c-Abl mediates angiotensin II-induced apoptosis in podocytes

Angiotensin II (Ang II) has been reported to cause podocyte apoptosis in rats both in vivo and in vitro studies. However, the underlying mechanisms are poorly understood. In the present study, we investigated the role of the nonreceptor tyrosine kinase c-Abl in Ang II-induced podocyte apoptosis. Male Sprague–Dawley rats in groups of 12 were administered either Ang II (400 kg/kg/min) or Ang II + STI-571 (50 mg/kg/day) by osmotic minipumps. In addition, 12 rats-receiving normal saline served as the control. Glomeruli c-Abl expression was carried out by real time PCR, Western blotting and immunolabeled, and occurrence of apoptosis was carried out by TUNEL staining and transmission electron microscopic analysis. In vitro studies, conditionally immortalized mouse podocytes were treated with Ang II (10^?9–10^?6 M) in the presence or absence of either c-Abl inhibitor, Src-I1, specific c-Abl siRNA, or c-Abl plasmid alone. Quantification of podocyte c-Abl expression and c-Abl phosphorylation at Y245 and Y412 was carried out by real time PCR, Western blotting and immunofluorescence imaging. The nuclear c-Abl and p53 were quantified by co-immunoprecipitation and Western blotting studies. Podocyte apoptosis was analysed by flow cytometry and Hoechst-33342 staining. c-Abl expression was demonstrated in rat kidney podocytes in vivo and cultured mouse podocytes in vitro. Ang II-receiving rats displayed enhanced podocyte c-Abl expression. And Ang II significantly stimulated c-Abl expression in cultured podocytes. Furthermore Ang II upregulated podocyte c-Abl phosphorylation at Y245 and Y412. Ang II also induced an increase of nuclear p53 protein and nuclear c-Abl-p53 complexes in podocytes and podocyte apoptosis. Down-regulation of c-Abl expression by c-Abl inhibitor (Src-I1) as well as specific siRNA inhibited Ang II-induced podocyte apoptosis; conversely, podoctyes transfected with c-Abl plasmid displayed enhanced apoptosis. These findings indicate that c-Abl may mediates Ang II-induced podocyte apoptosis, and inhibition of c-Abl expression can protect podocytes from Ang II-induced injury.     

Estrogen receptor alpha expression in podocytes mediates protection against apoptosis in-vitro and in-vivo

Context/Objective

Epidemiological studies have demonstrated that women have a significantly better prognosis in chronic renal diseases compared to men. This suggests critical influences of gender hormones on glomerular structure and function. We examined potential direct protective effects of estradiol on podocytes.

Methods

Expression of estrogen receptor alpha (ERα) was examined in podocytes in vitro and in vivo. Receptor localization was shown using Western blot of separated nuclear and cytoplasmatic protein fractions. Podocytes were treated with Puromycin aminonucleoside (PAN, apoptosis induction), estradiol, or both in combination. Apoptotic cells were detected with Hoechst nuclear staining and Annexin-FITC flow cytometry. To visualize mitochondrial membrane potential depolarization as an indicator for apoptosis, cells were stained with tetramethyl rhodamine methylester (TMRM). Estradiol-induced phosphorylation of ERK1/2 and p38 MAPK was examined by Western blot. Glomeruli of ERα knock-out mice and wild-type controls were analysed by histomorphometry and immunohistochemistry.

Results

ERα was consistently expressed in human and murine podocytes. Estradiol stimulated ERα protein expression, reduced PAN-induced apoptosis in vitro by 26.5±24.6% or 56.6±5.9% (flow cytometry or Hoechst-staining, respectively; both p<0.05), and restored PAN-induced mitochondrial membrane potential depolarization. Estradiol enhanced ERK1/2 phosphorylation. In ERα knockout mice, podocyte number was reduced compared to controls (female/male: 80/86 vs. 132/135 podocytes per glomerulus, p<0.05). Podocyte volume was enhanced in ERα knockout mice (female/male: 429/371 µm³ vs. 264/223 µm³ in controls, p<0.05). Tgfβ1 and collagen type IV expression were increased in knockout mice, indicating glomerular damage.

