Hyperosmotic stimuli activate polycystin proteins to aid in urine concentration
Karla M. Márquez-Nogueras, Ryne M. Knutila, Virdjinija Vuchkovska, Charlie Yang, Patricia Outeda, Darren P. Wallace, Ivana Y. Kuo
Karla M. Márquez-Nogueras, Ryne M. Knutila, Virdjinija Vuchkovska, Charlie Yang, Patricia Outeda, Darren P. Wallace, Ivana Y. Kuo
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Research Article Cell biology Nephrology

Hyperosmotic stimuli activate polycystin proteins to aid in urine concentration

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. These proteins are thought to form a signaling complex that can flux cations, including calcium. One of the earliest symptoms in ADPKD is a decline in the concentrating ability of the kidneys, occurring prior to cyst formation. We reasoned that hyperosmolality stimulates the polycystin complex, and that the loss of this function impairs water reabsorption. We found that hyperosmolality resulted in the phosphorylation of microtubule-associated protein 4 (MAP4) in a PC1-dependent manner, which then elicited ER-localized PC2 calcium signals. ER-localized PC2 hyperosmotic calcium signals were required for trafficking of the water channel aquaporin (AQP2). Precystic PC1-KO and PC2-KO murine kidneys had cytosol-localized AQP2 and diluted urine compared with their respective controls. Kidney tissue sections from ADPKD patients showed decreased AQP2 apical membrane localization in cystic and noncystic tubules. Our study demonstrates that osmolality is a physiological stimulus of the polycystin complex, and loss of polycystin osmosensing results in impaired water reabsorption via AQP2. This likely contributes to the declined concentrating ability of the kidneys and high circulating vasopressin levels in patients with ADPKD.

Authors

Karla M. Márquez-Nogueras, Ryne M. Knutila, Virdjinija Vuchkovska, Charlie Yang, Patricia Outeda, Darren P. Wallace, Ivana Y. Kuo

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Figure 1

Precystic PC1 and PC2-KO mice have decreased urine concentration and decreased calcium signaling upon hyperosmotic challenge.

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Precystic PC1 and PC2-KO mice have decreased urine concentration and dec...
(A) Model of precystic tubule–specific deletion of PC1 and PC2 by crossing Pax8-TetO-rtTA (CTL) mice with Pkd1fl/fl mice (PC1-KO) or Pkd2fl/fl mice (PC2-KO). (B) Representative images with decreased expression of PC2 in PC2-KO mouse. Residual PC2 staining in the PC2-KO mouse arises from non-epithelial cells. Scale bars: 10 μm. (C) Precystic PC2 (red) and PC1-KO (green) mice had decreased urine osmolality compared with CTL mice. Each dot represents an individual mouse. n = 8–9. Data analyzed by Mann-Whitney U test. (D) Representative time-lapse images of collecting duct (CD) tubules expressing gCaMP from kidney slices of CTL mice (top) and PC2-KO mice (bottom). Extracellular osmolality was increased from 250 to 400 mOsm with NaCl. Scale bars: 20 μm. (E) Representative trace of cytosolic calcium changes in CTL CD tubules (black line) and PC2-KO tubules (red line). (F) Quantification of area under the curve (AUC) of calcium transients comparing CTL to PC2-KO CD tubules; each dot is 1 mouse. n = 4–5. Data analyzed by Mann-Whitney U test. (G) Representative trace of cytosolic calcium increases in CTL IMCD3 cells (black line) with hyperosmotic stimuli not seen in PC1-KO IMCD3 cells (green line) and PC2-KO IMCD3 cells (red line). (H) AUC decreased in PC1-KO and PC2-KO IMCD3 cells. Data were analyzed to determine normality. Bar graphs represent mean ± SEM. Dark dots represent biological replicates, while light dots represent individual cells n = 30. P values listed in each panel.