Proliferation Of Tubular Epithelial Cells Research Articles

The role of desmoglein-2 in kidney disease

Desmosomes are multi-protein cell-cell adhesion structures supporting cell stability and mechanical stress resilience of tissues; best described in skin and heart. The kidney is exposed to various mechanical stimuli and stress, yet little is known about kidney desmosomes. In healthy kidneys, we found desmosomal proteins located at the apical-junctional complex in tubular epithelial cells. In four different animal models and patient biopsies with various kidney diseases, desmosomal components were significantly upregulated and partly miss-localized outside of the apical-junctional complexes along the whole lateral tubular epithelial cell membrane. The most upregulated component was desmoglein-2 (Dsg2). Mice with constitutive tubular epithelial cell-specific deletion of Dsg2 developed normally and other desmosomal components were not altered in these mice. When challenged with different types of tubular epithelial cell injury (unilateral ureteral obstruction, ischemia-reperfusion, and 2,8-dihydroxyadenine crystal nephropathy) we found increased tubular epithelial cell apoptosis, proliferation, tubular atrophy, and inflammation compared to wild type mice in all models and time points. In vitro, silencing DSG2 via siRNA weakened cell-cell adhesion in HK-2 cells and increased cell death. Thus, our data show a prominent upregulation of desmosomal components in tubular cells across species and diseases and suggest a protective role of Dsg2 against various injurious stimuli.

Molecular mechanism of Gan-song Yin inhibiting the proliferation of renal tubular epithelial cells by regulating miR-21-5p in adipocyte exosomes

Ethnopharmacological relevanceGan-song Yin is derived from the classic ancient prescription " Gan-song pill " for the treatment of wasting-thirst in Ningxia combined with the characteristic “fragrant medicine”. It is clinically used for the treatment of early renal fibrosis caused by diabetic nephropathy. Previous studies have shown that it has a good effect and great potential in the prevention and treatment of diabetic nephropathy, but its mechanism research is still limited. Aim of the studyTo investigate the mechanism of GSY to improve DN by interfering with miR-21-5p and glycolipid metabolism in adipocyte exosomes using 3T3-L1 and TCMK-1 co-culture system. Materials and methodsThe co-culture system of 3T3-L3 and TCMK-1 was established, the IR model was established, and the stability, lipid drop change, glucose consumption, triglyceride content, cell viability, cell cycle and apoptosis level, protein content and mRNA expression of the IR model were detected. ResultsGSY inhibited 3T3-L1 activity, increased glucose consumption and decreased TG content. Decreased TCMK-1 cell viability, inhibited apoptosis, cell cycle arrest occurred in G0/G1 phase and S phase. Adipocyte IR model and co-culture system were stable within 48 h. After GSY intervention, lipid droplet decomposition and glucose consumption increased. The TG content of adipocytes increased, while the TG content of co-culture system decreased. GSY can regulate the expression of TGF-β1/SMAD signaling pathway protein in IR state. After GSY intervention, the expression of miR-21-5p was increased in 3T3-L1 and Exo cells, and decreased in TCMK-1 cells. ConclusionsGSY can regulate TGF-β1/SMAD signaling pathway through the secretion of miR-21-5p from adipocytes, protect IR TCMK-1, regulate the protein and mRNA expression levels of PPARγ, GLUT4, FABP4, and improve glucose and lipid metabolism.

Ouabain enhances renal cyst growth in a slowly progressive mouse model of autosomal dominant polycystic kidney disease.

Renal cyst progression in autosomal dominant polycystic kidney disease (ADPKD) is highly dependent on agents circulating in blood. We have previously shown, using different in vitro models, that one of these agents is the hormone ouabain. By binding to Na+-K+-ATPase (NKA), ouabain triggers a cascade of signal transduction events that enhance ADPKD cyst progression by stimulating cell proliferation, fluid secretion, and dedifferentiation of the renal tubular epithelial cells. Here, we determined the effects of ouabain in vivo. We show that daily administration of ouabain to Pkd1RC/RC ADPKD mice for 1-5 mo, at physiological levels, augmented kidney cyst area and number compared with saline-injected controls. Also, ouabain favored renal fibrosis; however, renal function was not significantly altered as determined by blood urea nitrogen levels. Ouabain did not have a sex preferential effect, with male and female mice being affected equally. By contrast, ouabain had no significant effect on wild-type mice. In addition, the actions of ouabain on Pkd1RC/RC mice were exacerbated when another mutation that increased the affinity of NKA for ouabain was introduced to the mice (Pkd1RC/RCNKAα1OS/OS mice). Altogether, this work highlights the role of ouabain as a procystogenic factor in the development of ADPKD in vivo, that the ouabain affinity site on NKA is critical for this effect, and that circulating ouabain is an epigenetic factor that worsens the ADPKD phenotype.NEW & NOTEWORTHY This work shows that the hormone ouabain enhances the progression of autosomal dominant polycystic kidney disease (ADPKD) in vivo. Ouabain augments the size and number of renal cysts, the kidney weight to body weight ratio, and kidney fibrosis in an ADPKD mouse model. The Na+-K+-ATPase affinity for ouabain plays a critical role in these effects. In addition, these outcomes are independent of the sex of the mice.

