the role of FTO-mediated N6-methyladenosine (m6A) and its downstream targets in the pathogenesis of renal fibrosis. M6A modification, a prevalent mRNA internal modification, has been implicated in various organ fibrosis processes. We use a mouse model of unilateral ureteral obstruction (UUO) as an invivo model and treated tubular epithelial cells (TECs) with transforming growth factor (TGF)-β1 as invitro models. Our findings revealed increased FTO expression in UUO mouse model and TGF-β1-treated TECs. By modulating FTO expression through FTO heterozygous mutation mice (FTO+/- ) invivo and small interfering RNA (siRNA) invitro, we observed attenuation of UUO and TGF-β1-induced epithelial-mesenchymal transition (EMT), as evidenced by decreased fibronectin and N-cadherin accumulation and increased E-cadherin levels. Silencing FTO significantly improved UUO and TGF-β1-induced inflammation, apoptosis, and inhibition of autophagy. Further transcriptomic assays identified RUNX1 as a downstream candidate target of FTO. Inhibiting FTO was shown to counteract UUO/TGF-β1-induced RUNX1 elevation invivo and invitro. We demonstrated that FTO signaling contributes to the elevation of RUNX1 by demethylating RUNX1 mRNA and improving its stability. Finally, we revealed that the PI3K/AKT pathway may be activated downstream of the FTO/RUNX1 axis in the pathogenesis of renal fibrosis. In conclusion, identifying small-molecule compounds that target this axis could offer promising therapeutic strategies for treating renal fibrosis.

HIF-1 contributes to autophagy activation via BNIP3 to facilitate renal fibrosis in hypoxia in vitro and UUO in vivo.

The molecular basis of renal interstitial fibrosis, a major pathological feature of progressive kidney diseases, remains poorly understood. Autophagy has been implicated in renal fibrosis, but whether it promotes or inhibits fibrosis remains controversial. Moreover, it is unclear how autophagy is activated and sustained in renal fibrosis. The present study was designed to address these questions using the in vivo mouse model of unilateral ureteral obstruction and the in vitro model of hypoxia in renal tubular cells. Both models showed the activation of hypoxia-inducible factor-1 (HIF-1) and autophagy along with fibrotic changes. Inhibition of autophagy with chloroquine reduced renal fibrosis in unilateral ureteral obstruction model, whereas chloroquine and autophagy-related gene 7 knockdown decreased fibrotic changes in cultured renal proximal tubular cells, supporting a profibrotic role of autophagy. Notably, pharmacological and genetic inhibition of HIF-1 led to the suppression of autophagy and renal fibrosis in these models. Mechanistically, knock down of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a downstream target gene of HIF, decreased autophagy and fibrotic changes during hypoxia in BUMPT cells. Together, these results suggest that HIF-1 may activate autophagy via BNIP3 in renal tubular cells to facilitate the development of renal interstitial fibrosis.NEW & NOTEWORTHY Autophagy has been reported to participate in renal fibrosis, but its role and underlying activation mechanism is unclear. In this study, we report the role of HIF-1 in autophagy activation in models of renal fibrosis and further investigate the underlying mechanism.

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.

Cardiac transcriptomic changes induced by early CKD in mice reveal novel pathways involved in the pathogenesis of Cardiorenal syndrome type 4

BackgroundCardiorenal syndrome (CRS) type 4 is prevalent among the chronic kidney disease (CKD) population, with many patients dying from cardiovascular complications. However, limited data regarding cardiac transcriptional changes induced early by CKD is available. MethodsWe used a murine unilateral ureteral obstruction (UUO) model to evaluate renal damage, cardiac remodeling, and transcriptional regulation at 21 days post-surgery through histological analysis, RT-qPCR, RNA-seq, and bioinformatics. ResultsUUO leads to significant kidney injury, low uremia, and pathological cardiac remodeling, evidenced by increased collagen deposition and smooth muscle alpha-actin 2 expression. RNA-seq analysis identified 76 differentially expressed genes (DEGs) in UUO hearts. Upregulated DEGs were significantly enriched in cell cycle and cell division pathways, immune responses, cardiac repair, inflammation, proliferation, oxidative stress, and apoptosis. Gene Set Enrichment Analysis further revealed mitochondrial oxidative bioenergetic pathways, autophagy, and peroxisomal pathways are downregulated in UUO hearts. Vimentin was also identified as an UUO-upregulated transcript. ConclusionsOur results emphasize the relevance of extensive transcriptional changes, mitochondrial dysfunction, homeostasis deregulation, fatty-acid metabolism alterations, and vimentin upregulation in CRS type 4 development.

Delayed treatment with erythropoietin attenuates renal fibrosis in mouse model of unilateral ureteral obstruction.

Erythropoietin (EPO) exerts tissue-protective effects on various organs including the kidney. However, the effects of EPO on established renal fibrosis remain unclear. In this study, we aimed to examine the therapeutic potential of EPO against established renal fibrosis. Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) and the mice were treated with recombinant human EPO (rhEPO) daily during 7 and 13 days after UUO. The degrees of renal fibrosis, myofibroblast accumulation, and macrophage infiltration; the mRNA expression levels of transforming growth factor (TGF)-β1 and α1(I) collagen; and the protein levels of Kelch-like ECH-associated protein 1 (Keap1) and nuclear NF-E2-related factor 2 (Nrf2) in the kidneys were assessed on day 14 after UUO. Treatment with rhEPO significantly decreased fibrosis, myofibroblast accumulation, and α1(I) collagen mRNA expression, but it did not significantly affect TGF-β1 mRNA expression. Although treatment with rhEPO did not significantly affect the total number of interstitial macrophages, it significantly decreased the number of CD86-positive cells (M1 macrophages), while significantly increased the number of CD206-positive cells (M2 macrophages) in the interstitium. Treatment with rhEPO did not affect the Keap1/Nrf2 protein level or the peripheral blood hematocrit value. These results indicate for the first time that EPO exerts antifibrotic effects against the evolution of established renal fibrosis, possibly by influencing the polarization of infiltrating macrophages.

