Store-Operated Ca^(2+) Channels in Mesangial Cells Inhibit Matrix Protein Expression

Accumulation of extracellular matrix derived from glomerular mesangial cells is an early feature of diabetic nephropathy. Ca^(2+) signals mediated by store–operated Ca^(2+) channels regulate protein production in a variety of cell types. The aim of this study was to determine the effect of store–operated Ca^(2+) channels in mesangial cells on extracellular matrix protein expression. In cultured human mesangial cells, activation of store–operated Ca^(2+) channels by thapsigargin significantly decreased fibronectin protein expression and collagen IV mRNA expression in a dose-dependent manner. Conversely, inhibition of the channels by 2-aminoethyl diphenylborinate significantly increased the expression of fibronectin and collagen IV. Similarly, overexpression of stromal interacting molecule 1 reduced, but knockdown of calcium release–activated calcium channel protein 1 (Orai1) increased fibronectin protein expression. Furthermore, 2-aminoethyl diphenylborinate significantly augmented angiotensin II–induced fibronectin protein expression, whereas thapsigargin abrogated high glucose– and TGF-β1–stimulated matrix protein expression. In vivo knockdown of Orai1 in mesangial cells of mice using a targeted nanoparticle siRNA delivery system resulted in increased expression of glomerular fibronectin and collagen IV, and mice showed significant mesangial expansion compared with controls. Similarly, in vivo knockdown of stromal interacting molecule 1 in mesangial cells by recombinant adeno–associated virus–encoded shRNA markedly increased collagen IV protein expression in renal cortex and caused mesangial expansion in rats. These results suggest that store–operated Ca^(2+) channels in mesangial cells negatively regulate extracellular matrix protein expression in the kidney, which may serve as an endogenous renoprotective mechanism in diabetes

Store Operated Calcium Entry Suppressed TGF-β1/SMAD3 Signaling Pathway in Glomerular Mesangial Cells

Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca^(2+) entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca^(2+) channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La^(3+); 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca^(2+) channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca^(2+) channels, significantly augmented TGF-β1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-β1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the TGF-β1/Smad3 signaling pathway

Negative regulation of Smad1 pathway and collagen IV expression by store-operated Ca^(2+) entry in glomerular mesangial cells

Collagen IV (Col IV) is a major component of expanded glomerular extracellular matrix in diabetic nephropathy and Smad1 is a key molecule regulating Col IV expression in mesangial cells (MCs). The present study was conducted to determine if Smad1 pathway and Col IV protein abundance were regulated by store-operated Ca^(2+) entry (SOCE). In cultured human MCs, pharmacological inhibition of SOCE significantly increased the total amount of Smad1 protein. Activation of SOCE blunted high-glucose-increased Smad1 protein content. Treatment of human MCs with ANG II at 1 µM for 15 min, high glucose for 3 days, or TGF-β1 at 5 ng/ml for 30 min increased the level of phosphorylated Smad1. However, the phosphorylation of Smad1 by those stimuli was significantly attenuated by activation of SOCE. Knocking down Smad1 reduced, but expressing Smad1 increased, the amount of Col IV protein. Furthermore, activation of SOCE significantly attenuated high-glucose-induced Col IV protein production, and blockade of SOCE substantially increased the abundance of Col IV. To further verify those in vitro findings, we downregulated SOCE specifically in MCs in mice using small-interfering RNA (siRNA) against Orai1 (the channel protein mediating SOCE) delivered by the targeted nanoparticle delivery system. Immunohistochemical examinations showed that expression of both Smad1 and Col IV proteins was significantly greater in the glomeruli with positively transfected Orai1 siRNA compared with the glomeruli from the mice without Orai1 siRNA treatment. Taken together, our results indicate that SOCE negatively regulates the Smad1 signaling pathway and inhibits Col IV protein production in MCs

Impact of long term Yoga practice on sleep quality and quality of life in the elderly

Background: Sleep disturbances and decline in the physical functionality are common conditions associated with aging. Pharmacological treatment of sleep disturbances can be associated with various adverse effects. Short term trials of Yoga on sleep have shown beneficial effects. Objectives: To evaluate the effect of long-term Yoga exercises on sleep quality and quality of life (QOL) in the elderly. Materials and Methods: This was a cross-sectional study in which data were collected from elderly people aged 60 years or more living in Nagpur city. We employed two types of survey questionnaires: Pittsburgh sleep quality index (PSQI) and QOL Leiden-Padua (LEIPAD) Questionnaire. A total of 65 elderly men and women who signed an informed consent and completed questionnaires were included in the study. Sleep quality score PSQI and QOL (LEIPAD Questionnaire) score of the study group were evaluated and compared with the control group using Mann-Whitney U test. Results: Total PSQI score in Yoga group was lower than that of the control group. Also various QOL scores of the Yoga groups were higher than the control group. Conclusion: Addition of regular Yoga exercises in the daily routine of elderly people can help to achieve good sleep quality as well as improve the QOL

Store-Operated Calcium Entry Upregulated IL6 Expression in Glomerular Mesangial Cells through NF-κB Pathway

