A new model to predict acute kidney injury after cardiac surgery in patients with renal insufficiency.

OBJECTIVE: To establish a simple model for predicting postoperative acute kidney injury (AKI) requiring renal replacement therapy (RRT) in patients with renal insufficiency (CKD stages 3-4) who underwent cardiac surgery. METHODS: A total of 330 patients were enrolled. Among them, 226 were randomly selected for the development group and the remaining 104 for the validation group. The primary outcome was AKI requiring RRT. A nomogram was constructed based on the multivariate analysis with variables selected by the application of the least absolute shrinkage and selection operator. Meanwhile, the discrimination, calibration, and clinical power of the new model were assessed and compared with those of the Cleveland Clinic score and Simplified Renal Index (SRI) score in the validation group. Results: The rate of RRT in the development group was 10.6% (n = 24), while the rate in the validation group was 14.4% (n = 15). The new model included four variables such as postoperative creatinine, aortic cross-clamping time, emergency, and preoperative cystatin C, with a C-index of 0.851 (95% CI, 0.779-0.924). In the validation group, the areas under the receiver operating characteristic curves for the new model, SRI score, and Cleveland Clinic score were 0.813, 0.791, and 0.786, respectively. Furthermore, the new model demonstrated greater clinical net benefits compared with the Cleveland Clinic score or SRI score. CONCLUSIONS: We developed and validated a powerful predictive model for predicting severe AKI after cardiac surgery in patients with renal insufficiency, which would be helpful to assess the risk for severe AKI requiring RRT.

Anti-Phospholipase A2 Receptor (Anti-PLA2R) Antibody in Diagnosis and Treatment of Idiopathic Membranous Nephropathy: A Single-Center Observational Study in China.

BACKGROUND The aim of this study was to observe the concentration of serum anti-PLA2R antibody in idiopathic membranous nephropathy (IMN) patients and analyze its relationship with clinical and laboratory parameters. MATERIAL AND METHODS We treated 72 patients with idiopathic membranous nephropathy diagnosed by renal biopsy; all these patients who presented nephrotic syndrome were enrolled for investigation, and then underwent combination therapy with prednisone and cyclosporine A for 6 months. We collected data on 24-h total proteinuria (TUpro), creatinine clearance rate (Ccr), and serum albumin (Alb) levels before and after immunosuppressive treatment. Serum anti-PLA2R antibody was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS Fifty-six out of 72 IMN patients presented positive serum anti-PLA2R antibody. The titer of anti-PLA2R antibody was significantly correlated with both TUpro and serum Alb levels of pre- and post-therapeutic values in IMN (P<0.05), but did not have a relationship with Ccr (P>0.05). In comparison with the anti-PLA2R antibody-negative group, there were significantly higher TUpro and lower Alb levels in the anti-PLA2R antibody-positive group (P<0.05). However, Ccr was comparatively lower in the anti-PLA2R antibody-positive group, but the difference was not statistically significant (P>0.05). There were 24 patients with negative anti-PLA2R antibody and 14 patients had complete remission in the positive anti-PLA2R antibody group, while anti-PLA2R antibody of all 14 patients became negative. Eight out of 16 patients without anti-PLA2R antibody went into complete remission. CONCLUSIONS Serum anti-PLA2R antibody, as determined by non-invasive technique, is a specific biomarker for diagnosis of IMN. Our results suggest that serum anti-PLA2R antibody has great potential to guide clinical diagnosis and treatment, as well as prognosis determination, in IMN patients.

SB-216763, a GSK-3ß inhibitor, protects against aldosterone-induced cardiac, and renal injury by activating autophagy.

