Activation of the metabolic sensor AMP-activated protein kinase inhibits aquaporin-2 function in kidney principal cells

Aquaporin-2 (AQP2) is essential to maintain body water homeostasis. AQP2 traffics from intracellular vesicles to the apical membrane of kidney collecting duct principal cells in response to vasopressin [arginine vasopressin (AVP)], a hormone released with low intravascular volume, which causes decreased kidney perfusion. Decreased kidney perfusion activates AMP-activated kinase (AMPK), a metabolic sensor that inhibits the activity of several transport proteins. We hypothesized that AMPK activation also inhibits AQP2 function. These putative AMPK effects could protect interstitial ionic gradients required for urinary concentration during metabolic stress when low intravascular volume induces AVP release. Here we found that short-term AMPK activation by treatment with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 75 min) in kidney tissue prevented baseline AQP2 apical accumulation in principal cells, but did not prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCD(c14) cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopus oocytes expressing AQP2. We performed phosphorylation assays to elucidate the mechanism by which AMPK regulates AQP2. Although AMPK weakly phosphorylated immunoprecipitated AQP2 in vitro, no direct AMPK phosphorylation of the AQP2 COOH-terminus was detected by mass spectrometry. AMPK promoted Ser-261 phosphorylation and antagonized dDAVP-dependent phosphorylation of other AQP2 COOH-terminal sites in cells. Our findings suggest an increasing, time-dependent antagonism of AMPK on AQP2 regulation with AICAR-dependent inhibition of cAMP-dependent apical accumulation and AVP-dependent phosphorylation of AQP2. This inhibition likely occurs via a mechanism that does not involve direct AQP2 phosphorylation by AMPK

Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action.

The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif-containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2-suppressing antinatriuretic hormones to NCC phosphorylation status

L-WNK1 is Required for BK Channel Activation in Intercalated Cells

Large-conductance K+ (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K+ diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K+ diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K+ (5% K+, as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K+ diet have higher blood K+ concentration and reduced IC BK channel activity are consistent with impaired urinary K+ secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K+ diet.NEW & NOTEWORTHY When mice are placed on a high-K+ diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K+ channel, blunted large-conductance K+ channel currents in intercalated cells, and increased blood K+ concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K+ diet

Intercalated Cell BKα Subunit is Required for Flow-Induced K+ Secretion

BK channels are expressed in intercalated cells (ICs) and principal cells (PCs) in the cortical collecting duct (CCD) of the mammalian kidney and have been proposed to be responsible for flow-induced K+ secretion (FIKS) and K+ adaptation. To examine the IC-specific role of BK channels, we generated a mouse with targeted disruption of the pore-forming BK α subunit (BKα) in ICs (IC-BKα-KO). Whole cell charybdotoxin-sensitive (ChTX-sensitive) K+ currents were readily detected in control ICs but largely absent in ICs of IC-BKα-KO mice. When placed on a high K+ (HK) diet for 13 days, blood [K+] was significantly greater in IC-BKα-KO mice versus controls in males only, although urinary K+ excretion rates following isotonic volume expansion were similar in males and females. FIKS was present in microperfused CCDs isolated from controls but was absent in IC-BKα-KO CCDs of both sexes. Also, flow-stimulated epithelial Na+ channel-mediated (ENaC-mediated) Na+ absorption was greater in CCDs from female IC-BKα-KO mice than in CCDs from males. Our results confirm a critical role of IC BK channels in FIKS. Sex contributes to the capacity for adaptation to a HK diet in IC-BKα-KO mice