Urinary miR-185-5p is a biomarker of renal tubulointerstitial fibrosis in IgA nephropathy.

For IgA nephropathy (IgAN), tubular atrophy/interstitial fibrosis is the most important prognostic pathological indicator in the mesangial and endocapillary hypercellularity, segmental sclerosis, interstitial fibrosis/tubular atrophy, and presence of crescents (MEST-C) score. The identification of non-invasive biomarkers for tubular atrophy/interstitial fibrosis would aid clinical monitoring of IgAN progression and improve patient prognosis. The study included 188 patients with primary IgAN in separate confirmation and validation cohorts. The associations of miR-92a-3p, miR-425-5p, and miR-185-5p with renal histopathological lesions and prognosis were explored using Spearman correlation analysis and Kaplan-Meier survival curves. Bioinformatics analysis and dual luciferase experiments were used to identify hub genes for miR-185-5p. The fibrotic phenotypes of tubular epithelial cells were evaluated in vivo and in HK-2 cells. miRNA sequencing and cohort validation revealed that the expression levels of miR-92a-3p, miR-425-5p, and miR-185-5p in urine were significantly increased among patients with IgAN; these levels could predict the extent of tubular atrophy/interstitial fibrosis in such patients. The combination of the three biomarkers resulted in an area under the receiver operating characteristic curve of 0.742. The renal prognosis was significantly worse in the miR-185-5p high expression group than in the low expression group (P=0.003). Renal tissue in situ hybridization, bioinformatics analysis, and dual luciferase experiments confirmed that miR-185-5p affects prognosis in patients with IgAN mainly by influencing expression of the target gene tight junction protein 1 (TJP1) in renal tubular epithelial cells. In vitro experiment revealed that an miR-185-5p mimic could reduce TJP1 expression in HK-2 cells, while increasing the levels of α-smooth muscle actin, fibronectin, collagen I, and collagen III; these changes promoted the transformation of renal tubular epithelial cells to a fibrotic phenotype. An miR-185-5p inhibitor can reverse the fibrotic phenotype in renal tubular epithelial cells. In a unilateral ureteral obstruction model, the inhibition of miR-185-5p expression alleviated tubular atrophy/interstitial fibrosis. Urinary miR-185-5p, a non-invasive biomarker of tubular atrophy/interstitial fibrosis in IgAN, may promote the transformation of renal tubular epithelial cells to a fibrotic phenotype via TJP1.

Highly Sensitive Early Diagnosis of Kidney Damage Using Renal Clearable Zwitterion-Coated Ferrite Nanoprobe via Magnetic Resonance Imaging In Vivo.

Iron oxide nanoprobes exhibit substantial potential in magnetic resonance imaging (MRI) of kidney diseases and can eliminate the nephrotoxicity of gadolinium-based contrast agents (GBCAs). Nevertheless, there is an extreme shortage of highly sensitive and renal clearable iron oxide nanoprobes suitable for early kidney damage detection through MRI. Herein, a renal clearable ultra-small ferrite nanoprobe (UMFNPs@ZDS) is proposed for highly sensitive early diagnosis of kidney damage via structural and functional MRI in vivo for the first time. The nanoprobe comprises a ferrite core coated with a zwitterionic layer, and possesses a high T1 relaxivity (12.52 mm-1 s-1 ), a small hydrodynamic size (6.43nm), remarkable water solubility, excellent biocompatibility, and impressive renal clearable ability. In a rat model of unilateral ureteral obstruction (UUO), the nanoprobe-based MRI can not only accurately visualize the locations of renal injury, but also provide comprehensive functional data including peak value, peak time, relative renal function (RRF), and clearance percentage via MRI. The findings prove the immense potential of ferrite nanoprobes as a superior alternative to GBCAs for the early diagnosis of kidney damage.

Catalpol attenuates EMT by inhibiting Wnt/β-catenin and TGF-β/Smads signaling to alleviate kidney fibrosis

Purpose: To determine the anti-fibrosis effect and underlying mechanism of action of catalpol (CAT) in chronic kidney disease (CKD).
 Methods: Forty (40) rats were randomly divided into a sham group (10 rats) and a unilateral ureteral obstruction (UUO) model group (30  rats) which was further randomly subdivided into three groups of ten (10) rats each: the UUO model group, UUO + CAT low-dose group,  and UUO + CAT high-dose group. HK-2 cells were stimulated with TGF-β1 for in vitro studies. Renal injury and fibrotic lesions were determined by H&E and Masson’s staining. Key proteins of TGF-β/Smads and Wnt/β-catenin signaling pathways involved in epithelial- mesenchymal transition (EMT) were determined by immunohistochemistry, immunofluorescence staining and Western blotting.
 Results: Catalpol downregulated the expression of α-SMA (p < 0.05) and upregulated the expression of E-cadherin (p < 0.05) stimulated  by TGF-β1 and LiCl in HK-2 cells, which is consistent with the role of DKK1 in vitro. CAT ameliorated renal fibrosis and repressed the  expression of key proteins of TGFβ/Smads and Wnt/β-catenin pathways in UUO rats.
 Conclusion: Catalpol inhibits EMT and alleviates  kidney fibrosis by suppressing the hyperactivation of TGF-β/Smad and Wnt/β-catenin signaling pathways. Therefore, CAT is a promising  therapeutic drug for renal fibrosis.