Conclusions

Podocytes express ERα, whose activation leads to a significant protection against experimentally induced apoptosis. Possible underlying mechanisms include stabilization of mitochondrial membrane potential and activation of MAPK signalling. Characteristic morphological changes indicating glomerulopathy in ERα knock-out mice support the in vivo relevance of the ERα for podocyte viability and function. Thus, our findings provide a novel model for the protective influence of female gender on chronic glomerular diseases.     

Calcium entry via TRPC6 mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes

Albumin, which is the most abundant component of urine proteins, exerts injurious effects on renal cells in chronic kidney diseases. However, the toxicity of albumin to podocytes is not well elucidated. Here, we show that a high concentration of albumin triggers intracellular calcium ([Ca²⁺]_i) increase through mechanisms involving the intracellular calcium store release and extracellular calcium influx in conditionally immortalized podocytes. The canonical transient receptor potential-6 (TRPC6) channel, which is associated with a subset of familial forms of focal segmental glomerulosclerosis (FSGS) and several acquired proteinuric kidney diseases, was shown to be one of the important Ca²⁺ permeable ion channels in podocytes. Therefore we explored the role of TRPC6 on albumin-induced functional and structural changes in podocytes. It was found that albumin-induced increase in [Ca²⁺]_i was blocked by TRPC6 siRNA or SKF-96365, a blocker of TRP cation channels. Long-term albumin exposure caused an up-regulation of TRPC6 expression in podocytes, which was inhibited by TRPC6 siRNA. Additionally, the inhibition of TRPC6 prevented the F-actin cytoskeleton disruption that is induced by albumin overload. Moreover, albumin overload induced expression of the endoplasmic reticulum (ER) stress protein GRP78, led to caspase-12 activation and ultimately podocyte apoptosis, all of which were abolished by the knockdown of TRPC6 using TRPC6 siRNA. These results support the view that albumin overload may induce ER stress and the subsequent apoptosis in podocytes via TRPC6-mediated Ca²⁺ entry.     

ICOS mediates the development of insulin-dependent diabetes mellitus in nonobese diabetic mice

Initiation of diabetes in NOD mice can be mediated by the costimulatory signals received by T cells. The ICOS is found on Ag-experienced T cells where it acts as a potent regulator of T cell responses. To determine the function of ICOS in diabetes, we followed the course of autoimmune disease and examined T cells in ICOS-deficient NOD mice. The presence of ICOS was indispensable for the development of insulitis and hyperglycemia in NOD mice. In T cells, the deletion of ICOS resulted in a decreased production of the Th1 cytokine IFN-gamma, whereas the numbers of regulatory T cells remained unchanged. We conclude that ICOS is critically important for the induction of the autoimmune process that leads to diabetes.     

MiRNA-1271-5p regulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting forkhead box O1 (FOXO1)

Forkhead box O1 (FOXO1) is a key regulator of osteogenesis. The aim of this study was to identify the mechanisms of microRNAs (miRNAs) targeting FOXO1 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). Three miRNA target prediction programs were used to search for potential miRNAs that target FOXO1. Quantitative real-time polymerase chain reaction was conducted to detect the expression of miR-1271-5p and FOXO1 during osteogenic differentiation. Target gene prediction and screening, luciferase reporter assay was used to verify the downstream target gene of miR-1271-5p. The expression levels of FOXO1 and Runx2 were detected by RT-qPCR and Western blot analysis. Alkaline phosphatase (ALP) activity and matrix mineralization were detected by biochemical methods. The expression levels of Runx2, ALP, and osteocalcin were detected by RT-qPCR. Our results showed that miR-1271-5p was downregulated during osteogenic induction. And the expression levels of miR-1271-5p were higher in osteoporotic tissues than that in adjacent nonosteoporotic tissues. The expression levels of FOXO1 were lower in osteoporotic tissues than that in adjacent nonosteoporotic tissues. And a negative correlation was found between miR-1271-5p and FOXO1 in osteoporotic tissues. Overexpression of miR-1271-5p downregulated FOXO1 and inhibited osteogenic differentiation in hMSCs. Overexpression of miR-1271-5p downregulated the expression of osteogenic markers and reduced ALP activity. In addition, ectopic expression of FOXO1 reversed the effect of miR-1271-5p on osteogenic differentiation. In conclusion, miR-1271-5p functioned as a therapeutic target of osteogenic differentiation in hMSCs by inhibiting FOXO1, which provides valuable insights into the use of miR-1271-5p as a target in the treatment of osteoporosis and other bone metabolic diseases.     

