The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation.

The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na(+) and K(+) homeostasis. Endogenous NCC is highly ubiquitylated, but the role of individual ubiquitylation sites is not established. Here, we assessed the role of 10 ubiquitylation sites for NCC function. Transient transfections of HEK293 cells with human wildtype (WT) NCC or various K to R mutants identified greater membrane abundance for K706R, K828R and K909R mutants. Relative to WT-NCC, stable tetracycline inducible MDCKI cell lines expressing K706R, K828R and K909R mutants had significantly higher total and phosphorylated NCC levels at the apical plasma membrane under basal conditions. Low chloride stimulation increased membrane abundance of all mutants to similar or greater levels than WT-NCC. Under basal conditions K828R and K909R mutants had less ubiquitylated NCC in the plasma membrane, and all mutants displayed reduced NCC ubiquitylation following low chloride stimulation. Thiazide-sensitive sodium-22 uptakes were elevated in the mutants and internalization from the plasma membrane was significantly less than WT-NCC. K909R had increased half-life, whereas chloroquine or MG132 treatment indicated that K706 and K909 play roles in lysosomal and proteasomal NCC degradation, respectively. In conclusion, site-specific ubiquitylation of NCC plays alternative roles for NCC function

Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC

The sodium–chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho-specific antibodies targeting pS124-NCC demonstrated a band of 160 kDa in the kidney cortex, but not medulla, which was preabsorbed by a corresponding phosphorylated peptide. Confocal microscopy with kidney tubule segment-specific markers localized pS124-NCC to all distal convoluted tubule cells. Double immunogold electron microscopy demonstrated that pS124-NCC co-localized with total NCC in the apical plasma membrane of distal convoluted tubule cells and intracellular vesicles. Acute treatment of Munich–Wistar rats or vasopressin-deficient Brattleboro rats with the vasopressin type 2 receptor-specific agonist dDAVP significantly increased pS124-NCC abundance, with no changes in total NCC plasma membrane abundance. pS124-NCC levels also increased in abundance in rats after stimulation of the renin–angiotensin–aldosterone system by dietary low sodium intake. In contrast to other NCC phosphorylation sites, the STE20/SPS1-related proline–alanine-rich kinase and oxidative stress-response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and mTOR/FRAP) showed weak but significant phosphorylation activity at S124. In oocytes, (36)Cl uptake studies combined with biochemical analysis showed decreased activity of plasma membrane-associated NCC when replacing S124 with alanine (A) or aspartic acid (D). In novel tetracycline-inducible MDCKII-NCC cell lines, S124A and S124D mutants were able to traffic to the plasma membrane similarly to wildtype NCC

Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule

The thiazide-sensitive Na+–Cl− cotransporter (NCC) is important for renal electrolyte balance and its phosphorylation causes an increase in its transport activity and cellular localization. Here, we generated phospho-specific antibodies against two conserved N-terminal phosphorylation sites (Thr53, Thr58 and Thr53/Thr58) to assess the role of arginine vasopressin (AVP) in regulating NCC in rodent kidney in vivo. Immunohistochemistry showed distinct staining of phosphorylated NCC (pNCC) at the apical plasma membrane domain of distal convoluted tubule (DCT) cells. Unlike total NCC, pNCC was localized only to the apical plasma membrane as determined by immunogold electron microscopy. In AVP-deficient Brattleboro rats, acute deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) exposure significantly increased pNCC abundance at the apical plasma membrane by about threefold, whereas total NCC and its cellular distribution were not affected. dDAVP significantly increased the abundance of phosphorylated STE20/SPS1-related proline–alanine-rich kinase and oxidative stress-response kinase (SPAK and OSR1), kinases implicated in NCC phosphorylation. Intracellular calcium levels in early and late DCTs were increased in response to 1min superfusion of dDAVP, confirming that these segments are AVP responsive. In rats fed a high-salt diet with angiotensin (ANG) type 1-receptor blockade, similar increases in pNCC and active SPAK and OSR1 were detected following chronic or acute dDAVP, thus indicating the effects of AVP are independent of ANGII. Our results show that AVP is a potent regulator of NCC activity

Author Correction: The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation.

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CHIP regulates aquaporin-2 quality control and body water homeostasis

The importance of the kidney distal convoluted tubule (DCT) and cortical collecting duct (CCD) is highlighted by various water and electrolyte disorders that arise when the unique transport properties of these segments are disturbed. Despite this critical role, little is known about which proteins have a regulatory role in these cells and how these cells can be regulated by individual physiologic stimuli. By combining proteomics, bioinformatics, and cell biology approaches,we found that the E3 ubiquitin ligase CHIP is highly expressed throughout the collecting duct; is modulated in abundance by vasopressin; interacts with aquaporin-2 (AQP2), Hsp70, and Hsc70; and can directly ubiquitylate the water channel AQP2 in vitro. shRNA knockdown of CHIP in CCD cells increased AQP2 protein t 1/2 and reduced AQP2 ubiquitylation, resulting in greater levels of AQP2 andphosphorylatedAQP2.CHIP knockdown increased the plasma membrane abundance of AQP2 in these cells. Compared with wild-type controls, CHIP knockout mice or novel CRISPR/Cas9 mice without CHIPE3 ligase activity had greater AQP2 abundance and altered renal water handling, with decreased water intake and urine volume, alongside higher urine osmolality. We did not observe significant changes in other water- or sodium-transporting proteins in the gene-modified mice. In summary, these results suggest that CHIP regulates AQP2 and subsequently, renal water handling