#4976 DIFFERENTIAL ROLES OF REGNASE3 IN RESIDENT MACROPHAGE VERSUS TUBULAR EPITHELIAL CELLS IN THE HEALTHY OR POSTISCHEMIC KIDNEY

Abstract Background and Aims Acute kidney injury (AKI) is a prevalent yet severe condition that occurs in clinical settings. The prognosis for AKI is poor, with many patients progressing to AKD, CKD, and even ESKD especially in those who were admitted to the ICU. RNA-binding proteins (RBPs) are a class of proteins that play a vital role in regulating gene expression and have been implicated in a wide range of diseases. Regnase3 is a member of the Regnase RBPs family and has been shown to promote inflammation by increasing TNF in macrophages. Despite this knowledge, the full extent of the role played by Regnase3 in AKI remains unknown. Therefore, we hypothesized that Regnase3 plays a role in both macrophages and tubular epithelial cells, influencing inflammation and tubular repair in AKI. Method In order to investigate the role of Regnase3 in kidney injury, a series of genetically-modified mice were developed on the C57BL/6J genetic background. These mice included the paired box gene 8 (Pax8)-reverse tetracycline transactivator (rTtA, Pax8rTtA), tetracycline resistance protein (TetO)-Cre (TetOCre), transgenic receptor activator of nuclear factor kappa-Β (Rank)-Cre (RankCre), and Regnase3 floxed (Regnase-3fl/fl) mice. Using these genetically modified mice, we applied a range of experimental animal models to study the effects of Regnase3 on kidney injury, including unilateral kidney ischemia-reperfusion surgery with or without nephrectomy and nephrocalcinosis-related kidney injury. Additionally, we employed in vitro models utilizing primary cells to study Regnase3 on the cellular level. Furthermore, we utilized scRNA-seq and RNA-seq to investigate the underlying mechanisms of Regnase3’s function. Results We conducted a thorough investigation and discovered that the Regnase3 is highly expressed in macrophages located within the kidney after AKI. The scRNA-seq showed the expression Regnase3 positively correlated with the phagocytosis, chemokines, and monocyte maturation. Furthermore, Rank-Regnase3 mice suffered from more inflammation and kidney injury after AKI, characterized by an increase in CCR2 positive leukocyte infiltration. Additionally, in vitro experiments revealed that Regnase3 is involved in modulating macrophage behavior, acting as a regulator of macrophage polarization towards an M1 and M2 phenotype and influencing the capacity of cell migration. These findings indicate that Regnase3 is essential in the migration of macrophages after AKI, which contribute to both early phase inflammation and progression towards AKI- CKD. Next, we aimed to examine the role of Regnase3 in the context of renal tubule injury. We observed that Regnase3 is highly expressed in healthy renal tubules, but its expression is reduced following injury. Through in silico, in vivo and in vitro experiments, we found that Regnase3 controls early apoptosis, cell death, proliferation, and wound healing capability of tubular epithelial cells. These effects contribute to kidney injury in postischemic kidneys. Additionally, we discovered that the deletion of Regnase3 provides protection against ischemic AKI (Figure 1). Mechanistically, Regnase3 targets pre-RNA and modulates alternative splicing by increasing the proportion of targeted skipped exon events and decreasing the probability of targeted retained intron events. Overall, our findings indicate that Regnase3 plays a vital role in tubular epithelial cells in the response to kidney injury by regulating alternative splicing. Conclusion In summary, our findings indicate that Regnase3 contributes to kidney injury in postischemic kidneys. However, the impact of Regnase3 is contingent upon the specific cell lineage in question. The Rank-Regnase3 leads to an exacerbation of kidney injury by increasing macrophage recruitment, whereas the Pax8-Regnase3 leads to an improvement in kidney injury through its effects on cell death and wound healing capability of tubular epithelial cells. Therefore, the role of Regnase3 in kidney injury is dependent on the cell lineage, which ultimately determines the detrimental or beneficial impact on kidney injury.

