Bone loss in KLHL3 knock-in mice characterized by a pseudohypoaldosteronism type II-like phenotype is mediated by renal PTH resistance

This is the final versionPoster presented at the 43rd Annual European Calcified Tissue Society Congress, Rome, Italy, 14 - 17 May 2016Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by hypertension, hypercalciuria and osteopenia. PHAII is caused by mutations in with-no-lysine kinase 1 (WNK1), WNK4, or the cullin RING ligase family members kelch-like 3 (KLHL3) or cullin 3 (CUL3). All mutations result in up-regulation of the WNK signalling pathway which activates thiazide-sensitive Na-Cl cotransporters (NCC) in renal distal tubules, leading to salt retention and hypertension in PHAII. The mechanism underlying hypercalciuria in PHAII is unknown. To better understand the mechanisms leading to osteopenia in PHAII, we used KLHL3R528H/+ knock-in mice carrying the same mutation as some PHAII patients. As expected, KLHL3R528H/+ mutants exhibited hyperkalemia, hypernatremia, renal calcium wasting and increased phosphorylation of NCC in the kidney. Furthermore, KLHL3R528H/+ mutants showed elevated serum parathyroid hormone (PTH), increased bone resorption as demonstrated by elevated urinary collagen crosslinks excretion and increased osteoclast numbers in femoral cancellous bone, and reduced distal femoral cancellous bone BMD and volume as evidenced by pQCT and μCT analysis. Analysis of the expression of proteins involved in renal calcium transport revealed elevated membrane abundance of the fully glycosylated epithelial calcium channel TRPV5, decreased TRPV6 abundance, and unchanged calbindin D28k expression in KLHL3R528H/+ mutants. In contrast to the upregulated TRPV5 protein expression, TRPV5 phosphorylation was reduced in KLHL3R528H/+ mutants, suggesting downregulated TRPV5 activity. In line with a crosstalk between NCC activity and PTH-mediated TRPV5 activation, we found by 2-photon microscopy that the PTH-mediated increase in Ca2+ uptake in mouse distal tubular mpkDCT4 cells was enhanced by the NCC blocker chlorothiazide or by knockout of NCC. Taken together, our study provides a mechanistic explanation for the hypercalciuria and bone loss found in PHAII patients: elevated NCC activity in KLHL3R528H/+ mice blocks PTH-mediated TRPV5 activation, leading to renal PTH resistance with subsequent renal Ca wasting and a counter-regulatory PTH-induced bone loss

Toolkit for Simulation Modeling of Logistics Warehouse in Distributed Computing Environment

We address an important problem of an automation of logistics warehouses simulation modeling in distributed service-oriented computing environments. To this end, we propose new approach for adjusting management system parameters of real warehouse in production use. It is based on the integration of the conceptual, wireframe and service-oriented programming used to develop parameter sweep applications and data analysis in the simulation modeling process. We develop a toolkit for supporting modeling of warehouse logistics. The practical experiments are focused upon the refrigerated warehouse. The developed application demonstrates high efficiency and scalability for optimizing nine criteria to cope with different production demands.The study was supported by the Russian Foundation of Basic Research, projects no. 15-29-07955 and no. 16-07-00931, and Program 1.33P of fundamental research of Presidium RAS, project “Development of new approaches to creation and study of complex models of information-computational and dynamic systems with applications”

Klotho Lacks a Vitamin D Independent Physiological Role in Glucose Homeostasis, Bone Turnover, and Steady-State PTH Secretion In Vivo

Apart from its function as co-receptor for fibroblast growth factor-23 (FGF23), Klotho is thought to regulate insulin signaling, intracellular oxidative stress, and parathyroid hormone (PTH) secretion in an FGF23 independent fashion. Here, we crossed Klotho deficient (Kl−/−) mice with vitamin D receptor (VDR) mutant mice to examine further vitamin D independent functions of Klotho. All mice were fed a rescue diet enriched with calcium, phosphorus, and lactose to prevent hyperparathyroidism in VDR mutants, and were killed at 4 weeks of age after double fluorochrome labeling. Kl−/− mice displayed hypercalcemia, hyperphosphatemia, dwarfism, organ atrophy, azotemia, pulmonary emphysema, and osteomalacia. In addition, glucose and insulin tolerance tests revealed hypoglycemia and profoundly increased peripheral insulin sensitivity in Kl−/− mice. Compound mutants were normocalcemic and normophosphatemic, did not show premature aging or organ atrophy, and were phenocopies of VDR mutant mice in terms of body weight, bone mineral density, bone metabolism, serum calcium, serum phosphate, serum PTH, gene expression in parathyroid glands, as well as urinary calcium and phosphate excretion. Furthermore, ablation of vitamin D signaling in double mutants completely normalized glucose and insulin tolerance, indicating that Klotho has no vitamin D independent effects on insulin signaling. Histomorphometry of pancreas islets showed similar beta cell volume per body weight in all groups of animals. In conclusion, our findings cast doubt on a physiologically relevant vitamin D and Fgf23 independent function of Klotho in the regulation of glucose metabolism, bone turnover, and steady-state PTH secretion in vivo

Effects of vitamin D supplementation on endothelial function:a systematic review and meta-analysis of randomised clinical trials

Background: In addition to regulating calcium homoeostasis and bone health, vitamin D influences vascular and metabolic processes including endothelial function (EF) and insulin signalling. This systematic review and meta-analysis of randomised clinical trials (RCTs) were conducted to investigate the effect of vitamin D supplementation on EF and to examine whether the effect size was modified by health status, study duration, dose, route of vitamin D administration, vitamin D status (baseline and post-intervention), body mass index (BMI), age and type of vitamin D.  Methods: We searched the Medline, Embase, Cochrane Library and Scopus databases from inception until March 2015 for studies meeting the following criteria: (1) RCT with adult participants, (2) vitamin D administration alone, (3) studies that quantified EF using commonly applied methods including ultrasound, plethysmography, applanation tonometry and laser Doppler.  Results: Sixteen articles reporting data for 1177 participants were included. Study duration ranged from 4 to 52 weeks. The effect of vitamin D on EF was not significant (SMD: 0.08, 95 % CI −0.06, 0.22, p = 0.28). Subgroup analysis showed a significant improvement of EF in diabetic subjects (SMD: 0.31, 95 % CI 0.05, 0.57, p = 0.02). A non-significant trend was found for diastolic blood pressure (β = 0.02; p = 0.07) and BMI (β = 0.05; p = 0.06).  Conclusions: Vitamin D supplementation did not improve EF. The significant effect of vitamin D in diabetics and a tendency for an association with BMI may indicate a role of excess adiposity and insulin resistance in modulating the effects of vitamin D on vascular function. This remains to be tested in future studies