Cardiovascular and renal inflammation induced by Aldosterone (Aldo) plays a pivotal role in the pathogenesis of hypertension and renal fibrosis. GSK-3ß contributes to inflammatory cardiovascular and renal diseases, but its role in Aldo-induced hypertension, and renal damage is not clear. In the present study, rats were treated with Aldo combined with SB-216763 (a GSK-3ß inhibitor) for 4 weeks. Hemodynamic, cardiac, and renal parameters were assayed at the indicated time. Here we found that rats treated with Aldo presented cardiac and renal hypertrophy and dysfunction. Cardiac and renal expression levels of molecular markers attesting inflammation and fibrosis were increased by Aldo infusion, whereas the treatment of SB-216763 reversed these alterations. SB-216763 suppressed cardiac and renal inflammatory cytokines levels (TNF-a, IL-1ß, and MCP-1). Meanwhile, SB-216763 increased the protein levels of LC3-II in the cardiorenal tissues as well as p62 degradation, indicating that SB-216763 induced autophagy activation in cardiac, and renal tissues. Importantly, inhibition of autophagy by 3-MA attenuated the role of SB-216763 in inhibiting perivascular fibrosis, and tubulointerstitial injury. These data suggest that SB-216763 protected against Aldo-induced cardiac and renal injury by activating autophagy, and might be a therapeutic option for salt-sensitive hypertension and renal fibrosis.

Down-regulation of IRAK1 attenuates podocyte apoptosis in diabetic nephropathy through PI3K/Akt signaling pathway.

Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus and often results in chronic renal failure. Here, we found that Interleukin 1 receptor associated kinases (IRAK1) was up-regulated in kidney in both DN patients and high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice. In vivo, down regulation of IRAK1 ameliorated renal injury and function, with lower podocyte apoptosis, increased expression of Nephrin, attenuated thickness of the glomerular basement membrane and podocyte footprocess effacement. Furthermore, in vitro, down regulation of IRAK1 in podocytes treated with high glucose (HG), podocyte apoptosis and inflammatory cytokines were significantly decreased, but Nephrin increased. Meanwhile, apoptosis-related genes caspase-3/-9 were inhibited and phosphorylation levels of PI3K/Akt were dramatically down regulated. Thus, IRAK1 is one of the critical components involved in podocyte apoptosis in DN.

Associations of serum soluble klotho and fibroblast growth factor 23 with carotid artery calcification in patients undergoing continuous ambulatory peritoneal dialysis: A retrospective study.

ABSTRACT: This study aimed to assess the associations of serum soluble klotho and fibroblast growth factor 23 (FGF-23) with the occurrence of carotid artery calcification. Peritoneal dialysis patients treated from June 2018 to June 2019 were retrospectively analyzed. They were divided into the carotid artery calcification and non-carotid artery calcification groups according to color Doppler ultrasound findings. Basic indicators in both groups were compared, and the influencing factors of carotid artery calcification were analyzed by logistic regression. Among the 73 continuous ambulatory peritoneal dialysis (CAPD) patients enrolled, 40 (54.8%) had carotid artery calcification. Significant differences were found in age (68.85 ±â€Š7.45 vs 46.62 ±â€Š5.51 years), dialysis time (8.15 ±â€Š1.42 vs 6.02 ±â€Š1.14 months), klotho amounts (325.56 ±â€Š41.15 vs 436.65 ±â€Š45.58 pg/mL) and FGF-23 levels (114.45 ±â€Š15.56 vs 70.15 ±â€Š12.23 pg/mL) between the carotid artery calcification and non-carotid artery calcification groups (all P < .001). The above factors were associated with carotid artery calcification occurrence in univariate analysis. Multivariate analysis showed that elevated age (odds ratio [OR] = 1.55, 95% confidence interval [CI] 1.13-1.74; P = .025) and FGF-23 (OR = 2.16, 95% CI 2.01-2.44; P = .042), and lower klotho (OR = 0.66, 95% CI 0.47-0.85; P = .036) were independent risk factors for carotid artery calcification in CAPD. Serum FGF-23 and age are risk factors for carotid artery calcification in patients with CAPD, whereas klotho is a protective factor.

Elevated soluble CD163 predicts renal function deterioration in lupus nephritis: a cohort study in Eastern China.

