Comprehensive Pan-Cancer Analysis of Connexin 43 as a Potential Biomarker and Therapeutic Target in Human Kidney Renal Clear Cell Carcinoma (KIRC).

Background and Objectives: Connexin 43 (Cx43) is involved in the transfer of small signaling molecules between neighboring cells, thereby exerting a major influence on the initiation and progression of tumorigenesis. However, there is a lack of systematic research on Cx43 expression and its predictive role in clinical diagnosis and prognosis in pan-cancer. Materials and Methods: Several biological databases were used to evaluate the expression levels of GJA1 (encoding Cx43) and its diagnostic and prognostic significance in pan-cancer. We targeted kidney renal clear cell carcinoma (KIRC) and investigated the relationship between GJA1 expression and different clinical features of KIRC patients. Then, we performed cell-based experiments to partially confirm our results and predicted several proteins that were functionally related to Cx43. Results: The expression of GJA1 has a high level of accuracy in predicting KIRC. High GJA1 expression was remarkably correlated with a favorable prognosis, and this expression was reduced in groups with poor clinical features in KIRC. Cell experiments confirmed the inhibitory effects of increased GJA1 expression on the migratory capacity of human renal cancer (RCC) cell lines, and protein-protein interaction (PPI) analysis predicted that CDH1 and CTNNB1 were closely related to Cx43. Conclusions: GJA1 could be a promising independent favorable prognostic factor for KIRC, and upregulation of GJA1 expression could inhibit the migratory capacity of renal cancer cells.

The same heterozygous Col4A4 mutation triggered different renal pathological changes in Chinese family members.

Background: Mutations in the collagen components of the glomerular basement membrane (GBM) often lead to hereditary glomerulonephritis. Previous studies have identified that autosomal dominant mutations of Col4A3, Col4A4 or Col4A5 are associated with thin basement membrane nephropathy (TBMN), Alport syndrome and other hereditary kidney diseases. However, the genetic mutations underlying other glomerulonephritis types have not been elucidated. Methods: In this study, we investigated a Chinese family with hereditary nephritis using the methods of genetic sequencing and renal biopsy. Genomic DNA was extracted from peripheral blood of the proband and her sister, and subsequently was performed genetic sequencing. They were found to have the similar mutation sites. Other family members were then validated using Sanger sequencing. The proband and her sister underwent renal puncture biopsies, and experienced pathologists performed PAS, Masson, immunofluorescence, and immunoelectron microscopic staining of the kidney tissue sections. Results: Through genetic sequencing analysis, we detected a novel heterozygous frameshift mutation c.1826delC in the COL4A4 (NM_000092.4) gene coding region, and 1 hybrid missense variation c.86G>A (p. R29Q) was also detected in the TNXB (NM_019105.6) gene coding region in several members of this Chinese family. Interestingly, we found that the same mutations caused different clinical features and distinct pathological changes in individual family members, which confirmed that pathological and genetic testing are crucial for the diagnosis and treatment of hereditary kidney diseases. Conclusion: In this study, we found a novel heterozygous mutation in Col4A4 and co-mutations of the TNXB gene in this Chinese family. Our study indicated that the same Col4A4 mutated variants produced different pathological and clinical changes in different family members. This discovery may provide novel insights into the study of hereditary kidney disease. In addition, new genetic biology techniques and renal biopsy of individual family members are essential.

Blocking connexin 43 and its promotion of ATP release from renal tubular epithelial cells ameliorates renal fibrosis.

Whether metabolites derived from injured renal tubular epithelial cells (TECs) participate in renal fibrosis is poorly explored. After TEC injury, various metabolites are released and among the most potent is adenosine triphosphate (ATP), which is released via ATP-permeable channels. In these hemichannels, connexin 43 (Cx43) is the most common member. However, its role in renal interstitial fibrosis (RIF) has not been fully examined. We analyzed renal samples from patients with obstructive nephropathy and mice with unilateral ureteral obstruction (UUO). Cx43-KSP mice were generated to deplete Cx43 in TECs. Through transcriptomics, metabolomics, and single-cell sequencing multi-omics analysis, the relationship among tubular Cx43, ATP, and macrophages in renal fibrosis was explored. The expression of Cx43 in TECs was upregulated in both patients and mice with obstructive nephropathy. Knockdown of Cx43 in TECs or using Cx43-specific inhibitors reduced UUO-induced inflammation and fibrosis in mice. Single-cell RNA sequencing showed that ATP specific receptors, including P2rx4 and P2rx7, were distributed mainly on macrophages. We found that P2rx4- or P2rx7-positive macrophages underwent pyroptosis after UUO, and in vitro ATP directly induced pyroptosis by macrophages. The administration of P2 receptor or P2X7 receptor blockers to UUO mice inhibited macrophage pyroptosis and demonstrated a similar degree of renoprotection as Cx43 genetic depletion. Further, we found that GAP 26 (a Cx43 hemichannel inhibitor) and A-839977 (an inhibitor of the pyroptosis receptor) alleviated UUO-induced fibrosis, while BzATP (the agonist of pyroptosis receptor) exacerbated fibrosis. Single-cell sequencing demonstrated that the pyroptotic macrophages upregulated the release of CXCL10, which activated intrarenal fibroblasts. Cx43 mediates the release of ATP from TECs during renal injury, inducing peritubular macrophage pyroptosis, which subsequently leads to the release of CXCL10 and activation of intrarenal fibroblasts and acceleration of renal fibrosis.