COL28 promotes proliferation, migration, and EMT of renal tubular epithelial cells

Type XXVIII collagen (COL28) is involved in cancer and lung fibrosis. COL28 polymorphisms and mutations might be involved in kidney fibrosis, but the exact role of COL28 in renal fibrosis is unknown. This study explored the function of COL28 in renal tubular cells by examining the expression of COL28 mRNA and the effects of COL28 overexpression in human tubular cells. COL28 mRNA expression and localization were observed in normal and fibrotic kidney tissues from humans and mice using real-time PCR, western blot, immunofluorescence, and immunohistochemistry. The consequences of COL28 overexpression on cell proliferation, migration, cell polarity, and epithelial-to-mesenchymal transition (EMT) induced by TGF-β1 were examined in human tubular HK-2 cells. COL28 expression was low in human normal renal tissues, mainly observed in the renal tubular epithelial cells and especially in proximal renal tubules. COL28 protein expression in human and mouse obstructive kidney disease was higher than in normal tissues (p < 0.05) and more significant in the UUO2-Week than the UUO1-Week group. The overexpression of COL28 promoted HK-2 cell proliferation and enhanced their migration ability (all p < 0.05). TGF-β1 (10 ng/ml) induced COL28 mRNA expression in HK-2 cells, decreased E-cadherin and increased α-SMA in the COL28-overexpression group compared with controls (p < 0.05). ZO-1 expression decreased while COL6 increased in the COL28-overexpression group compared with controls (p < 0.05). In conclusion, COL28 overexpression promotes the migration and proliferation of renal tubular epithelial cells. The EMT could also be involved. COL28 could be a therapeutic target against renal- fibrotic diseases.

Exosomes from high glucose-treated macrophages promote epithelial–mesenchymal transition of renal tubular epithelial cells via long non-coding RNAs

BackgroundMacrophages contribute to epithelial–mesenchymal transition (EMT) in diabetic nephropathy (DN). Exosomal long non-coding RNAs (lncRNAs) derived from macrophages play a major role in transmitting biological information, whereas related studies on DN are rare. Here we investigated the effects of exosomal lncRNAs from high glucose-treated macrophages on EMT.MethodsHigh glucose-treated macrophage exosomes (HG-exos) were extracted by coprecipitation and stabilized. Then, mouse renal tubular epithelial cells were treated with HG-exos for 24 h. Expression of E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin was detected by western blotting, qPCR, and immunofluorescence. High-throughput sequencing was then applied to analyze the bioinformatics of HG-exos.ResultsHG-exos inhibited the proliferation of tubular epithelial cells. Additionally, HG-exos markedly upregulated α-SMA and fibronectin expression and downregulated E-cadherin expression in tubular epithelial cells, indicating EMT induction. A total of 378 differentially expressed lncRNAs and 674 differentially expressed mRNAs were identified by high-throughput sequencing of HG-exos. Bioinformatics analysis and subsequent qPCR validation suggested 27 lncRNAs were enriched in the EMT-related MAPK pathway. Among them, ENSMUST00000181751.1, XR_001778608.1, and XR_880236.2 showed high homology with humans.ConclusionExosomes from macrophages induce EMT in DN and lncRNAs in exosomes enriched in the MAPK signaling pathway may be the possible mechanism.

Ustiloxin A inhibits proliferation of renal tubular epithelial cells in vitro and induces renal injury in mice by disrupting structure and respiratory function of mitochondria

Recently, we found that Ustiloxin A (UA, a mycotoxin) was widely detected in paddy environment and rice samples from several countries, and was also detected in human urine samples from China. However, the current knowledge about the health risks of UA are limited. In this research, the cytotoxicity of UA in mice renal tubular epithelial cells (mRTECs) was evaluated, and the results indicated that UA arrested cell cycle in G2/M phase via altering cellular morphology and microtubule, and inhibited the proliferation and division of mRTECs. Furthermore, UA could inhibit mitochondrial respiration via binding to the CoQ-binding site in dihydro-orotate dehydrogenase (DHODH) protein, and resulted in mitochondrial damage. These adverse effects of UA on mitochondria might be responsible for the cytotoxicity observed in vitro. In vivo, UA at concentrations that were comparable to the realistic concentrations of human exposure induced renal insufficiency in mice, and this might be associated with the renal mitochondrial damage in mice. However, exposure to UA at those realistic concentrations did not promote the progression from renal insufficiency to renal fibrosis and chronic kidney disease was not observed in mice.

Targeting endogenous kidney regeneration using anti-IL11 therapy in acute and chronic models of kidney disease

The kidney has large regenerative capacity, but this is compromised when kidney damage is excessive and renal tubular epithelial cells (TECs) undergo SNAI1-driven growth arrest. Here we investigate the role of IL11 in TECs, kidney injury and renal repair. IL11 stimulation of TECs induces ERK- and p90RSK-mediated GSK3β inactivation, SNAI1 upregulation and pro-inflammatory gene expression. Mice with acute kidney injury upregulate IL11 in TECs leading to SNAI1 expression and kidney dysfunction, which is not seen in Il11 deleted mice or in mice administered a neutralizing IL11 antibody in either preemptive or treatment modes. In acute kidney injury, anti-TGFβ reduces renal fibrosis but exacerbates inflammation and tubule damage whereas anti-IL11 reduces all pathologies. Mice with TEC-specific deletion of Il11ra1 have reduced pathogenic signaling and are protected from renal injury-induced inflammation, fibrosis, and failure. In a model of chronic kidney disease, anti-IL11 therapy promotes TEC proliferation and parenchymal regeneration, reverses fibroinflammation and restores renal mass and function. These data highlight IL11-induced mesenchymal transition of injured TECs as an important renal pathology and suggest IL11 as a therapeutic target for restoring stalled endogenous regeneration in the diseased kidney.

Fisetin reduces the senescent tubular epithelial cell burden and also inhibits proliferative fibroblasts in murine lupus nephritis.

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease characterized by the involvement of multiple organs. Lupus nephritis (LN) is a major risk factor for overall morbidity and mortality in SLE patients. Hence, designing effective drugs is pivotal for treating individuals with LN. Fisetin plays a senolytic role by specifically eliminating senescent cells, inhibiting cell proliferation, and exerting anti-inflammatory, anti-oxidant, and anti-tumorigenic effects. However, limited research has been conducted on the utility and therapeutic mechanisms of fisetin in chronic inflammation. Similarly, whether the effects of fisetin depend on cell type remains unclear. In this study, we found that LN-prone MRL/lpr mice demonstrated accumulation of Ki-67-positive myofibroblasts and p15INK4B-positive senescent tubular epithelial cells (TECs) that highly expressed transforming growth factor β (TGF-β). TGF-β stimulation induced senescence of NRK-52E renal TECs and proliferation of NRK-49F renal fibroblasts, suggesting that TGF-β promotes senescence and proliferation in a cell type-dependent manner, which is inhibited by fisetin treatment in vitro. Furthermore, fisetin treatment in vivo reduced the number of senescent TECs and myofibroblasts, which attenuated kidney fibrosis, reduced senescence-associated secretory phenotype (SASP) expression, and increased TEC proliferation. These data suggest that the effects of fisetin vary depending on the cell type and may have therapeutic effects in complex and diverse LN pathologies.

404.7: Study on the Regulatory Effect of iPS-Derived Exosomes on the Local Immune Microenvironment in DCD Kidney Transplanted Rats

Introduction: Ischemia reperfusion injury (IRI) of donation After Cardiac death (DCD) transplanted kidney is the key factor of acute renal injury, acute and chronic rejection of renal transplantation and transplanted kidney disease.The latest researches show that mesenchymal stem cells (MSCs) have the function of protecting damaged tissues and organs and strong immune regulation. Induced pluripotent stem (iPS) cells have higher proliferative capacity and exosome secretion potential than MSCs. In addition to the functions of stem cells, exosomes have the unique ability to cross the blood-brain barrier and deliver remotely, which means that exosomes have better advantages in clinical disease treatment compared with the therapeutic potential of stem cells. Therefore, the main purpose of this study is to clarify the role of IPS derived exosomes in protecting the IRI of DCD transplanted kidney and regulating its local immune microenvironment. Methods: The iPS cells were passaged to the third passage and massively expanded. After 48 hours of culture, 100 mL of cell culture supernatant was collected and exosomes were extracted from the supernatant by the kit method, and the morphology and surface membrane marker proteins of the exosomes were identified. The SD rats were established with DCD renal transplantation model and 10, 50, and 100 μg of exosomes were infused into that SD rats respectively, and a PBS control group was set at the same time. On the 3rd, 5th, 7th, 14th and 28th days after transplantation, the content of cytokines in the serum of the recipient rats, the proportion of lymphocyte subsets CD4+CD25+Foxp3+Tregs in the spleen and the relative expression of Foxp3 mRNA were detected. The proliferation of renal tubular cells was detected by PCNA immunofluorescence. The pathological changes of renal tissue were determined by HE staining, and the apoptosis of renal tubular epithelial cells was detected by Tunel staining. The infiltration of inflammatory cells was detected by immunohistochemistry. The expression changes of regulatory T cells in renal tissue and macrophage phenotypic characteristics were analyzed by flow cytometry. Results: The experimental amount of exosomes was successfully obtained, and the morphology was observed by transmission electron microscope. The exosomes were round or oval membranous vesicles with uniform size, and the edges were clearly visible. The TSG101, CD63, and CD9 proteins were highly expressed on the exosome membrane surface by western-blot. A DCD rat kidney transplantation model was successfully established. The expression levels of IL-12, IL-2, IFN-γ, and TNF-α in the serum of each group were increased to varying degrees from the 3rd day after operation. On the 3rd and 7th days, the levels of IL-12, IL-2, IFN-γ, and TNF-α in the 100 μg exosome infusion group were significantly lower than those in the 10 μg, 50 μg and PBS groups (P<0.05), and the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than 10μg, 50μg and PBS groups (P<0.05). On the 28th day, the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than other groups (P<0.01). Compared with other groups, the CD4+CD25+Tregs and CD4+CD25+Foxp3+Tregs accounted for the highest proportion of CD4+T cells in the spleen of the 100μg infusion group (P<0.05). On the 28th day, the expression level of Foxp3 mRNA in the 100 μg infusion group was significantly higher than that in the 50 μg group (P<0.05). The stainings of PCNA, HE and Tunel showed that SD rats infused with 100μg exosomes could significantly reduce the pathological damage, inflammatory response and apoptosis of renal tubular epithelial cells in the transplanted kidney, promote the proliferation of renal tubular epithelial cells, and have a protective effect on IRI kidney. On the 28th day, the expression of CD4+CD25+Foxp3+Tregs cells in the transplanted kidney tissue was significantly increased, which further promoted the differentiation of macrophages into the immunomodulatory M2 subset. Conclusions: The infusion of iPS-derived high-dose exosomes is involved in the protection of the DCD kidney transplant during IRI process, and alleviates the acute and chronic injury of the DCD kidney transplant, and promotes the formation of the local immunosuppressive microenvironment in the DCD kidney transplantation. The National Natural Science Foundation of China (No.81900686).

NGAL release from peripheral blood mononuclear cells protects against acute kidney injury and prevents AKI induced fibrosis

We propose the use of a peripheral blood mononuclear cell therapy based on cell NGAL release to be used in the clinical setting for acute kidney injury (AKI) and the derived fibrosis. First, we designed a procedure whereby PBMC overexpress NGAL and anti-inflammatory agents when subjected to repetitive anoxia/reoxygenation (PBMC (A/R)). Using an in vivo AKI model, we observed that PBMC(A/R) reduces BUN and creatinine levels in blood and inflammation, enhances anti-inflammation, induces proliferation of tubular epithelial cells and reduces AKI-induced fibrosis. Flow cytometry analysis evidenced that monocytes are the only cells accumulated in the injured kidney and phenotype analysis of freshly isolated kidney macrophages, revealed that the healing phenotype is maintained the time needed for recovery. NGAL release from PBMC(A/R) determines the beneficial effect of the therapy since administration of a NGAL antibody previous to the therapy or injection of PBMC(A/R) obtained from NGAL KO animals abolished the beneficial effects. CD11b–NGAL positive cells were enhanced in tissue after PBMC (A/R) therapy and were produced by the injected monocytes. In an in vitro model with tubular epithelial cells (NRK52e) we proved that NGAL release by PBMC(A/R) induced epithelial proliferation and activation of PI3K/Akt pathway.

The overexpressed transcription factor RGPR-p117 suppresses the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells: Involvement of diverse signaling pathways

AimsRGPR-p117 was originally discovered as a novel transcription factor, which specifically binds to a nuclear factor I (NFI) consensus motif TTGGC(N)6CC in the promoter region of the regucalcin gene. RGPR-p117 is also called as Lztr2 and SEC16B. The role of RGPR-p117 in cell regulation is poorly understood. This study was undertaken to determine whether the overexpression of RGPR-p117 impacts the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells in vitro. Main methodsThe NRK-52E wild-type cells and RGPR-p117-overexpressing NRK-52E cells were cultured in DMEM containing fetal bovine serum. Key findingsThe overexpression of RGPR-p117 repressed colony formation and proliferation of NRK-52E cells. Interestingly, RGPR-p117 overexpression blocked cell proliferation promoted by culturing with Bay K 8644, a calcium-entry agonist, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. The depressive effects of RGPR-p117 overexpression on cell proliferation were not occurred by culturing with various inhibitors of cell cycle and intracellular signaling processes. RGPR-p117 overexpression increased the translocation of RGPR-p117 into the nucleus of NRK-52E cells. Mechanistically, RGPR-p117 overexpression diminished the levels of Ras, PI3 kinase, Akt, mitogen-activated protein kinase, and mTOR, while it raised the levels of p53, Rb, p21, and regucalcin. Furthermore, RGPR-p117 overexpression protected cell death caused by apoptosis-inducing factors, suggesting that the suppressive effects of RGPR-p117 on cell growth are independent of cell death. SignificanceThe present study demonstrates that the overexpressed transcription factor RGPR-p117 suppresses cell proliferation via targeting diverse signaling processes, suggesting a role of RGPR-p117 in cell regulation.