OBJECTIVE: This study investigated the association between soluble scavenger receptor differentiation antigen 163 (sCD163) and the severity and prognosis of renal injury in lupus nephritis (LN). METHODS: Serum sCD163 levels in 121 Eastern Chinese patients with LN who underwent renal biopsy were determined by enzyme-linked immunosorbent assays. Clinical data were collected, and the glomerular filtration rate and disease activity score of lupus were calculated. Pathological classification was performed, and renal pathological scores were assessed by the activity index (AI) and chronic index (CI). Kaplan-Meier survival curves were drawn to evaluate prognosis. RESULTS: The pathological classification, AI and CI scores in the high sCD163 group were increased. The sCD163 levels were positively correlated with serum creatinine, blood urea nitrogen, AI scores and CI scores and negatively correlated with the estimated glomerular filtration rate. Kaplan-Meier survival analysis showed that the incidence of renal endpoint events was increased in the high sCD163 group compared with the normal sCD163 group. CONCLUSION: The serum sCD163 level correlates with the severity of LN and is an important indicator of poor renal prognosis in patients with LN.

Coexistence of micro-inflammatory and macrophage phenotype abnormalities in chronic kidney disease.

The heterogeneity of macrophages promotes renal fibrosis and plays an important role in the repair of kidney damage. The "microinflammation state" is closely related to accelerated mortality in patients with chronic kidney disease (CKD). The aim of this study was to investigate the relationship between microinflammation and macrophage polarization in CKD. The levels of high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in peripheral blood of 30 non-dialysis CKD-5 patients (CKD group) and 20 healthy subjects (Con group) were measured. Peripheral mononuclear cells (PBMC) of each group were obtained, induced to differentiate into mature macrophages, and the expression of CD206 on the surface of macrophage M2 was detected. The expression of IL-10, TGF-ß1 and TNF-α in the supernatant of macrophage culture medium was detected by real time RCR and ELISA. We found that the levels of hs-CRP, IL-6 and TNF-α in peripheral blood of patients with CKD were significantly higher than those of the control group. The expression of CD206 in macrophages was significantly decreased in CKD patients. The anti-inflammatory cytokines IL-10 and TGF-ß1 in the supernatant of CKD macrophages decreased significantly, while the pro-inflammatory factor TNF-α did not change significantly. Our results demonstrate that the expressions of macrophage phenotype and anti-inflammatory cytokine in CKD patients are abnormal, which may be related to the microinflammation state prevalent in CKD patients.

BAFF is involved in the pathogenesis of IgA nephropathy by activating the TRAF6/NF­κB signaling pathway in glomerular mesangial cells.

The aim of the present study was to investigate the involvement of B cell­activating factor (BAFF) in the pathogenesis of IgA nephropathy by activating the tumor necrosis factor receptor­associated factor 6 (TRAF6)/NF­κB signaling pathway in glomerular mesangial cells. For the clinical analysis, blood, urine and kidney tissue samples were collected from 58 patients diagnosed with primary IgA nephropathy by renal biopsy. For the in vitro study, glomerular mesangial cells were divided into five groups: Control (con)­short hairpin RNA (shRNA) (control group); con­shRNA + BAFF (20 ng/ml); con­shRNA + BAFF + BAFF­RFc chimera protein (500 µg/ml); TRAF6­shRNA; and TRAF6­shRNA + BAFF (20 ng/ml). For the in vivo experiments, 60 Sprague­Dawley rats were randomly divided into four groups: Con­small interfering RNA (siRNA) (control group); con­siRNA + IgA (IgA nephropathy group), BAFF­RFc chimera protein (2 µg/ml) + IgA, and TRAF6­siRNA (0.2 µM) + IgA. Reverse transcription­quantitative PCR was performed to evaluate the mRNA expression levels of TRAF6, connective tissue growth factor (CTGF), fibronectin (FN) and NF­κBP65. Western blot analysis was used to detect the protein expression levels of TRAF6, FN, CTGF and phosphorylated­NF­κBP65 in glomerular mesangial cells and kidney tissues. The results revealed that plasma BAFF levels were positively correlated with the severity of pathological damage in patients with IgA nephropathy. In vitro, BAFF induced the mRNA and protein expression of TRAF6, CTGF, FN and NF­κBP65 in glomerular mesangial cells. After the BAFF­RFc chimera protein was added to inhibit the binding of BAFF and BAFF­receptor (­R), this effect was reduced. In vivo, inhibition of the effects of BAFF via injection with the BAFF­R Fc chimera protein reduced kidney damage in rats suffering from IgA nephropathy. The effect on the expression of signaling pathway­associated proteins was also alleviated. In conclusion, BAFF enhanced the expression of fibroblast factors in the kidneys by activating the TRAF6/NF­κB signaling pathway.

