Water and Salt So030 Amousemodelof Saltwasting, Hypercalciuria and Kidney Stones

Introduction and Aims: Calcium nephrolithiasis is a complex disease with multiple pathogenetic mechanisms. The role of salt wasting and the subsequent volume depletion in the pathogenesis of hypercalciuria and kidney stone formation remains speculative. The only known monogenic disorder associated with salt wasting/volume depletion, hypercalciuria and nephrolithiasis/ nephrocalcinosis is Bartter Syndrome. We have developed a model of distal tubule salt wasting, which is caused by the simultaneous deletion of the Cl/HCO3 exchanger pendrin and the Na-Cl co-transporter NCC (PNAS 2012). These animals become severely volume depleted and have nephrogenic DI (PNAS 2012). We hypothesized that salt wasting followed by volume depletion, irrespective of the etiology or the nephron segment(s) involved, activates a cascade of events that lead to increased generation of the arachidonic acid metabolites Prostaglandin E2 (PGE2) and 20-hydroxyeicosatetraenoic acid (20-HETE) that impair salt and calcium reabsorption in the thick ascending limb and the proximal tubule, and block the action of AVP and aldosterone on water and salt reabsorption in the collecting duct, leading to the worsening of volume depletion and calcium wasting. We hypothesize that this self-propagating disadvantageous cycle can lead to super-saturation of calcium crystals in the urine and result in nephrocalcinosis and/or nephrolithiasis. Methods:DNAmicroarray, northern hybridization, western blotting, immunofluorescence labeling, H and E staining, Von Kossa stain, functional studies and appropriate treatment with various chemicals were performed on kidneys of wt and double pendrin/NCC KOmice. Results: DNAmicroarray demonstrated the activation of arachidonic acid metabolites PGE2 and 20-HETE-generating cytochrome p450 isoforms (Cyp4a12a and 12b) in kidneys of dKOmice. The 24 hr urine collection indicated significant increases in PGE2 and 20-HETE excretion in dKOmice (p<0.01 vs WT or single KO mice). In addition to profound salt wasting, the 24 hr urine analysis showed a 3-fold increase in calcium excretion in dKO mice. The histological analysis of kidneys demonstrated multiple calcium stones in the medullary collecting ducts in dKO mice but not in pendrin or NCC single KO mice. The stones were comprised of calcium based on strong staining with Von Kossa stain. Phosphate excretion increased by 2 folds in dKO mice and correlated with a significant reduction in the expression of NaPi-IIa, the major phosphate absorbing transporter in the proximal tubule. Serum calcium and phosphate levels were mildly reduced in dKO mice but remained normal in single KO orWTmice. Blood levels for PTH, 1,25 Vitamin D and FGF23 in dKO mice were comparable to WT or single KO mice. dKOmice had low urine osmolality despite severe volume depletion and had impaired response to exogenous dDAVP, consistent with the presence of nephrogenic DI. Treatment with the PG inhibitor indomethacin for 3 days significantly decreased PGE2 excretion and calcium wasting and caused a significant decrease in urine output and an increase in urine osmolality in dKO mice. Conclusions:We conclude that salt wasting followed by volume depletion can trigger a cascade of events that includes the activation of arachidonic acid metabolites PGE2 and 20-HETE, which in turn exacerbate salt wasting in multiple nephron segments and cause hypercalciuria, which in the context of volume depletion can result in the precipitation of calcium crystals and calcium stone formation.