Protease‐activated receptor‐1 contributes to renal injury and interstitial fibrosis during chronic obstructive nephropathy

End‐stage renal disease, the final stage of all chronic kidney disorders, is associated with renal fibrosis and inevitably leads to renal failure and death. Transition of tubular epithelial cells (TECs) into mesenchymal fibroblasts constitutes a proposed mechanism underlying the progression of renal fibrosis and here we assessed whether protease‐activated receptor (PAR)‐1, which recently emerged as an inducer of epithelial‐to‐mesenchymal transition (EMT), aggravates renal fibrosis. We show that PAR‐1 activation on TECs reduces the expression of epithelial markers and simultaneously induces mesenchymal marker expression reminiscent of EMT. We next show that kidney damage was reduced in PAR‐1‐deficient mice during unilateral ureter obstruction (UUO) and that PAR‐1‐deficient mice develop a diminished fibrotic response. Importantly, however, we did hardly observe any signs of mesenchymal transition in both wild‐type and PAR‐1‐deficient mice suggesting that diminished fibrosis in PAR‐1‐deficient mice is not due to reduced EMT. Instead, the accumulation of macrophages and fibroblasts was significantly reduced in PAR‐1‐deficient animals which were accompanied by diminished production of MCP‐1 and TGF‐β. Overall, we thus show that PAR‐1 drives EMT of TECs in vitro and aggravates UUO‐induced renal fibrosis although this is likely due to PAR‐1‐dependent pro‐fibrotic cytokine production rather than EMT.     

B‐cell‐activating factor code and human cytomegalovirus infection in renal transplant recipients

The objective of the present study was to explore the correlation between the BAFF signal and HCMV‐TLR activation in RTx recipients complicated by HCMV. Peripheral blood (anticoagulated by EDTA‐Na₂) and urine of 113 RTx recipients were collected; healthy volunteers were controlled. Urine HCMV‐DNA was detected by real‐time PCR. Recipients were classified into a positive group (>10,000 copies/mL urine) and a negative group (<10,000 copies/mL urine). ELISA results showed that sBAFF, sera anti‐HCMV pp65 immunoglobulin (Ig)G antibody, and total IgG all significantly increased in recipients with positive HCMV‐DNA (>10,000 copies/mL urine) (P < 0.05) compared with negative recipients (<10,000 copies/mL urine). In the positive group, HCMV‐DNA copies and total IgG positively correlated with sBAFF (r = 0.988 and 0.625, respectively) (P < 0.05). Luminex assay results suggested that the incidence of anti‐HLA I and II and MICA antibody obviously increased in positive recipients. The expression level of BAFF and BAFF‐R increased in positive recipients. A total of 88 particular genes—involved in TLR signaling pathways, NF‐κB signaling pathways, and cytokine‐cytokine receptor signaling pathways—were detected in real‐time PCR chip assay. A total of 46 genes were differentially expressed greater than two‐fold, and the expression characteristic of BAFF‐R was concordant with FACS results. Our findings are that activation of HCMV would induce or enhance the activation of BAFF code in RTx recipients, which may independently or cooperatively participate in renal allograft injury and decrease the long‐term outcome of renal allografts.     

Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma

H. Ohsaki, E. Hirakawa, K. Kagawa, M. Nakamura, H. Kiyomoto and R. Haba Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma Objective: To assess whether computer‐assisted quantitative morphological parameters can be an effective tool for objectively distinguishing reactive renal tubular cells from low‐grade urothelial carcinoma cells (LG‐UCs) in voided urine. Methods: Nuclear morphometry was performed by a computer‐assisted image analyser system on Papanicolaou‐stained cytological specimens. The circumference of reactive renal tubular cells (n = 40) or LG‐UC (n = 20) nuclei were manually traced, and the following nuclear morphometric parameters were analysed: (i) area, (ii) perimeter, (iii) roundness factor, (iv) maximum length, and (v) linear factor. For each nuclear measurement, we calculated the maximum, minimum, mean and standard deviation. Results: The mean nuclear area and nuclear perimeter were higher in reactive renal tubular cells compared to the LG‐UCs. The mean of roundness and linear factors (reflecting a tendency for the nuclear outline to be regular and oval, respectively) were higher in LG‐UCs compared with reactive renal tubular cells. Among nuclear areas, the nuclear perimeter, roundness factors and maximum length did not show any significant differences between reactive renal tubular cells and LG‐UCs. On the other hand, the linear factor showed a mean higher value among LG‐UCs than reactive renal tubular cells (P = 0.023). Conclusions: Of five quantitative nuclear morphological parameters, only linear factor was statistically significant in differentiating reactive renal tubular cells in renal disease from LG‐UCs.     

Knockout of receptor for advanced glycation end‐products attenuates age‐related renal lesions

Pro‐aging effects of endogenous advanced glycation end‐products (AGEs) have been reported, and there is increasing interest in the pro‐inflammatory and ‐fibrotic effects of their binding to RAGE (the main AGE receptor). The role of dietary AGEs in aging remains ill‐defined, but the predominantly renal accumulation of dietary carboxymethyllysine (CML) suggests the kidneys may be particularly affected. We studied the impact of RAGE invalidation and a CML‐enriched diet on renal aging. Two‐month‐old male, wild‐type (WT) and RAGE^?/? C57Bl/6 mice were fed a control or a CML‐enriched diet (200 μg CML/g_food) for 18 months. Compared to controls, we observed higher CML levels in the kidneys of both CML WT and CML RAGE^?/? mice, with a predominantly tubular localization. The CML‐rich diet had no significant impact on the studied renal parameters, whereby only a trend to worsening glomerular sclerosis was detected. Irrespective of diet, RAGE^?/? mice were significantly protected against nephrosclerosis lesions (hyalinosis, tubular atrophy, fibrosis and glomerular sclerosis) and renal senile apolipoprotein A‐II (ApoA‐II) amyloidosis (p < 0.001). A positive linear correlation between sclerosis score and ApoA‐II amyloidosis score (r = 0.92) was observed. Compared with old WT mice, old RAGE^?/? mice exhibited lower expression of inflammation markers and activation of AKT, and greater expression of Sod2 and SIRT1. Overall, nephrosclerosis lesions and senile amyloidosis were significantly reduced in RAGE^?/? mice, indicating a protective effect of RAGE deletion with respect to renal aging. This could be due to reduced inflammation and oxidative stress in RAGE^?/? mice, suggesting RAGE is an important receptor in so‐called inflamm‐aging.     

Advanced glycation end‐products activate extracellular signal‐regulated kinase via the oxidative stress‐EGF receptor pathway in renal fibroblasts

Advanced glycation end‐products (AGEs), epidermal growth factor receptor (EGFR), reactive oxygen species (ROS), and extracellular signal‐regulated kinases (ERK) are implicated in diabetic nephropathy (DN). Therefore, we asked if AGEs‐induced ERK protein phosphorylation and mitogenesis are dependent on the receptor for AGEs (RAGE)–ROS–EGFR pathway in normal rat kidney interstitial fibroblast (NRK‐49F) cells. We found that AGEs (100 µg/ml) activated EGFR and ERK1/2, which was attenuated by RAGE short‐hairpin RNA (shRNA). AGEs also increased RAGE protein and intracellular ROS levels while RAGE shRNA and N‐acetylcysteine (NAC) attenuated AGEs‐induced intracellular ROS. Hydrogen peroxide (5–25 µM) increased RAGE protein level while activating both EGFR and ERK1/2. Low‐dose hydrogen peroxide (5 µM) increased whereas high‐dose hydrogen peroxide (100 µM) decreased mitogenesis at 3 days. AGEs‐activated EGFR and ERK1/2 were attenuated by an anti‐oxidant (NAC) and an EGFR inhibitor (Iressa). Moreover, AGEs‐induced mitogenesis was attenuated by RAGE shRNA, NAC, Iressa, and an ERK1/2 inhibitor (PD98059). In conclusion, it was found that AGEs‐induced mitogenesis is dependent on the RAGE–ROS–EGFR–ERK1/2 pathway whereas AGEs‐activated ERK1/2 is dependent on the RAGE–ROS–EGFR pathway in NRK‐49F cells. J. Cell. Biochem. 109: 38–48, 2010. © 2009 Wiley‐Liss, Inc.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