Resveratrol improves left ventricular remodeling in chronic kidney disease via Sirt1-mediated regulation of FoxO1 activity and MnSOD expression.

Left ventricular remodeling commonly complicates end-stage renal disease following chronic kidney disease (CKD). This study investigated the therapeutic efficacy of resveratrol (RSV), a polyphenolic compound, on left ventricular remodeling in subtotal nephrectomy rats and sought to uncover the underlying molecular mechanisms. Subtotal nephrectomy caused renal dysfunction, such as gradual increases in serum creatinine and blood urea nitrogen, glomerular sclerosis, and tubulointerstitial fibrosis. In addition, subtotal nephrectomy also resulted in significant increases in myocyte cross-sectional area, interstitial and perivascular fibrosis, and left ventricular dilatation. All these detrimental effects were alleviated in the presence of RSV. Mechanistically, RSV treatment led to the upregulation of manganese-containing superoxide dismutase (MnSOD) in the heart. Coimmunoprecipitation studies showed that silent information regulator 1 (Sirt1) bound forkhead box protein O1 (FoxO1) and thus reduced acetylated FoxO1. RSV strengthened this interaction between Sirt1 and FoxO1. Loss of one allele of Sirt1 aggravated renal damage, myocyte hypertrophy, and interstitial fibrosis in nephrectomized mice. Taken together, our data show that Sirt1 is an important mediator for the protective roles of RSV on renal and heart damage in CKD rodent model, and FoxO1 and MnSOD are likely downstream targets of Sirt1. Therefore, Sirt1 might be a potential therapeutic target for the treatment of left ventricular remodeling caused by CKD.

Low triiodothyronine syndrome is associated with platelet function in patients with nephrotic syndrome.

OBJECTIVE: The objective of this study was to investigate the effects of low triiodothyronine syndrome (LT3S) on platelet function and clotting factors in patients with nephrotic syndrome(NS). METHODS: Patients with primary nephrotic syndrome were divided into two groups, normal thyroid function (group A) and LT3S (group B), based on whether they had LT3S or not. Healthy subjects were selected as the control group (group C). Blood coagulation function was detected in each group. The platelet activation function (CD62P, CD63) was determined by flow cytometry. The platelet aggregation rate was detected by an optical method using adenosine diphosphate and arachidonic acid as inducers. RESULTS: The proportion of primary nephrotic syndrome with LT3S was 23.2% (69/298). Compared with group C, group A had higher CD62P and PAgTADP, and group B had higher CD62P, CD63, PAgTAA, and PAgTADP; the difference was statistically significant (all P < 0.05). There was no significant difference in renal pathology between group A and group B (X2 = 4.957, P = 0.421). Compared with group A, the 24-hour urine protein, CD63, PAgTAA, and PAgTADP were higher in group B, and APTT and Alb were lower. The difference was statistically significant (P < 0.05). Logistic regression analysis showed that LT3S was associated with CD36 (OR: 3.516; 95% CI: 1.742~8.186; P = 0.004) and PAgTAA (OR: 0.442; 95% CI: 1.001~1.251; P = 0.037). CONCLUSION: NS patients are prone to LT3S. Patients with LT3S may have abnormal platelet activation and increase of platelet aggregation.

EP4 knockdown alleviates glomerulosclerosis through Smad and MAPK pathways in mesangial cells.

