Modulation of Tubular pH by Acetazolamide in a Ca2+ Transport Deficient Mice Facilitates Calcium Nephrolithiasis

Proximal tubular (PT) acidosis, which alkalinizes the urinary filtrate, together with Ca2+ supersaturation in PT can induce luminal calcium phosphate (CaP) crystal formation. While such CaP crystals are known to act as a nidus for CaP/calcium oxalate (CaOx) mixed stone formation, the regulation of PT luminal Ca2+ concentration ([Ca2+]) under elevated pH and/or high [Ca2+] conditions are unknown. Since we found that transient receptor potential canonical 3 (TRPC3) knockout (KO; -/-) mice could produce mild hypercalciuria with CaP urine crystals, we alkalinized the tubular pH in TRPC3-/- mice by oral acetazolamide (0.08%) to develop mixed urinary crystals akin to clinical signs of calcium nephrolithiasis (CaNL). Our ratiometric (λ340/380) intracellular [Ca2+] measurements reveal that such alkalization not only upsurges Ca2+ influx into PT cells, but the mode of Ca2+ entry switches from receptor-operated to store-operated pathway. Electrophysiological experiments show enhanced bicarbonate related current activity in treated PT cells which may determine the stone-forming phenotypes (CaP or CaP/CaOx). Moreover, such alkalization promotes reactive oxygen species generation, and upregulation of calcification, inflammation, fibrosis, and apoptosis in PT cells, which were exacerbated in absence of TRPC3. Altogether, the pH-induced alteration of the Ca2+ signaling signature in PT cells from TRPC3 ablated mice exacerbated the pathophysiology of mixed urinary stone formation, which may aid in uncovering the downstream mechanism of CaNL.

Hepatic endoplasmic reticulum calcium fluxes: effect of free fatty acids and KATP channel involvement

As a common sequel to obesity, plasma and intracellular free fatty acid (FFA) concentrations are elevated and, as a consequence, manifold disturbances in metabolism may ensue. Biochemical processes in the cytosol and organelles, such as mitochondria and endoplasmic reticulum (ER), can be disturbed. In the ER, the maintenance of a high calcium gradient is indispensable for viability. In sarcoplasmic reticulum, selective FFA can induce ER stress by disrupting luminal calcium homeostasis; however, there are limited studies in hepatic microsomes. Our studies found that FFA has a noxious effect on rat hepatic microsomal calcium flux, and the extent of which depended on the number of double bonds and charge. Furthermore, insofar as the FFA had no effect on microsomal calcium efflux, their inhibitory action primarily involves calcium influx. Finally, other cationic channels have been found in hepatic ER, and evidence is presented of their interaction with the Ca2+ ATPase pump.

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.

Comparative Analysis of Atherosclerotic Disease in the Carotid and Femoral Arteries

K Hurst1, R Choudhury2, A Handa1 1. Nuffield Department of Surgical Sciences, University of Oxford 2. Radcliffe Department of Medicine, University of Oxford Introduction The outcomes for patients with PAOD have remained unchanged for over 25 years, whereas primary prevention has made a marked difference to the incidence of stroke and MI. Why is treatment more effective for certain types of atherosclerotic plaques than others? Why do certain patients experience a stroke but never an amputation? Methods The study aims to use in vivo and ex vivo analysis of carotid and femoral atherosclerotic plaque from patients enrolled in the OxPVD study. Each participant will undergo pre-operative in vivo carotid and femoral vessel (ipsilateral) imaging using T1/T2 weighted MRI; inclusive of plaque, and ex vivo plaque analysis using micro CT, micro MRI and histological staining. Comparative analysis of the carotid versus femoral plaques will be completed using above techniques. Results Primary results from this study have shown that novel MRI DANTE sequences can successfully characterise femoral atherosclerotic plaque in vivo. Using micro CT and mass spectrometry techniques from micro MRI, we have further quantified the differences in composition of carotid and femoral disease. Finally, histological staining has implied that the disease processes behind femoral and carotid atherosclerosis may be different; with carotid narrowing by fat infiltration and statistically significant reduction in nuclei, compared to femoral plaque showing intra luminal calcium deposition without intimal destruction. Conclusion These preliminary results are beginning to highlight the key compositional differences between carotid and femoral atherosclerosis, and with these results it may be possible to trial different therapeutic targets, particularly in the context of PAOD.

Dynamic formation of ER–PM junctions presents a lipid phosphatase to regulate phosphoinositides

Endoplasmic reticulum–plasma membrane (ER–PM) contact sites play an integral role in cellular processes such as excitation–contraction coupling and store-operated calcium entry (SOCE). Another ER–PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER–PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2. Activation of G protein–coupled receptors that deplete PM PI(4,5)P2 disrupts E-Syt2–mediated ER–PM junctions, reducing Sac1’s access to the PM and permitting PM PI(4)P and PI(4,5)P2 to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER–PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes.