Hypercalciuria switches Ca^(2+) signaling in proximal tubular cells,induces oxidative damage to promote calcium nephrolithiasis

Proximal tubule(PT)transports most of the renal Ca^(2+),which was usually described as paracellular(passive).We found a regulated Ca^(2+)entry pathway in PT cells via the apical transient receptor potential canonical 3(TRPC3)channel,which initiates transcellular Ca^(2+)transport.Although TRPC3 knockout(−/−)mice were mildly hypercalciuric and displayed luminal calcium phosphate(CaP)crystals at Loop of Henle(LOH),no CaP+calcium oxalate(CaOx)mixed urine crystals were spotted,which are mostly found in calcium nephrolithiasis(CaNL).Thus,we used oral calcium gluconate(CaG;2%)to raise the PT luminal[Ca^(2+)]o further in TRPC3−/−mice for developing such mixed stones to understand the mechanistic role of PT-Ca^(2+)signaling in CaNL.Expectedly,CaG-treated mice urine samples presented with numerous mixed crystals with remains of PT cells,which were pronounced in TRPC3−/−mice,indicating PT cell damage.Notably,PT cells from CaG-treated groups switched their mode of Ca^(2+)entry from receptor-operated to store-operated pathway with a sustained rise in intracellular[Ca^(2+)]([Ca^(2+)]i),indicating the stagnation in PT Ca^(2+)transport.Moreover,those PT cells from CaG-treated groups demonstrated an upregulation of calcification,inflammation,fibrotic,oxidative stress,and apoptotic genes;effects of which were more robust in TRPC3 ablated condition.Furthermore,kidneys from CaG-treated groups exhibited fibrosis,tubular injury and calcifications with significant reactive oxygen species generation in the urine,thus,indicating in vivo CaNL.Taken together,excess PT luminal Ca^(2+)due to escalation of hypercalciuria in TRPC3 ablated mice induced surplus CaP crystal formation and caused stagnation of PT[Ca^(2+)]i,invoking PT cell injury,hence mixed stone formation.