Prostaglandin E2 has exhibited pleiotropic effects in the regulation of glomerulosclerosis progression through its four receptors. The current study aimed to evaluate the effect of prostaglandin receptor EP4 on mesangial cell proliferation. In vivo, 5/6 nephrectomy was introduced into EP4+/­ and wild­type (WT) mice. Clinical parameters were monitored post­surgery. At 8 weeks post­surgery, glomerular fibrosis­associated indicators were measured by immunohistochemical staining and trichrome staining. In vitro, mesangial cells in different groups (transfected with green fluorescent protein, AD­EF4 or AD­CRE) were exposed to transforming growth factor (TGF)­ß1 for 24 h to detect the level of downstream signaling. Corresponding signaling inhibitors were also used to validate the signaling effects. Following surgery, EP4+/­ mice presented a higher survival rate and normal urine volume compared with the WT group, and serum creatinine level and 24 h urine protein were lower in the EP4+/­ mice. Furthermore, associated profibrotic indicators were identified to have decreased at 8 weeks post­surgery along with less tubule­interstitial fibrosis. In vivo, the inhibition of extracellular signal­regulated kinase and P38 phosphorylation alleviated the accumulation of mesangial matrix, and these signals were enhanced when EP4 was overexpressed. EP4 enhancement aggravated imbalanced mesangial cell proliferation stimulated by TGF­ß1 and GS of mice treated with 5/6 nephrectomy through the Smad and mitogen­activated protein kinase pathways.

The protective effect of the EP2 receptor on TGF-ß1 induced podocyte injury via the PI3K / Akt signaling pathway.

Transforming growth factor ß1 (TGF-ß1) plays a central role in chronic kidney diseases. TGF-ß1 induction causes podocyte injury, which results in proteinuria and renal failure. However, the effect of the prostaglandin E2 /E-prostanoid receptor (EP2) on TGF-ß1-induced podocyte injury remains unknown. Previous studies have shown that phosphoinositide 3-OH kinase (PI3K)/Akt is widespread in cells, and is vital for the regulation of cell proliferation, differentiation, apoptosis and metabolism. In this study, we cultured immortalized mouse podocytes in vitro in different groups: control group; TGF-ß1 (5ng/ml) group; EP2 agonist Butaprost treatment (10-7, 10-6, or 10-5mol/L) +TGF-ß1 group; EP2 antagonist AH6809 treatment (10-7, 10-6, or 10-5mol / L) + TGF-ß1 group. We found that compared with the control group, proliferation of podocytes in the TGF-ß1 group significantly decreased and apoptosis increased. Expression of cAMP decreased, whereas PGE2 increased. Meanwhile, expressions of nephrin, podocin and CD2AP mRNA and protein were dramatically downregulated, activated caspase-3 was increased, and activated PI3K/Akt activity were depressed. Butaprost intervention promoted podocyte proliferation with reduced apoptosis. Conversely, AH6809 intervention led to opposite results (P<0.05). Our findings suggested that EP2 agonist protects podocytes by increasing expression of cAMP, which creates feedback of inhibiting PGE2 expression. This causes the interaction of nephrin, podocin and CD2AP resulting the inhibition of apoptosis induced by activation of the PI3K / Akt signaling pathway.

Low triiodothyronine syndrome is associated with platelet function in patients with nephrotic syndrome