Yishen Capsule Alleviated Symptoms of Diabetic Nephropathy via NOD-like Receptor Signaling Pathway

PurposeTo explore the mechanism of Yishen capsule against diabetic nephropathy (DN) based on the analysis of transcriptomics.Material and MethodsSD rats (Male, SPF grade) were randomly divided into four groups, the normal group, the DN group, the Yishen capsule group and the resveratrol group. Urine and renal tissue samples were collected after feeding with physiological saline and above drugs for 8 weeks. 24-hour urine microalbumin protein was detected by ELISA. HE staining and PAS staining were performed on renal tissues. Differential gene expression in renal tissues was analyzed by transcriptome sequencing. The differentially expressed genes were analyzed by GO enrichment and KEGG enrichment, and verified by RT-PCR and immunohistochemistry staining.ResultsThe level of 24-hour urinary microalbumin in DN group was increased, while Yishen capsule treatment reversed the increasement of urinary microalbumin. Mesangial cell proliferation, matrix accumulation, edema and vacuolar degeneration of renal tubular epithelial cells and glycogen accumulation were observed in DN group. However, pathological phenotypes mentioned above were alleviated after Yisen capsule administration. This result indicates that Yishen capsule reversed pathological phenotypes of DN in rats. The expression of 261 genes were changed in Yishen capsule group compared with DN group. GO enrichment analysis and KEGG pathway analysis showed that these genes were implicated in pathways, including mineral absorption, adipocytokine signaling pathway, fatty acid biosynthesis, thyroid hormone synthesis, renin–angiotensin system, and NOD-like receptor signaling pathway. Based on previous reported study, the expression of key factors in NOD-like receptor signaling pathway was verified. RT-PCR and immunohistochemistry staining showed that the expression of NLRP3, Caspase-1 and IL-1β in renal tissues of DN group were increased (P < 0.05), which were decreased in Yishen capsule group (P < 0.05).ConclusionYishen capsule reduced microalbuminuria and alleviated pathological changes in DN rats, which may be achieved by regulating NOD-like receptor signaling pathway.

Cyclin-dependent kinase 4-related tubular epithelial cell proliferation is regulated by Paired box gene 2 in kidney ischemia-reperfusion injury

Paired box 2 (Pax2) is a transcription factor essential for kidney development and is reactivated in proximal tubular epithelial cells (PTECs) during recovery from kidney injury. However, the role of Pax2 in this process is still unknown. Here the role of Pax2 reactivation during injury was examined in the proliferation of PTECs using an ischemia-reperfusion injury (IRI) mouse model. Kidney proximal tubule-specific Pax2 conditional knockout mice were generated by mating kidney androgen-regulated protein-Cre and Pax2 flox mice. The degree of cell proliferation and fibrosis was assessed and a Pax2 inhibitor (EG1) was used to evaluate the role of Pax2 in the hypoxic condition of cultured PTECs (O2 5%, 24 hours). The number of Pax2-positive cells and Pax2 mRNA increased after IRI. Sirius red staining indicated that the area of interstitial fibrosis was significantly larger in knockout mice 14 days after IRI. The number of Ki-67-positive cells (an index of proliferation) was significantly lower in knockout than in wild-type mice after IRI, whereas the number of TUNEL-positive cells (an index of apoptotic cells) was significantly higher in knockout mice four days after IRI. Expression analyses of cell cycle-related genes showed that cyclin-dependent kinase 4 (CDK4) was significantly less expressed in the Pax2 knockout mice. Invitro data showed that the increase in CDK4 mRNA and protein expression induced by hypoxia was attenuated by EG1. Thus, Pax2 reactivation may be involved in PTEC proliferation by activating CDK4, thereby limiting kidney fibrosis.

Comparison of the treatment efficacy of umbilical mesenchymal stem cell transplantation via renal subcapsular and parenchymal routes in AKI-CKD mice

BackgroundMesenchymal stem cells (MSCs) have emerged as a promising cell-based therapy for acute kidney injury (AKI). However, the optimal route of MSC transplantation remains controversial, and there have been no comparisons of the therapeutic benefits of MSC administration through different delivery routes.MethodsIn this study, we encapsulated MSCs into a collagen matrix to help achieve local MSC retention in the kidney and assessed the survival of MSCs in vitro and in vivo. After transplanting collagen matrix-encapsulated-MSCs (Col-MSCs) under the renal capsule or into the parenchyma using the same cell dose and suspension volume in an ischemia/reperfusion injury model, we evaluated the treatment efficacy of two local transplantation routes at different stages of AKI.ResultsWe found that Col-MSCs could be retained in the kidney for at least 14 days. Both local MSC therapies could reduce tubular injury, promote the proliferation of renal tubular epithelial cells on Day 3 and alleviate renal fibrosis on Day 14 and 28. MSC transplantation via the subcapsular route exerts better therapeutic effects for renal functional and structural recovery after AKI than MSC administration via the parenchymal route.ConclusionsSubcapsular MSC transplantation may be an ideal route of MSC delivery for AKI treatment, and collagen I can provide a superior microenvironment for cell–cell and cell–matrix interactions to stabilize the retention rate of MSCs in the kidney.