Platelet factor 4 (PF4) and heparin released platelet factor 4 (HR-PF4) in diabetes mellitus. Effect of the duration of the disease

Several investigators have reported an altered platelet function in diabetes mellitus as measured by elevated levels of platelet specific proteins platelet factor 4 (PF4) and B-thromboglobulin (BTG). We studied 20 insulin dependent (IDD), 20 non insulin dependent (NIDD) diabetic males without overt clinical symptoms of cardiovascular disorders and 30 normal controls. We evaluated PF4, BTG and heparin released platelet factor 4 (HR-PF4) as measured 2.5 minutes after a bolus injection of 5,000 I.U. of a commercial mucous heparin. The patients showed normal levels of both PF4 and BTG. Furthermore HR-PF4 failed to show statistically significant variation between patients and controls. However when the diabetics were divided on the basis of the duration of the disease, the IDD had an increased HR-PF4 mean level and the trend became statistically significant when diabetes existed more than 17 years (patients HR-PF4 149.1 ng/ml, range 17.3-194; controls HR-PF4 110.9 ng/ml range 50-160, less than p less than 0.05). NIDD failed to reveal the same pattern. Although the significance of HR-PF4 is unknown, insulin dependent diabetes mellitus after many years could cause a potentially dangerous, silent vascular damage with enhanced platelet vessel wall interaction as measured by an elevated HR-PF4.     

Immune dysfunction in patients with diabetes mellitus (DM)

Patients with diabetes mellitus (DM) have infections more often than those without DM. The course of the infections is also more complicated in this patient group. One of the possible causes of this increased prevalence of infections is defects in immunity. Besides some decreased cellular responses in vitro, no disturbances in adaptive immunity in diabetic patients have been described. Different disturbances (low complement factor 4, decreased cytokine response after stimulation) in humoral innate immunity have been described in diabetic patients. However, the clinical relevance of these findings is not clear. Concerning cellular innate immunity most studies show decreased functions (chemotaxis, phagocytosis, killing) of diabetic polymorphonuclear cells and diabetic monocytes/macrophages compared to cells of controls. In general, a better regulation of the DM leads to an improvement of these cellular functions. Furthermore, some microorganisms become more virulent in a high glucose environment. Another mechanism which can lead to the increased prevalence of infections in diabetic patients is an increased adherence of microorganisms to diabetic compared to nondiabetic cells. This has been described for Candida albicans. Possibly the carbohydrate composition of the receptor plays a role in this phenomenon.     

Cellular immunity in insulin-dependent diabetes mellitus (type 1)

Cell-mediated immunity was investigated with T-cell blastic transformation stimulated by phytohaemagglutinin and/or insulin in patients with diabetes mellitus type 1. T-cell blastic transformation was determined in the whole blood by the intake of labelled thymidine intake by the lymphocytic DNA. Healthy individuals and patients with diabetes mellitus type 2 served as control groups. It was found that T-cell blastic transformation stimulated with phytohaemagglutinin is markedly diminished in patients with diabetes mellitus type 1 and to a lesser degree in patients with diabetes mellitus type 2. Insulin increased T-cell blastic transformation in insulin-dependent diabetic patients but has no effect in diabetes mellitus type 2. The obtained results suggest that induction and central phases of the cell-mediated immunological response are diminished in diabetes mellitus independently on its type. Such disorders may have different etiology depending on the type of diabetes mellitus.