Background: IL6 is a pleotropic cytokine and functions as a pro‐ as well as anti‐inflammatory mechanism depending on the cell type or the specific pathological environment. It is unclear if a particular signaling pathway confers IL6 with this property. Immunological and inflammatory mechanisms play a significant role in the development of diabetic nephropathy (DN). Locally produced cytokines/chemokines contributes to the early changes and histological impairment in DN. Glomerular mesangial cell (MC) is a major cell type in glomerulus to produce cytokines/chemokines in response to diabetes and a major contributor to mesangial expansion in DN. We have previously demonstrated that the Orai‐1 mediated store‐operated calcium entry (SOCE) suppressed ECM protein production by MCs. The aim of this study was to determine whether and how SOCE in MCs regulated IL6 production by MCs. Methods: Orai1, the channel protein mediating SOCE in MCs was knocked down using the targeted nanoparticle‐siRNA delivery system in wild type C57BLKS/J mice at the age of 16 weeks. Immunohistochemistry was performed on the paraffin embedded kidney sections to examine glomerular IL6 expression. In cultured human MCs, the expression of IL6 was examined in culture media and whole cell lysates using ELISA and Western blot analysis, respectively in the presence of normal glucose (5 mM D‐glucose + 20 mM L‐glucose) with/without an activator (thapsigargin at 1 μM) or inhibitor (GSK‐7975A 10 μM) of SOCE for 15 hrs. To determine the role of NF‐κB in SOCE effects on IL6 production, human MCs were treated with an inhibitor of NF‐κB, helenaline or siRNA against p65 subunit of NF‐κB. Furthermore, the effect of SOCE on the nuclear translocation of p65 was examined by assessing abundance of nuclear p65 protein in response to activation and inhibition of SOCE. Results: The IL6 expression level was reduced in the glomeruli of the mice treated with nanoparticle/Orai1 siRNA for 2 weeks compared to that in the control mice. In cultured human MCs, thapsigargin significantly increased IL‐6 level in supernatant media and in whole cell lysates of MCs, and this effect was attenuated by GSK 7975‐A, a selective inhibitor of SOCE. However, tunicamycin, an ER stress inducer did not alter the amount of IL6 as thapsigargin did. Treatment with helenaline, an inhibitor of NF‐κB pathway, or knockdown of p65, a subunit of NF‐κB significantly blunted the thapsigargin‐induced increase in IL6 protein abundance. Moreover, thapsigargin stimulated the nuclear translocation of p65 in human MCs. Conclusion: Orai1‐mediated SOCE positively regulates IL6 production by MCs through activation of NF‐κB pathway

Increased glomerular filtration rate and impaired contractile function of mesangial cells in TRPC6 knockout mice

The present study was conducted to determine if TRPC6 regulates glomerular filtration rate (GFR) and the contractile function of glomerular mesangial cells (MCs). GFR was assessed in conscious TRPC6 wild type and knockout mice, and in anesthetized rats with and without in vivo knockdown of TRPC6 in kidneys. We found that GFR was significantly greater, and serum creatinine level was significantly lower in TRPC6 deficient mice. Consistently, local knockdown of TRPC6 in kidney using TRPC6 specific shRNA construct significantly attenuated Ang II-induced GFR decline in rats. Furthermore, Ang II-stimulated contraction and Ca2+ entry were significantly suppressed in primary MCs isolated from TRPC6 deficient mice, and the Ca2+ response could be rescued by re-introducing TRPC6. Moreover, inhibition of reverse mode of Na+-Ca2+ exchange by KB-R7943 significantly reduced Ca2+ entry response in TRPC6-expressing, but not in TRPC6-knocked down MCs. Ca2+ entry response was also significantly attenuated in Na+ free solution. Single knockdown of TRPC6 and TRPC1 resulted in a comparable suppression on Ca2+ entry with double knockdown of both. These results suggest that TRPC6 may regulate GFR by modulating MC contractile function through multiple Ca2+ signaling pathways.Fil: Li, Weizu. Anhui Medical University; ChinaFil: Ding, Yanfeng. University of North Texas; Estados UnidosFil: Smedley, Crystal. University of North Texas; Estados UnidosFil: Wang, Yanxia. University of North Texas; Estados UnidosFil: Chaudhari, Sarika. University of North Texas; Estados UnidosFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. National Institutes of Health; Estados UnidosFil: Ma, Rong. University of North Texas; Estados Unido

Add-on Ayurveda Treatment for early stage COVID-19: A single centre retrospective cohort study from Gujarat, India

The retrospective cohort study aimed to evaluate the clinical outcomes of Ayurveda treatment exposure as an add-on to conventional care in early stage COVID-19 patients admitted at Samaras COVID care centre, Ahmedabad, India. Conventional care included Vitamin-c, Azithromycin, and Paracetamol. Ayurveda formulations used as add-on were Dashamula and Pathyadi decoctions along with Trikatu powder, Sanshamani tablet, AYUSH -64 tablet AND Yastimadhu Ghana tablet for oral administration. Considering Add-on Ayurveda medicines as exposure of interest, patients who received Add-on Ayurveda medicines at least for seven days were included in the exposed group while those who received only conventional care in unexposed group. Data was collected through record review and telephonic interviews. The outcomes of interest were the development of symptoms, duration of symptomatic phase in those progressing to symptomatic stage and mortality. Total 762 participants were included [541 (71%) in the exposed group and 221 (29%) in the unexposed]. Progression to symptomatic phase did not differ significantly between groups [27.6 % in exposed, 24.6% in unexposed, adjusted RR 0.85; 95% CI 0.6-1.2]. The total duration of symptomatic phase among those progressing to the symptomatic stage was significantly decreased in the exposed group (x̅= 3.66±1.55 days in exposed (n=133); x̅= 5.34±3.35 days in unexposed (n=61), p<0.001). No mortality was observed in either of the groups. Ayurveda Treatment as adjunctive to conventional care reduced the duration of symptomatic phase in early stage COVID 19 as compared to standalone conventional care. Add-on Ayurveda treatment has promising potential for management of early stage COVID-19