Salvia miltiorrhiza Bunge (Danshen) and Bioactive Compound Tanshinone IIA Alleviates Cisplatin-Induced Acute Kidney Injury Through Regulating PXR/NF-κB Signaling.

Objective: The present study aims to provide evidence on the potential protective role of Salvia miltiorrhiza Bunge (Danshen) and its bioactive compound Tanshinone IIA (TanIIA) in AKI and to reveal the specific regulatory function of PXR/NF-κB signaling in AKI-induced renal inflammation. Methods: A network pharmacological analysis was used to study target genes and regulatory networks in the treatment of Salvia miltiorrhiza on AKI. Further experiments with in vivo AKI mouse model and in vitro studies were applied to investigate the renal protective effect of TanIIA in AKI. The mechanisms of TanIIA regulating PXR/NF-κB signaling in renal inflammation were also studied. Results: Network pharmacology had suggested the nuclear receptor family as new therapeutic targets of Salvia miltiorrhiza in AKI treatment. The in vivo studies had demonstrated that TanIIA improved renal function and inflammation by reducing necrosis and promoting the proliferation of tubular epithelial cells. Improved renal arterial perfusion in AKI mice with TanIIA treatment was also recorded by ultrasonography. In vitro studies had shown that TanIIA ameliorated renal inflammation by activating the PXR while inhibiting PXR-mediated NF-κB signaling. The results had suggested a role of PXR activation against AKI-induced renal inflammation. Conclusion: Salvia miltiorrhiza Bunge (Danshen) may protect the kidneys against AKI by regulating nuclear receptors. TanIIA improved cell necrosis proliferation and reduced renal inflammation by upregulating the expression of the PXR and inhibiting NF-κB signaling in a PXR-dependent manner. The PXR may be a potential therapeutic target for AKI treatment.

Effect of BDNF-AS/miR-145-5p axis on renal tubular epithelial cell injury induced by high glucose

To investigate the effect and possible mechanism of BDNF-AS on renal tubular epithelial cell injury induced by high glucose. Human renal tubular epithelial cells HK-2 were cultured in vitro and transfected with BDNF-AS small interfering RNA or miR-145-5p mimic, or co-transfected with BDNF-AS small interfering RNA and miR-145-5p inhibitor, respectively. The cells were then intervened with 30 mmol/L glucose for 24 hours. The expression of BDNF-AS and miR-145-5p were detected by RT-qPCR. Cell proliferation was detected by CCK-8, and apoptosis was detected by flow cytometry. The expression of Bcl-2 and Bax proteins were detected by Western blotting, and the levels of IL-1β and IL-6 in cell culture supernatant were detected by enzyme-linked immunosorbent assay. Dual luciferase reporter gene experiment was used to verify the regulatory relationship of BDNF-AS with miR-145-5p. High glucose promoted the expression of BDNF-AS in HK-2 cells (P<0.05), but inhibited that of miR-145-5p (P<0.05). Interfering with BDNF-AS or overexpression of miR-145-5p decreased the inhibition rate, apoptosis rate and expression of Bax protein, IL-1β and IL-6 of HK-2 cells induced by high glucose (P<0.05), but promoted the expression of Bcl-2 protein (P<0.05). Interfering with miR-145-5p reversed the effect of interfering with BDNF-AS on the proliferation, apoptosis rate and the expression of IL-1β and IL-6 of HK-2 cells induced by high glucose. BDNF-AS could target and down-regulate miR-145-5p. Interfering with BDNF-AS may promote the proliferation of renal tubular epithelial cells induced by high glucose and inhibit cell apoptosis and the expression of inflammatory factor by down-regulating miR-145-5p.

Icariin Attenuation of Diabetic Kidney Disease Through Inhibition of Endoplasmic Reticulum Stress via G Protein-Coupled Estrogen Receptors.

