Xuejing Zhu

Low-dose paclitaxel ameliorates fibrosis in the remnant kidney model by down-regulating miR-192

Transforming growth factor (TGF)‐β has been shown to play a central role in the development of tubulointerstitial fibrosis, which can be corrected via treatment with paclitaxel. The biology of microRNA (miR) can be modulated by paclitaxel. We hypothesized that paclitaxel may attenuate renal fibrosis in a rat model of remnant kidney disease by inhibiting TGF‐β induced‐miRs. Rats in groups of 12 were subjected to 5/6 nephrectomy and received low‐dose intraperitoneal injection of paclitaxel. Renal functions were assessed at 8 weeks. The TGF‐β signalling cascade and ECM proteins were evaluated by real‐time polymerase chain reaction (TRT–PCR) and immunofluorescence microscopy. Animals with remnant kidneys developed hypertension, which was not relieved with paclitaxel treatment. However, paclitaxel treatment resulted in dampening the proteinuric response, reduction in serum BUN, creatinine levels and urine protein : creatinine ratio and normalization of creatinine clearance. These effects were accompanied by the inhibition of Smad2/3 activation, attenuation of renal fibrosis and normalization of integrin‐linked kinase (ILK), COL(I)A1, COL(IV)A2 and α‐SMA expression. Also, paclitaxel down‐regulated the expression of miR‐192, miR‐217 and miR ‐377, while miR‐15 was up‐regulated in the remnant kidney. In vitro, in tubular epithelial cells (NRK‐52E), paclitaxel also inhibited TGF‐β1‐induced Smad2/3 activation and normalized ILK, COL(I)A1, COL(IV)A2 and α‐SMA expression. Furthermore, ChIP analyses indicated that Taxol suppressed Smad3‐mediated miR‐192 transcriptional activity. Over‐expression of miR‐192 in NRK‐52E mimicked the changes seen in the remnant kidney, while inclusion of miR‐192 inhibitor in the culture medium blocked TGF‐β1‐induced COL(I)A1 and COL(IV)A2 expression, while ILK and α‐SMA were unaffected. These data suggest that low‐dose paclitaxel ameliorates renal fibrosis via modulating miR‐192 pathobiology and TGF‐β/Smad signalling. Copyright

Rap1 ameliorates renal tubular injury in diabetic nephropathy

Rap1b ameliorates high glucose (HG)-induced mitochondrial dysfunction in tubular cells. However, its role and precise mechanism in diabetic nephropathy (DN) in vivo remain unclear. We hypothesize that Rap1 plays a protective role in tubular damage of DN by modulating primarily the mitochondria-derived oxidative stress. The role and precise mechanisms of Rap1b on mitochondrial dysfunction and of tubular cells in DN were examined in rats with streptozotocin (STZ)-induced diabetes that have Rap1b gene transfer using an ultrasound microbubble-mediated technique as well as in renal proximal epithelial tubular cell line (HK-2) exposed to HG ambiance. The results showed that Rap1b expression decreased significantly in tubules of renal biopsies from patients with DN. Overexpression of a constitutively active Rap1b G12V notably ameliorated renal tubular mitochondrial dysfunction, oxidative stress, and apoptosis in the kidneys of STZ-induced rats, which was accompanied with increased expression of transcription factor C/EBP-β and PGC-1α. Furthermore, Rap1b G12V also decreased phosphorylation of Drp-1, a key mitochondrial fission protein, while boosting the expression of genes related to mitochondrial biogenesis and antioxidants in HK-2 cells induced by HG. These effects were imitated by transfection with C/EBP-β or PGC-1α short interfering RNA. In addition, Rap1b could modulate C/EBP-β binding to the endogenous PGC-1α promoter and the interaction between PGC-1α and catalase or mitochondrial superoxide dismutase, indicating that Rap1b ameliorates tubular injury and slows the progression of DN by modulation of mitochondrial dysfunction via C/EBP-β–PGC-1α signaling.