SUMMARY OBJECTIVE The objective of this study was to investigate the effects of low triiodothyronine syndrome (LT3S) on platelet function and clotting factors in patients with nephrotic syndrome(NS). METHODS Patients with primary nephrotic syndrome were divided into two groups, normal thyroid function (group A) and LT3S (group B), based on whether they had LT3S or not. Healthy subjects were selected as the control group (group C). Blood coagulation function was detected in each group. The platelet activation function (CD62P, CD63) was determined by flow cytometry. The platelet aggregation rate was detected by an optical method using adenosine diphosphate and arachidonic acid as inducers. RESULTS The proportion of primary nephrotic syndrome with LT3S was 23.2% (69/298). Compared with group C, group A had higher CD62P and PAgTADP, and group B had higher CD62P, CD63, PAgTAA, and PAgTADP; the difference was statistically significant (all P < 0.05). There was no significant difference in renal pathology between group A and group B (X2 = 4.957, P = 0.421). Compared with group A, the 24-hour urine protein, CD63, PAgTAA, and PAgTADP were higher in group B, and APTT and Alb were lower. The difference was statistically significant (P < 0.05). Logistic regression analysis showed that LT3S was associated with CD36 (OR: 3.516; 95% CI: 1.742~8.186; P = 0.004) and PAgTAA (OR: 0.442; 95% CI: 1.001~1.251; P = 0.037). CONCLUSION NS patients are prone to LT3S. Patients with LT3S may have abnormal platelet activation and increase of platelet aggregation.

RESUMO OBJETIVO O objetivo deste estudo foi investigar os efeitos da síndrome do baixo triiodotironina (LT3S) na função plaquetária e nos fatores de coagulação em pacientes com síndrome nefrótica (SN). MÉTODOS Pacientes com síndrome nefrótica primária foram divididos em dois grupos, função tireoidiana normal (grupo A) e LT3S (grupo B), com base na presença ou não de LT3S. Indivíduos saudáveis foram selecionados como grupo de controle (grupo C). A função de coagulação do sangue foi analisada em cada grupo. A função de ativação plaquetária (CD62P, CD63) foi determinada por citometria de fluxo. A taxa de agregação plaquetária foi detectada por um método óptico usando adenosina difosfato e ácido araquidônico como indutores. RESULTADOS A proporção de síndrome nefrótica primária com LT3S foi de 23,2% (69/298). Em comparação com o grupo C, o grupo A apresentou níveis mais altos de CD62P e PAgTADP, e o grupo B apresentou maiores CD62P, CD63, PAgTAA e PAgTADP; a diferença teve significância estatística (P < 0,05). Não houve diferença significativa na patologia renal entre o grupo A e o grupo B (X2 = 4,957, P = 0,421). Em comparação com o grupo A, a proteína em urina de 24 horas, CD63, PAgTAA e PAgTADP foram maiores no grupo B, já APTT e Alb foram mais baixos. A diferença apresentou significância estatística (P < 0,05). A análise de regressão logística mostrou uma associação entre LT3S e CD36 (OR: 3,516; 95% IC: 1,742~8,186; P = 0,004) e PAgTAA (OR: 0,442; 95% IC: 1,001~1,251; P = 0,037). CONCLUSÃO Pacientes com síndrome nefrótica estão propensos à síndrome do baixo triiodotironina (LT3S). Pacientes com LT3S podem ter ativação plaquetária anormal e aumento da agregação plaquetária.

Effects of all-trans retinoic acid on signal pathway of cyclooxygenase-2 and Smad3 in transforming growth factor-ß-stimulated glomerular mesangial cells.

All-trans retinoic acid (ATRA) has been used for the treatment of acute promyelocytic leukemia. It remains unclear, however, whether ATRA affects cyclooxygenase-2 (COX-2; an enzyme involved in prostaglandin production), PGE2, and thromboxane A2 (TXA2) (metabolic products of COX-2) by a transforming growth factor-ß/Smad-signaling pathway, which plays important roles in mesangial-cell proliferation and renal fibrosis. In this study, the mRNA and protein of Smad3, Smad7, and COX-2 were detected by reverse transcription-polymerase chain reaction and Western blot, respectively, in mesangial cells stimulated by transforming growth factor-ß (TGF-ß) and treated with ATRA at various concentrations and times. The protein level of PGE2 and TXA2 was also measured by enzyme-linked immunosorbent assay. The localization of Smad3 and Smand7 was observed by confocal microscope. Cell proliferation was detected by MTT assay, while apoptosis was determined using Hoechest staining. The expression of Smad3, Smad7, and COX-2 mRNA and protein was increased by exogenous TGF-ß, but inhibited by pretreatment of ATRA, in dose and time-dependent manners. In addition, the expression of Smad3 and Smad7 was significantly reduced not only by staurosporine, an inhibitor of threonine/serine protein kinases as well as smad, but also by NS-398, an inhibitor of COX-2. PGE2 and TXA2 were raised by TGF-ß, but also decreased by ATRA, staurosporine, and NS-398. Moreover, ATRA reversed the translocation of Smad3 and Smad7 induced by TGF-ß. Compared with the control, TGF-ß also significantly enhanced proliferation and inhibited apoptosis of mesangial cells. ATRA dose-dependently inhibited TGF-ß-induced cell proliferation, but had no significant effect on apoptosis in rat mesangial cells. Therefore, ATRA repressed COX-2, PGE2, and TXA2 via the TGF-ß/Smad-signaling pathway and inhibited mesangial-cell proliferation, which might subsequently prevent renal fibrosis.

