Hypercalcemia induces targeted autophagic degradation of aquaporin-2 at the onset of nephrogenic diabetes insipidus

Abstract

Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI.

From Molecules to Mechanisms: Functional Proteomics and Its Application to Renal Tubule Physiology

“…In addition to increased knowledge of normal kidney physiology, more studies are also needed to advance understanding of renal pathophysiology. For example, two recent proteomicdriven studies revealed a prominent role for selective autophagy in the downregulation of specific proteins during development of nephrogenic diabetes insipidus due to electrolyte imbalance disorders [hypokalemia (84) and hypercalcemia (82)]. Another proof-of-concept study recently demonstrated an application of proteomic analysis in exploring the molecular pathophysiology in an animal model of chronic tubulointerstitial fibrosis (137).…”

Dysregulated Mineral Metabolism in AKI

“…Hypercalcemia can cause AKI directly through a variety of mechanisms: afferent arteriolar vasoconstriction, leading to decreased glomerular filtration rate 73 ; binding of calcium to the CaSR on the basolateral membrane of the thick ascending limb, resulting in down-regulation of the sodium-potassium-chloride cotransporter, leading to natriuresis and volume depletion 74 ; and nephrogenic diabetes insipidus, which occurs as a result of enhanced autophagic degradation of aquaporin-2 channels in the inner medullary collecting ducts. 75 In addition, hypercalcemia can cause hypercalciuria, nephrolithiasis, and nephrocalcinosis, which can cause both acute and chronic renal impairment.…”

CaSR signaling down‐regulates AQP2 expressionviaa novel microRNA pathway in pendrin and NaCl cotransporter knockout mice

“…Therefore, although we do not provide direct in vivo data that CaSR activation contributes to vasopressin resistance and polyuria in dKO mice, indirect in vivo and direct ex vivo data strongly support this hypothesis. In line with this, recent data indicate that hypercalcemia‐induced hypercalciuria, by exposing cells to sustained elevated extracellular calcium, causes AQP2 autophagic degradation that is mediated by CaSR activation (66). Cell‐surface AQP2 abundance is crucial to the maintenance of body water homeostasis; however, it should be considered that dKO mice may also have a moderate defect in the generation of the cortical‐medullary gradient as a result of the down‐regulation of NKCC2 (20), which is also critical for the production of concentrated urine.…”