Interleukin-22 regulates gastric cancer cell proliferation through regulation of the JNK signaling pathway.

Inflammation is considered as one of the major hallmarks of cancer and is associated with gastric cancer. Interleukin-22 (IL-22), a member of the IL-10 family, serves an important role in inflammatory diseases and tumors. The aim of the present study was to examine the effects of IL-22 on the proliferation of gastric cancer cells (AGS cells) in vitro and explore the associated molecular mechanism. The results of a Cell Counting kit-8 assay using AGS cells transfected with an IL-22-plasmid indicated that IL-22 could promote AGS cell viability. However, when IL-22 was knocked down by IL-22-short hairpin (sh)RNA, the viability of AGS cells was significantly impaired. Western blotting results indicated that IL-22 decreased the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, IL-22-shRNA transfection increased the activation of MAPK, as evidenced by the upregulated phosphorylation of ERK and JNK. Taken together, the results of the present study suggest that IL-22 regulated the viability of gastric cancer cells through the JNK signaling pathway, suggesting a therapeutic approach for gastric cancer via targeting IL-22.

Protective Effects of PGC-1α on the Blood Brain Barrier After Acute Kidney Injury.

Blood brain barrier (BBB) disruption plays an important role in brain injury after acute kidney injury (AKI). However, its underlying mechanisms remain poorly understood. Recent evidence has revealed that proper mitochondrial function is essential for BBB permeability. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a key factor in mitochondrial biogenesis and function. This study was designed to investigate the role of PGC-1α in BBB injury after AKI and its related mechanisms. Mice received recombinant adenovirus encoding murine PGC-1α (100 µl, 1.0 × 109PFU/ml) or vehicle 5 days before renal I/R or sham operation. Twenty-four hours after the operation, brain, kidney and serum samples were collected for assessments. We found that mice suffering from renal I/R injury showed decreased PGC-1α levels in both the kidney and BBB. PGC-1α transfection resulted in increased PGC-1α level and mitochondrial transcripts in BBB at 24 h after AKI. PGC-1α transfection improved renal function, systemic inflammation and BBB permeability via both the paracellular and transcellular pathways. Further study suggested that PGC-1α overexpression elevated fatty acid oxidation related gene expression. Our findings demonstrate the importance of PGC-1α in AKI-induced BBB injury and suggest that it could be a therapeutic target for BBB repair via the regulation of mitochondrial function.

Putative endothelial progenitor cells do not promote vascular repair but attenuate pericyte-myofibroblast transition in UUO-induced renal fibrosis.

BACKGROUND: Putative endothelial progenitor cells (pEPCs) have been confirmed to participate in alleviation of renal fibrosis in several ischaemic diseases. However, their mechanistic effect on renal fibrosis, which is characterized by vascular regression and further rarefaction-related pathology, remains unknown. METHODS: To explore the effect and molecular mechanisms by which pEPCs act on unilateral ureteral obstruction (UUO)-induced renal fibrosis, we isolated pEPCs from murine bone marrow. In vivo, pEPCs (2 × 105 cells/day) and pEPC-MVs (microvesicles) were injected into UUO mice via the tail vein. In vitro, pEPCs were co-cultured with renal-derived pericytes. Pericyte-myofibroblast transition was evaluated using the myofibroblast marker α-smooth muscle actin (α-SMA) and pericyte marker platelet-derived growth factor receptor ß (PDGFR-ß). RESULTS: Exogenous supply of bone marrow-derived pEPCs attenuated renal fibrosis by decreasing pericyte-myofibroblast transition without significant vascular repair in the UUO model. Our results indicated that pEPCs regulated pericytes and their transition into myofibroblasts via pEPC-MVs. Co-culture of pericytes with pEPCs in vitro suggested that pEPCs inhibit transforming growth factor-ß (TGF-ß)-induced pericyte-myofibroblast transition via a paracrine pathway. CONCLUSION: pEPCs effectively attenuated UUO-induced renal fibrosis by inhibiting pericyte-myofibroblast transition via a paracrine pathway, without promoting vascular repair.