Circular RNA circ_001842 plays an oncogenic role in renal cell carcinoma by disrupting microRNA‐502‐5p‐mediated inhibition of SLC39A14

Renal cell carcinoma (RCC) is a common urologic malignancy, and up to 30% of RCC patients present with locally advanced or metastatic disease at the time of initial diagnosis. Increasing evidence suggests that circular RNAs (circRNAs) serve as genomic regulatory molecules in various human cancers. Our initial in silico microarray‐based analysis identified that circRNA circ_001842 was highly expressed in RCC. Such up‐regulation of circ_001842 in RCC was experimentally validated in tissues and cell lines using RT‐qPCR. Thereafter, we attempted to identify the role of circ_001842 in the pathogenesis of RCC. Through a series of gain‐ and loss‐of function assays, cell biological functions were examined using colony formation assay, Transwell assay, annexin V‐FITC/PI‐labelled flow cytometry and scratch test. A high expression of circ_001842 in tissues was observed as associated with poor prognosis of RCC patients. circ_001842 was found to elevate SLC39A14 expression by binding to miR‐502‐5p, consequently resulting in augmented RCC cell proliferation, migration and invasion, as well as EMT in vitro and tumour growth in vivo. These observations imply the involvement of circ_001842 in RCC pathogenesis through a miR‐502‐5p‐dependent SLC39A14 mechanism, suggesting circ_001842 is a potential target for RCC treatment.     

Class I HDACs specifically regulate E‐cadherin expression in human renal epithelial cells

Epithelial‐mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti‐fibrotic effect in various diseases. However, the pathophysiological role of isoform‐specific HDACs or class‐selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK‐2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor β1 (TGF‐β1)‐induced HK2 cells. However, restoration of TGF‐β1‐induced E‐cadherin down‐regulation was only seen in HK‐2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF‐β1‐induced N‐cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E‐cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective.     

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N‐terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP‐like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β‐ d ‐galactosidase‐labeled Gly‐γAbu‐SDKP as a marker antigen, an anti‐rabbit IgG‐coated immunoplate as a bound/free separator and 4‐methylumbelliferyl‐β‐ d ‐galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP‐IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter‐assay and 13.3 and 7.8% for intra‐assay comparisons. Plasma AcSDKP‐IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

YB‐1 orchestrates onset and resolution of renal inflammation via IL10 gene regulation

The Y‐box‐binding protein (YB)‐1 plays a non‐redundant role in both systemic and local inflammatory response. We analysed YB‐1‐mediated expression of the immune regulatory cytokine IL‐10 in both LPS and sterile inflammation induced by unilateral renal ischaemia–reperfusion (I/R) and found an important role of YB‐1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis‐regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB‐1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB‐1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL‐10 mRNA expression in mice following LPS challenge and in I/R. Mice with half‐maximal expression of YB‐1 (Yb1^+/?) had diminished IL‐10 expression upon LPS challenge. In I/R, Yb1^+/? mice exhibited ameliorated kidney injury/inflammation in the early‐phase (days 1 and 5), however showed aggravated long‐term damage (day 21) with increased expression of IL‐10 and other known mediators of renal injury and inflammation. In conclusion, these data support the notion that there are context‐specific decisions concerning YB‐1 function and that a fine‐tuning of YB‐1, for example, via a post‐translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.     

Circular RNA MYLK promotes tumour growth and metastasis via modulating miR‐513a‐5p/VEGFC signalling in renal cell carcinoma

Growing evidence indicates that circular RNAs (circRNAs) are promising biomarkers, as they play significant roles in the development of various cancers. The circular RNA MYLK (circMYLK) has been reported to be involved in the development of malignant tumours, including liver, prostate and bladder cancers. Nevertheless, the biological function of circMYLK in renal cell carcinoma (RCC) remains unclear. In this study, we observed that circMYLK is notably up‐regulated in RCC. Increased circMYLK expression led to a larger tumour size, distant metastasis and poor prognosis of RCC patients. Moreover, circMYLK silencing repressed RCC growth and metastasis in vitro and in vivo. Mechanistically, circMYLK can capture miR‐513a‐5p to facilitate VEGFC expression and further promote the tumorigenesis of RCC cells. In summary, our findings demonstrate that circMYLK has an oncogenic role in RCC growth and metastasis by modulating miR‐513a‐5p/VEGFC signalling. Thus, circMYLK has potential as a diagnostic biomarker and therapeutic target in the treatment of RCC.     

PI3K inhibition potentiates Bcl‐2‐dependent apoptosis in renal carcinoma cells

Inhibitors of PI3‐K/Akt are currently being assessed clinically in patients with advanced RCC. Identification of therapeutic strategies that might enhance the efficacy of PI3‐K/Akt inhibitors is therefore of great interest. As PI3‐K inhibition would be expected to have many pro‐apoptotic effects, we hypothesized that there may be unique synergy between PI3‐K inhibitors and BH3‐mimetics. Towards this end, we assessed the combination of the PI3K inhibitor LY 294002 and the Bcl‐2 family inhibitor ABT‐737 in RCC cell lines. We found that the combinatorial treatment with these agents led to a significant increase in PARP cleavage and cell death in all RCC cell lines. The synergized cell death was correlated with decreased levels of Mcl‐1 and XIAP, and increased levels in Bim, and appears critically dependent upon the activation of caspase 3 and 8. The enhanced lethality observed with the combination also appears dependent upon the regulation of XIAP, Mcl‐1 and Bim levels. Our results suggest that the combination of PI3‐K inhibitors with BH3‐mimetics may be a viable therapeutic strategy in RCC.     

Signalling pathways involved in hypoxia‐induced renal fibrosis

Renal fibrosis is the common pathological hallmark of progressive chronic kidney disease (CKD) with diverse aetiologies. Recent researches have highlighted the critical role of hypoxia during the development of renal fibrosis as a final common pathway in end‐stage kidney disease (ESKD), which joints the scientist's attention recently to exploit the molecular mechanism underlying hypoxia‐induced renal fibrogenesis. The scaring formation is a multilayered cellular response and involves the regulation of multiple hypoxia‐inducible signalling pathways and complex interactive networks. Therefore, this review will focus on the signalling pathways involved in hypoxia‐induced pathogenesis of interstitial fibrosis, including pathways mediated by HIF, TGF‐β, Notch, PKC/ERK, PI3K/Akt, NF‐κB, Ang II/ROS and microRNAs. Roles of molecules such as IL‐6, IL‐18, KIM‐1 and ADO are also reviewed. A comprehensive understanding of the roles that these hypoxia‐responsive signalling pathways and molecules play in the context of renal fibrosis will provide a foundation towards revealing the underlying mechanisms of progression of CKD and identifying novel therapeutic targets. In the future, promising new effective therapy against hypoxic effects may be successfully translated into the clinic to alleviate renal fibrosis and inhibit the progression of CKD.     

