Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca2+ entry (SOCE). Paradoxically, despite enhancing Ca2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca2+, despite limiting CRAC channel activity.

Abbreviations mtTFs, mitochondrial transcription factors; OXPHOS, oxidative phosphorylation; ATP, adenosine triphosphate; ETC, electron transport chain, TiH, tubulointerstitial hypoxia; PGC-1αperoxisome proliferator activated receptor gamma coactivator 1-alpha; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rat; HIF-1α, Hypoxia Inducible factor 1α; HREs, hypoxia response elements; mtDNA, mitochondrial DNA; ChIP, chromatin immunoprecipitation; bp, base pair. AbstractIntroduction: Kidneys are organs with high resting metabolic rate and low tissue pO 2 due to enhanced mitochondrial oxygen consumption and ATP production for active solute transport.Such enhanced mitochondrial activity leads to progressive hypoxia from the renal cortex to medulla. Renal tubulointerstitial hypoxia (TiH) is severe in hypertensive rats due to increased sodium reabsorption within their nephrons. However, expression of mitochondrial transcription factors (mtTFs) (viz. Tfam, Tfb1m and Tfb2m) and mitochondrial biogenesis is not reported during hypoxic conditions. Materials and Methods:Transcriptional regulation of Tfam, Tfb1m and Tfb2m under acute hypoxia was studied using promoter-driven luciferase assays, qPCR, western blotting and Chromatin Immunoprecipitation. Results:The expression of HIF-1α, PGC-1α, Tfam, Tfb1m, Tfb2m and OXPHOS proteins were higher in hypertensive rats as compared to the normotensive ones. Additionally, studies on NRK52e cells show that acute hypoxia increases the expression of these genes. We also observed a positive correlation between HIF-1α and mtTFs in human tissues. Furthermore, we report for the first time, that HIF-1α binds to promoters of Tfam, Tfb1m and Tfb2m genes and augments their promoter activity.Conclusion: Hypertensive rats, with increased TiH show enhanced expression of mitochondrial proteins. HIF-1α directly binds to and increases promoter activity of mtTFs.Acute hypoxia induces the expression of mtTFs and probably promotes mitochondrial biogenesis in NRK52e cells.