High renal DC-SIGN+ cell density is associated with severe renal lesions and poor prognosis in patients with immunoglobulin A nephropathy.

BACKGROUND AND AIMS: In this observational cohort study, we assessed the prognostic value of DC-SIGN+ cells in the pathogenesis and progression of IgA nephropathy (IgAN). METHODS AND RESULTS: A total of 139 adult IgAN patients were enrolled into this study from June 2009 to June 2010. We characterised DC-SIGN+ cells by immunohistochemistry or immunofluorescence in renal biopsy tissue. Correlations between the DC-SIGN, intercellular adhesion molecule 3 (ICAM-3), CD4 and CD8 were evaluated. Patients were classified into the DC-SIGNhigh and DC-SIGNlow groups. Depending on an average of 100-month follow-up, the predictive value of DC-SIGN+ cells in IgAN progression was analysed. DC-SIGN+ cells were found frequently in IgAN kidneys while rarely observed in normal kidneys, and almost all DC-SIGN+ cells expressed MHC-II. We also found that DC-SIGN+ cells were adjacent to ICAM-3-positive CD4+ and CD8+ lymphocytes. The density of DC-SIGN+ cells was positively and linearly correlated with the density of ICAM-3+ cells, CD4+ cells and CD8+ cells in renal biopsy tissues. In the DC-SIGNhigh group, the degree of renal lesion and inflammatory cell infiltration was more severe compared to the DC-SIGNlow group. Patients in the DC-SIGNhigh group also had increased incidences of deteriorating renal function during the follow up compared to patients in the DC-SIGNlow group. CONCLUSIONS: DC-SIGN+ cells probably served as a potential contributor to exacerbate local inflammatory response. The density of DC-SIGN+ cells was associated with the severity of renal lesions of the patients. High renal DC-SIGN+ cell density might be used as a predictor of poor prognosis in patients with IgAN.

Evaluation of the safety and tolerability of tamoxifen for ischemia-incited renal injury in mice.

Tamoxifen is used to activate tamoxifen-dependent Cre recombinase (CreER) to generate time- and tissue-specific genetically mutant mice. However, tamoxifen is also an active estrogen analogue that binds with higher affinity to estrogen receptors and exhibits anti-apoptosis, anti-inflammation, and antifibrotic properties. Renal ischemia reperfusion (I/R) injury is characterized by increased apoptosis and inflammation, so optimal utility of tamoxifen-inducible CreER genetic systems in I/R model is important. The purpose of this study was to optimize the tamoxifen dose and evaluate its safety and tolerability in the development of mouse I/R injury. Seven-week-old C57/B6 mice were subjected to moderate reversible unilateral I/R and then injected intraperitoneally daily for 5 days with tamoxifen at doses of 50, 100, or 200 mg/kg/day. Regardless of the time of sacrifice, at 5 day or 28 day after I/R injury, there were no differences in pathological damage, apoptosis, inflammation, or the extent of fibrosis between untreated and treated mice from the time point of acute kidney injury (AKI) to subsequently chronic kidney disease. Data above indicated that tamoxifen with a dose among 0 to 200 mg/kg/day was safe and tolerable for mice, without influencing I/R induced kidney injury in mice. The results suggest that tamoxifen-inducible CreER genetic systems can be safely used in the mouse I/R model.

Pretreatment of Huaiqihuang extractum protects against cisplatin-induced nephrotoxicity.

Cisplatin is a commonly used chemotherapeutic agent in the treatment of different types of malignant tumors, but nephrotoxicity limits its usage. Therefore, in this study, we aimed to determine the possible protective effect of Huaiqihuang (HQH) extractum, a kind of Chinese herbal complex that consists of Trametes robiniophila Murr., Lycium barbarum and Polygonatum sibiricum, against nephrotoxicity induced by cisplatin in mice. We found that pretreatment with HQH significantly attenuated the cisplatin-induced increase in blood urea nitrogen (BUN), interstitial congestion, acute renal tubular injury and tubular cell apoptosis and necroptosis. It was further shown that HQH administration reduced cisplatin-induced release and nuclear-cytoplasmic translocation of HMGB1 and inactivated its downstream signaling molecules, TLR4 and NFκB, in renal tubular cells; as a result, HQH repressed cisplatin-induced TNF-α production. As dexamethasone (Dex) exerts renoprotective effects in severe Acute kidney injury (AKI), we compared it with HQH and found that HQH showed similar renoprotective effects to dexamethasone via similar mechanisms. Considering the potential side effects of corticosteroids, reducing the effectiveness of treatment and shortening survival in solid tumor patients, we suggest administration of HQH as a potential adjuvant for cisplatin therapy in solid tumor patients to preserve renal function.