MicroRNA-129-5p modulates epithelial-to-mesenchymal transition by targeting SIP1 and SOX4 during peritoneal dialysis

Peritoneal dialysis (PD) is the most readily feasible home-dialysis method for renal replacement therapy. However, repeated use of PD can lead to induction of mesothelial/epithelial–mesenchymal transition (MMT/EMT) and fibrosis, eventually leading to ultrafiltration failure and discontinuation of PD. MicroRNA-129-5p (miR-129-5p) is believed to be a potent downstream inhibitor of TGF-β1 in renal fibrosis, but the effect of miR-129-5p on MMT/EMT relevant to PD is unknown. In this study, as determined by microRNA array analysis and confirmed by northern blot analysis and real-time PCR, we demonstrate that miRNA-129-5p is decreased in mesothelial cells isolated from effluent of patients having PD for more than 6 months extending to several years compared with those who have undergone PD for less than 6 months. The decreased expression of miR-129-5p was accompanied with alterations in EMT-related genes and the expression of respective proteins in vivo. In addition, in in vitro studies we noted that the expression of E-cadherin and claudin-1 were significantly reduced with increased cell migration in HMrSV5, a human peritoneal mesothelial cell line (HPMC), treated with TGF-β1, whereas expression of vimentin, fibronectin and transcription factors SIP1 and SOX4 increased significantly, as assessed by real-time PCR, western blot analysis and immunofluorescence microscopy. Furthermore, alteration in EMT-related genes and proteins were reversed by overexpression of miR-129-5p. No effect was observed in cells treated with miR-negative control. Meanwhile, inhibition of SIP1 and SOX4 with their respective siRNA also could decrease the expression of EMT-related genes and protein levels in HPMCs induced with TGF-β1. Finally, we demonstrate that SIP1 can inhibit the promoter activity of E-cadherin while enhancing the promoter activity of vimentin. We also observed that miR-129-5p could directly target the 3′UTR of SIP1 and SOX4 genes, and repressed their post-transcriptional activities. These data suggest that there is a novel TGF-β1/miR-129-5p/SIP-1 or SOX4 pathway that has a significant role in MMT and fibrosis in the setting of PD.

p66Shc: A novel biomarker of tubular oxidative injury in patients with diabetic nephropathy

Increased p66Shc expression has been associated with diabetic nephropathy (DN). However, whether p66Shc can serve as a potential biomarker for tubular oxidative injury in DN is unknown. We measured the expression of p66Shc in peripheral blood monocytes (PBMs) and renal biopsy tissues from DN patients and then analysed the relationship between p66Shc expression and the clinical characteristics of patients with DN. Patients were divided into 4 groups (class IIa, class IIb, class III and the control group). qPCR, Western blotting and immunohistochemistry were performed. The results showed that both p66Shc and p-p66Shc expression significantly increased in PBMs and kidney tissues of DN patients. Moreover, Spearman’s correlation and multiple regression analyses were carried out. A positive relationship between the p66Shc expression and oxidative stress was found. p66Shc and oxidative stress were significant predictors of the degree of tubular damage. In addition, p66Shc expression was positively correlated with the concentrations of β-NAG, UACR and 8-OHdG, low-density lipoprotein and blood glucose levels, and duration of diabetes in patients with DN from class IIa to class III. These data indicated that increased expression of p66Shc may serve as a therapeutic target and a novel biomarker of DN.

Smad Anchor for Receptor Activation Regulates High Glucose-Induced EMT via Modulation of Smad2 and Smad3 Activities in Renal Tubular Epithelial Cells.

Background: SARA is an essential adaptor protein in TGF-β1 signaling and it is also involved in the process of Epithelial-mesenchymal transition (EMT) and fibrosis. Our aim was to investigate the effect of SARA on high glucose (HG)-induced EMT and extracellular matrix (ECM) synthesis in renal tubular epithelial cells. Methods: The cells were transfected with the following plasmids: wild-type SARA (SARA-WT), SARA-dSBD (SARA with Smad binding domain deletion) and then subjected to HG ambience (30 mM). The expression levels were assessed by Western-blot, real-time PCR and immunofluorescence. Results: HG-induced EMT phenotype with increased expression of ECM protein in HK-2 cells. This was associated with the decreased expression of SARA and Smad2. In comparison with the HG group, overexpression of SARA in HK-2 cells, a relatively high upregulation of ZO-1 expression was seen, while that of Vimentin, fibronectin and collagen I was decreased. The Smad2 protein expression was increased in HK-2 cells after transfection with SARA (WT) plasmid. Interestingly, the overexpression of SARA prolonged the activity period of Smad2 and shortened that of Smad3, which seemed consistent with the change of EMT phenotype and ECM changes in HK-2 cell induced by HG. Conclusions: SARA regulates HG-induced EMT and ECM excretion via modulation of the activation of Smad2 and Smad3 in renal tubular epithelial cells. In view of these findings, it is conceivable that SARA may serve as a potential novel target in pre-EMT states for the amelioration renal fibrosis seen in chronic kidney diseases.