EYA1 mutations associated with the branchio‐oto‐renal syndrome result in defective otic development in Xenopus laevis

Background information. The BOR (branchio‐oto‐renal) syndrome is a dominant disorder most commonly caused by mutations in the EYA1 (Eyes Absent 1) gene. Symptoms commonly include deafness and renal anomalies. Results. We have used the embryos of the frog Xenopus laevis as an animal model for early ear development to examine the effects of different EYA1 mutations. Four eya1 mRNAs encoding proteins correlated with congenital anomalies in human were injected into early stage embryos. We show that the expression of mutations associated with BOR, even in the presence of normal levels of endogenous eya1 mRNA, leads to morphologically abnormal ear development as measured by overall otic vesicle size, establishment of sensory tissue and otic innervation. The molecular consequences of mutant eya1 expression were assessed by QPCR (quantitative PCR) analysis and in situ hybridization. Embryos expressing mutant eya1 showed altered levels of multiple genes (six1, dach, neuroD, ngnr‐1 and nt3) important for normal ear development. Conclusions. These studies lend support to the hypothesis that dominant‐negative effects of EYA1 mutations may have a role in the pathogenesis of BOR.     

Urinary exosome miR‐30c‐5p as a biomarker of clear cell renal cell carcinoma that inhibits progression by targeting HSPA5

Exosome‐derived miRNAs are regarded as biomarkers for the diagnosis and prognosis of many human cancers. However, its function in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, differentially expressed miRNAs from urinal exosomes were identified using next‐generation sequencing (NGS) and verified using urine samples of ccRCC patients and healthy donors. Then, the exosomes were analysed in early‐stage ccRCC patients, healthy individuals and patients suffering from other urinary system cancers. Thereafter, the target gene of the miRNA was detected. Its biological function was investigated in vitro and in vivo. The results showed that miR‐30c‐5p could be amplified in a stable manner. Its expression pattern was significantly different only between ccRCC patients and healthy control individuals, but not compared with that of other urinary system cancers, which indicated its specificity for ccRCC. Additionally, the overexpression of miR‐30c‐5p inhibited ccRCC progression in vitro and in vivo. Heat‐shock protein 5 (HSPA5) was found to be a direct target gene of miR‐30c‐5p. The depletion of HSPA5 caused by miR‐30c‐5p inhibition reversed the promoting effect of ccRCC growth. In conclusion, urinary exosomal miR‐30c‐5p acts as a potential diagnostic biomarker of early‐stage ccRCC and may be able to modulate the expression of HSPA5, which is correlated with the progression of ccRCC.     

Bmi‐1 plays a critical role in protection from renal tubulointerstitial injury by maintaining redox balance

To determine whether Bmi-1 deficiency could lead to renal tubulointerstitial injury by mitochondrial dysfunction and increased oxidative stress in the kidney, 3-week-old Bmi-1^-/- mice were treated with the antioxidant N-acetylcysteine (NAC, 1 mg mL⁻¹) in their drinking water, or pyrro-quinoline quinone (PQQ, 4 mg kg⁻¹ diet) in their diet for 2 weeks, and their renal phenotypes were compared with vehicle-treated Bmi1^-/- and wild-type mice. Bmi-1 was knocked down in human renal proximal tubular epithelial (HK2) cells which were treated with 1 mm NAC for 72 or 96 h, and their phenotypes were compared with control cells. Five-week-old vehicle-treated Bmi-1^-/- mice displayed renal interstitial fibrosis, tubular atrophy, and severe renal function impairment with decreased renal cell proliferation, increased renal cell apoptosis and senescence, and inflammatory cell infiltration. Impaired mitochondrial structure, decreased mitochondrial numbers, and increased oxidative stress occurred in Bmi-1^-/- mice; subsequently, this caused DNA damage, the activation of TGF-β1/Smad signaling, and the imbalance between extracellular matrix synthesis and degradation. Oxidative stress-induced epithelial-to-mesenchymal transition of renal tubular epithelial cells was enhanced in Bmi-1 knocked down HK2 cells. All phenotypic alterations caused by Bmi-1 deficiency were ameliorated by antioxidant treatment. These findings indicate that Bmi-1 plays a critical role in protection from renal tubulointerstitial injury by maintaining redox balance and will be a novel therapeutic target for preventing renal tubulointerstitial injury.