Macrophages Regulate Unilateral Ureteral Obstruction-Induced Renal Lymphangiogenesis through C-C Motif Chemokine Receptor 2-Dependent Phosphatidylinositol 3-Kinase-AKT-Mechanistic Target of Rapamycin Signaling and Hypoxia-Inducible Factor-1α/Vascular Endothelial Growth Factor-C Expression.

Lymphangiogenesis occurs during renal fibrosis in patients with chronic kidney diseases and vascular endothelial growth factor (VEGF)-C is required for the formation of lymphatic vessels; however, the underlying mechanisms remain unclear. We demonstrate that macrophages can regulate unilateral ureteral obstruction (UUO)-induced renal lymphangiogenesis by expressing high levels of VEGF-C by C-C motif chemokine receptor 2 (CCR2)-mediated signaling. Mice deficient in Ccr2 manifested repressed lymphangiogenesis along with attenuated renal injury and fibrosis after UUO induction. The infiltrated macrophages after UUO induction generated a microenvironment in favor of lymphangiogenesis, which likely depended on Ccr2 expression. Mechanistic studies revealed that CCR2 is required for macrophages to activate phosphatidylinositol 3-kinase (PI3K)-AKT-mechanistic target of rapamycin (mTOR) signaling in response to its ligand monocyte chemoattractant protein 1 stimulation, whereas hypoxia-inducible factor (HIF)-1α is downstream of PI3K-AKT-mTOR signaling. HIF-1α directly bound to the VEGF-C promoter to drive its expression to enhance lymphangiogenesis. Collectively, we characterized a novel regulatory network in macrophages, in which CCR2 activates PI3K-AKT-mTOR signaling to mediate HIF-1α expression, which then drives VEGF-C expression to promote lymphangiogenesis.

Continuous AMD3100 Treatment Worsens Renal Fibrosis through Regulation of Bone Marrow Derived Pro-Angiogenic Cells Homing and T-Cell-Related Inflammation.

AMD3100 is a small molecule inhibitor of chemokine receptor type 4 (CXCR4), which is located in the cell membranes of CD34+ cells and a variety of inflammatory cells and has been reported to reduce organ fibrosis in the lung, liver and myocardium. However, the effect of AMD3100 on renal fibrosis is unknown. This study investigated the impact of AMD3100 on renal fibrosis. C57bl/6 mice were subjected to unilateral ureteral obstruction (UUO) surgery with or without AMD3100 administration. Tubular injury, collagen deposition and fibrosis were detected and analyzed by histological staining, immunocytochemistry and Western Blot. Bone marrow derived pro-angiogenic cells (CD45+, CD34+ and CD309+ cells) and capillary density (CD31+) were measured by flow cytometry (FACS) and immunofluorescence (IF). Inflammatory cells, chemotactic factors and T cell proliferation were characterized. We found that AMD3100 treatment did not alleviate renal fibrosis but, rather, increased tissue damage and renal fibrosis. Continuous AMD3100 administration did not improve bone marrow derived pro-angiogenic cells mobilization but, rather, inhibited the migration of bone marrow derived pro-angiogenic cells into the fibrotic kidney. Additionally, T cell infiltration was significantly increased in AMD3100-treated kidneys compared to un-treated kidneys. Thus, treatment of UUO mice with AMD3100 led to an increase in T cell infiltration, suggesting that AMD3100 aggravated renal fibrosis.

Fenofibrate attenuated glucose-induced mesangial cells proliferation and extracellular matrix synthesis via PI3K/AKT and ERK1/2.

Excess mesangial extracellular matrix (ECM) and mesangial cell proliferation is the major pathologic feature of diabetic nephropathy (DN). Fenofibrate, a PPARα agonist, has been shown to attenuate extracellular matrix formation in diabetic nephropathy. However, the mechanisms underlying this effect remain to be elucidated. In this study, the effect of fenofibrate on high-glucose induced cell proliferation and extracellular matrix exertion and its mechanisms were investigated in cultured rat mesangial cells by the methylthiazoletetrazolium (MTT) assay, flow cytometry and western blot. The results showed that treatment of mesangial cells (MCs) with fenofibrate repressed high-glucose induced up-regulation of extracellular matrix Collagen-IV, and inhibited entry of cell cycle into the S phase. This G1 arrest and ECM inhibition was caused by the reduction of phosphorylation and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT. On the contrary, PPARα siRNA accelerated high glucose-induced cell cycle progression by ERK1/2 and AKT activation. Taken together, fenofibrate ameliorated glucose-induced mesangial cell proliferation and matrix production via its inhibition of PI3K/AKT and ERK1/2 signaling pathways. Such mechanisms may contribute to the favorable effects of treatment using fenofibrate in diabetic nephropathy.