[Isolation, primary culture and characterization of mouse glomerular mesangial cells].

OBJECTIVE: To establish a method for the isolation, primary culture and characterization of mouse mesangial cells (MCs). METHODS: We used two-layer micropore filter device to isolate glomeruli, and the MCs were cultured with eugenic selection method. Cell structures of MCs were observed by inverted phase contrast microscope, HE staining, scanning electron microscope and transmission electron microscope. Expressions of α-smooth muscle actin (α-SMA and nephrin were investigated by immunofluorescent cytochemistry. We used angiotensin II to stimulate MCs and observed the biological characteristics of the cells. The growth curve of MCs was examined by CCK-8 assay. RESULTS: After 20-30 days, the primary cultured MCs gradually covered the bottom of the dish. The mouse MCs usually attached to the surface 6-10 hours after passage, followed by the exponential phase in 2-3 days and the platform period in 3-5 days. Immunofluorescent staining showed that the expression of α-SMA was positive and nephrin was negative. MCs contraction was observed after the cells were stimulated by angiotensin II for 15 minutes. CONCLUSION: A method for the isolation, culture and characterization of mouse mesangial cells has been successfully established.

All-trans retinoic acid inhibits mesangial cell proliferation by up-regulating p21Waf1/Cip1 and p27Kip1 and down-regulating Skp2.

BACKGROUND: The aim of this study was to examine effects of all-trans retinoic acid (ATRA) on rat mesangial cell proliferation, apoptosis and underlying mechanisms. METHODS: Cultured HBZY-1 rat mesangial cells received the following treatments: group 1: controls (DMSO); group 2: TGF-beta(1) (10 µg/L); groups 3-5: ATRA (0.1, 1.0 and 10 µmol/L) + TGF-beta(1) (10 µg/L). After treatments, the cells were studied by CCK-8 assay, cell cycle assay and TUNEL staining. p21(Waf1/Cip1), p27(Kip1) and Skp2 mRNA levels were measured by real-time PCR. p21(Waf1/Cip1), p27(Kip1) and Skp2 protein levels were detected by Western blot. The localization of p21(Waf1/Cip1) and p27(Kip1) proteins was observed by immunofluorescence and confocal microscopy. RESULTS: Compared with controls, TGF-beta(1) significantly enhanced proliferation and inhibited apoptosis of mesangial cells. ATRA dose-dependently reversed both TGF-beta(1)-induced decreases in p21(Waf1/Cip1) mRNA and protein levels and p27(Kip1) protein level and increases in Skp2 mRNA and protein levels, and inhibited TGF-beta(1)-induced proliferation through G(1) arrest. Meanwhile, after ATRA treatments, p21(Waf1/Cip1) and p27(Kip1) proteins mainly localized in the nucleus, and their concentrations in cytoplasm decreased. CONCLUSION: ATRA inhibited TGF-beta(1)-induced mesangial cell proliferation by up-regulating p21(Waf1/Cip1) mRNA and protein levels, and p27(Kip1) protein level, and down-regulating Skp2 mRNA and protein levels, but it had no significant effect on apoptosis in rat mesangial cells.