Validation of the interstitial fibrosis and tubular atrophy on the new pathological classification in patients with diabetic nephropathy: A single-center study in China.

OBJECTIVES The association between interstitial fibrosis and tubular atrophy (IFTA) and the clinical outcomes in diabetic nephropathy (DN) remains unclear. This study is to evaluate the clinical predictors and renal prognosis of IFTA score in patients with DN. METHODS 52 cases with DN with renal biopsy were divided into three groups according to IFTA score. The χ2 test or Fishers exact test, Mann-Whitney U-test, Kruskal-Wallis H-test and Spearmans correlation analysis were used in this subject. Ordinal regression mode was utilized to evaluate which clinical factors might be the predictors of IFTA score. RESULTS Compared to IFTA score 1 group, the patients in score 3 were younger and have greatly lower level of eGFR and hemoglobin and higher serum creatinine (p<0.01). A close relationship between the clinical findings and IFTA was observed, such as IFTA with eGFR(r=-0.58, P<0.01), triglyceride(r=-0.29, P=0.04), Hb (r=-0.38, P<0.01), systolic blood pressure (r=0.29, P=0.04) and urinary protein level (r=0.46, P<0.01); in addition, eGFR (OR 0.31 (95%Cl -1.883 to -0.485) p=0.001) showed statistical significance with IFTA. The 5-year renal survival rate was estimated as 100% in IFTA score 0, 88.9% in score 1, 76.9% in score 2, and 20.0% in score 3. Furthermore, greatly lower level of eGFR, and higher serum creatinine and BUN in the glomerular class IV were seen (p<0.01 vs class II), with positive correlation with IFTA (r=0.51, P<0.01). CONCLUSION The renal pathologic diagnosis in IFTA score was a good predictor for renal prognosis in type II DM. eGFR might be a predictor of IFTA in patients with DN.

Probucol ameliorates renal injury in diabetic nephropathy by inhibiting the expression of the redox enzyme p66Shc

Aims Probucol is an anti-hyperlipidemic agent and a potent antioxidant drug that can delay progression of diabetic nephropathy (DN) and reverses renal oxidative stress in diabetic animal models; however, the mechanisms underlying these effects remain unclear. p66Shc is a newly recognized mediator of mitochondrial ROS production in renal cells under high-glucose (HG) ambience. We previously showed that p66Shc can serve as a biomarker for renal oxidative injury in DN patients and that p66Shc up-regulation is correlated with renal damage in vivo and in vitro. Here, we determined whether probucol ameliorates renal injury in DN by inhibiting p66Shc expression. Results We found that the expression of SIRT1, Ac-H3 and p66Shc in kidneys of DN patients was altered. Also, probucol reduced the levels of serum creatinine, urine protein and LDL-c and attenuated renal oxidative injury and fibrosis in STZ induced diabetic mice. In addition, probucol reversed p-AMPK, SIRT1, Ac-H3 and p66Shc expression. Correlation analyses showed that p66Shc expression was correlated with p-AMPK and Sirt1 expression and severity of renal injury. In vitro pretreatment of HK-2 cells with p-AMPK and SIRT1 siRNA negated the beneficial effects of probucol. Furthermore, we noted that probucol activates p-AMPK and Sirt1 and inhibits p66shc mRNA transcription by facilitating the binding of Sirt1 to the p66Shc promoter and modulation of Ac-H3 expression in HK-2 cells under HG ambience. Innovation and conclusion Our results suggest for the first time that probucol ameliorates renal damage in DN by epigenetically suppressing p66Shc expression via the AMPK-SIRT1-AcH3 pathway.

Normoalbuminuric diabetic kidney disease

Diabetic kidney disease (DKD) is one of the primary causes of end-stage renal disease (ESRD). Early diagnosis is very important in preventing the development of DKD. Urinary albumin excretion rate (UAER) and glomerular filtration rate (GFR) are widely accepted as criteria for the diagnosis and clinical grading of DKD, and microalbuminuria has been recommended as the first clinical sign of DKD. The natural history of DKD has been divided into three stages: normoalbuminuria, microalbuminuria, and macroalbuminuria. However, this clinical paradigm has been questioned recently, as studies have shown that a portion of diabetes mellitus (DM) patients with normoalbuminuria have progressive renal insufficiency, referred to as normoalbuminuric diabetic kidney disease (NADKD) or nonalbuminuric diabetic nephropathy. Epidemiologic research has demonstrated that normoalbuminuric diabetic kidney disease is common, and the large number of NADKD patients suggests that the traditional paradigm needs to be shifted. Currently, the pathogenesis of NADKD remains unclear, but many clinical studies have identified some clinical and pathological features of NADKD. In addition, the long-term outcomes of NADKD patients remain controversial. In this article, we reviewed the latest studies addressing the pathogenesis, pathology, treatment and prevention of NADKD.

Evaluation of Renal Clinicopathological Changes in IgA Nephropathy by Urinary Podocytes Excretion and Podocalyxin Expression

Objective: To explore the association of urinary podocyte excretion and renal expression of podocyte-specific marker podocalyxin (PCX) with clinicopathological changes in immunoglobulin A nephropathy (IgAN). Methods: Morning urine samples from IgAN patients and healthy controls were collected. The expression of glomerular PCX was quantified in 50 IgAN patients diagnosed by renal biopsy. IgAN was classified based on the Lee’s Grading system and scored according to the Katafuchi semiquantitative criteria. Morphological evaluation of podocyte was determined by electron microscopy. Results: The amount of urinary podocytes in the IgAN patients was significantly higher than that in the healthy controls (p < 0.01). Pairwise comparison among Lee’s grades of IgAN showed that the median of urinary podocytes in Lee’s I–II group was lower than that in Lee’s III, IV, and V groups (p < 0.05); group III lower than group V (p < 0.05). The positive rate of urinary podocytes was the highest in Lee’s IV and V groups (100%), and lowest in Lee’s I–II group (55%). Multiple comparison among groups of Lee’s grades of IgAN showed that the glomerular PCX expression in Lee’s I–II group was higher than that in Lee’s III, IV, and V groups (p < 0.05); groups III and IV higher than group V (p < 0.05). The amount of urinary podocytes in IgAN patients was negatively correlated with PCX expression (r = −0.702, p < 0.01), but positively correlated with 24-h urinary protein (r = 0.465, p < 0.01) and glomerular (r = 0.233, p < 0.01) and renal tubular pathological scores (r = 0.307, p < 0.05). The glomerular PCX expression was negatively correlated with 24-h urinary protein (r = −0.367, p < 0.05) and glomerular (r = −0.560, p < 0.05) and tubular pathological scores (r = −0.377, p < 0.05). Electron microscopy showed significant changes in podocytes of IgAN, especially in the foot process. Conclusion: The amount of urinary podocyte can reflect the loss of podocytes in renal tissue, which may be a marker of IgAN progression.

Cognitive Emotion Regulation Questionnaire in hypertensive patients

OBJECTIVE To examine the reliability,validity,and practicability of Cognitive Emotion Regulation Questionnaire (CERQ) in hypertensive patients in China. METHODS Altogether 434 hypertensive patients and 462 healthy subjects were recruited. All the subjects were assessed with the CERQ-Chinese version (CERQ-C), Dysfunctional Attitude Scale (DAS), Mood and Anxiety Symptom Questionnaire-Short Form (MASQ-SF), and Center for Epidemiologic Studies Depression Scale (CES-D). We calculated the mean inter-item correlations for the total CERQ and for each of the subscales. Cronbachs alpha coefficient was used to analyze the inter-correlation and reliability, and confirmatory factor analysis was used to examine the 9-factor model. RESULTS 1) Hypertension group reported significantly higher score than that of healthy ones on rumination (12.19 ± 2.51 vs. 11.51 ± 2.60, P<0.001), catastrophizing(8.82 ± 2.19 vs.8.11 ± 2.70,P<0.001),and blaming others(10.76 ± 2.11 vs. 9.88 ± 2.48,P<0.001), and had significantly lower score than that of healthy ones on positive reappraisal(13.80 ± 3.55 vs.14.71 ± 4.11,P<0.001).2)Reliability:In the hypertension group the Cronbachs alpha for the total CERQ was 0.80, and that for the 9 subscales ranged from 0.71 (self-blame) to 0.90 (rumination). In the healthy group the Cronbachs alpha for the total CERQ was 0.79, and that for the 9 subscales ranged from 0.71 (positive reappraisal) to 0.90 (rumination). The mean inter-item correlation coefficient for the 9 subscales was 0.21-0.42(the hypertension group)/0.19-0.32 (the healthy group). In the hypertension group,the test-retest reliability of the total scale was 0.82, the test-retest reliability of the 9 subscales ranged from 0.73 to 0.92. The confirmatory factor analysis showed that the 9 first-order factor data fitted both 2 samples well. CONCLUSION CERQ meets the psychometric standard and it is reliable and valid for cognitive emotion regulation strategies, which may be regarded as an appropriate assessment tool.