Diabetic kidney disease (DKD) is the most common complication of diabetes mellitus and has become the primary cause of End-Stage Renal Disease (ESRD) globally. Icariin (ICA), an effective component extracted from Epimedium, has antiosteoporosis effect, antitumor effects, anti-ischemia effects, and other effects. In this study, a mouse DKD model was established, and Icariin solid nanoliposomes were administered to determine whether ICA had a protective effect on the renal function of DKD mice by regulating estrogen level and endoplasmic reticulum (ER) stress pathway. The results showed that the microalbumin/creatinine in urine, serum urea nitrogen, and CHOL in ICA cultured DKD mice significantly decreased, and mice nephropathy improved significantly. rat renal tubule epithelial cells were further tested, and the rat renal tubule epithelial cells were modeled by cultured cells with high glucose. The results showed that high glucose could promote the proliferation of renal tubular epithelial cells. Simultaneously, ICA can inhibit the proliferation of renal tubular epithelial cells and induce cell apoptosis. Furthermore, the expression of ER stress-related proteins IRE1 and XBP-1S was further detected. Additionally, to ICA intervention, a GPER antagonist (G-15) was added for intervention, the inhibitory effects of IRE1 and XBP-1S were reversed, and the ER stress pathway was activated. Cell experiments showed that ICA could promote GPER expression, while inhibiting GPER expression promoted the activation of ER stress pathway, and GPER expression was negatively correlated with ER stress protein expression. Therefore, the experiment proved that in DKD tissues, a high concentration of ICA can inhibit the ER stress response by promoting the expression of GPER, reducing the proliferation of diabetic nephropathy, and increasing the rate of tissue apoptosis.

Activation of Notch3 in Renal Tubular Cells Leads to Progressive Cystic Kidney Disease.

Background: Polycystic kidney disease (PKD) is a genetic disorder affecting millions of people worldwide that is characterized by fluid-filled cysts and leads to end-stage renal disease (ESRD). The hallmarks of PKD are proliferation and dedifferentiation of tubular epithelial cells, cellular processes known to be regulated by Notch signaling. Methods: We found increased Notch3 expression in human PKD and renal cell carcinoma biopsies. To obtain insight into the underlying mechanisms and the functional consequences of this abnormal expression, we developed a transgenic mouse model with conditional overexpression of the intracellular Notch3 (ICN3) domain specifically in renal tubules. We evaluated the alterations in renal function (creatininemia, BUN) and structure (cysts, fibrosis, inflammation) and measured the expression of several genes involved in Notch signaling and the mechanisms of inflammation, proliferation, dedifferentiation, fibrosis, injury, apoptosis and regeneration. Results: After one month of ICN3 overexpression, kidneys were larger with tubules grossly enlarged in diameter, with cell hypertrophy and hyperplasia, exclusively in the outer stripe of the outer medulla. After three months, mice developed numerous cysts in proximal and distal tubules. The cysts had variable sizes and were lined with a single- or multilayered, flattened, cuboid or columnar epithelium. This resulted in epithelial hyperplasia, which was observed as protrusions into the cystic lumen in some of the renal cysts. The pre-cystic and cystic epithelium showed increased expression of cytoskeletal filaments and markers of epithelial injury and dedifferentiation. Additionally, the epithelium showed increased proliferation with an aberrant orientation of the mitotic spindle. These phenotypic tubular alterations led to progressive interstitial inflammation and fibrosis. Conclusions: In summary, Notch3 signaling promoted tubular cell proliferation, the alignment of cell division, dedifferentiation and hyperplasia, leading to cystic kidney diseases and pre-neoplastic lesions.

Inhibiting Focal Adhesion Kinase Ameliorates Cyst Development in Polycystin-1-Deficient Polycystic Kidney Disease in Animal Model.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by numerous cysts originating from renal tubules and is associated with significant tubular epithelial cell proliferation. Focal adhesion kinase (FAK) promotes tumor growth by regulating multiple proliferative pathways. We established the forskolin (FSK)-induced three-dimensional (3D) Madin-Darby Canine Kidney cystogenesis model and 8-bromoadenosine-3`,5`-cyclic monophosphate-stimulated cyst formation in ex vivo embryonic kidney culture. Cultured human renal cyst-lining cells (OX-161) and normal tubular epithelial cells were treated with FAK inhibitors or transfected with green fluorescent protein-tagged FAK mutant plasmids for proliferation study. Furthermore, we examined the role of FAK in two transgenic ADPKD animal models, the kidney-specific Pkd1 knockout and the collecting duct-specific Pkd1 knockout mouse models. FAK activity was significantly elevated in OX-161 cells and in two ADPKD mouse models. Inhibiting FAK activity reduced cell proliferation in OX-161 cells and prevented cyst growth in ex vivo and 3D cyst models. In tissue-specific Pkd1 knockout mouse models, FAK inhibitors retarded cyst development and mitigated renal function decline. Mechanically, FSK stimulated FAK activation in tubular epithelial cells, which was blocked by a protein kinase A (PKA) inhibitor. Inhibition of FAK activation by inhibitors or transfected cells with mutant FAK constructs interrupted FSK-mediated Src activation and upregulation of ERK and mTOR pathways. Our study demonstrates the critical involvement of FAK in renal cyst development, suggests that FAK is a potential therapeutic target in treating patients with ADPKD, and highlights the role of FAK in cAMP-PKA-regulated proliferation.