Cullin-Ring ubiquitin ligases in kidney health and disease.

PURPOSE OF REVIEW: Members of the Cullin family act as scaffolds in E3 ubiquitin ligases and play a central role in mediating protein degradation. Interactions with many different substrate-binding adaptors permit Cullin-containing E3 ligases to participate in diverse cellular functions. In the kidney, one well established target of Cullin-mediated degradation is the transcription factor Nrf2, a key player in responses to oxidative stress. The goal of this review is to discuss more recent findings revealing broader roles for Cullins in the kidney. RECENT FINDINGS: Cullin 3 acts as the scaffold in the E3 ligase regulating Nrf2 abundance, but was more recently shown to be mutated in the disease familial hyperkalemic hypertension. Studies seeking to elucidate the molecular mechanisms by which Cullin 3 mutations lead to dysregulation of renal sodium transport will be discussed. Disruption of Cullin 3 in mice unexpectedly causes polyuria and fibrotic injury suggesting it has additional roles in the kidney. We will also review recent transcriptomic data suggesting that other Cullins are also likely to play important roles in renal function. SUMMARY: Cullins form a large and diverse family of E3 ubiquitin ligases that are likely to have many important functions in the kidney.

Deficient acid handling with distal RTA in the NBCe2 knockout mouse.

In many circumstances, the pathogenesis of distal renal tubular acidosis (dRTA) is not understood. In the present study, we report that a mouse model lacking the electrogenic Na(+)-HCO3 (-) cotransporter [NBCe2/Slc4a5; NBCe2 knockout (KO) mice] developed dRTA after an oral acid challenge. NBCe2 expression was identified in the connecting tubule (CNT) of wild-type mice, and its expression was significantly increased after acid loading. NBCe2 KO mice did not have dRTA when on a standard mouse diet. However, after acid loading, NBCe2 KO mice exhibited complete features of dRTA, characterized by insufficient urinary acidification, hyperchloremic hypokalemic metabolic acidosis, and hypercalciuria. Additional experiments showed that NBCe2 KO mice had decreased luminal transepithelial potential in the CNT, as revealed by micropuncture. Further immunofluorescence and Western blot experiments found that NBCe2 KO mice had increased expression of H(+)-ATPase B1 in the plasma membrane. These results showed that NBCe2 KO mice with acid loading developed increased urinary K(+) and Ca(2+) wasting due to decreased luminal transepithelial potential in the CNT. NBCe2 KO mice compensated to maintain systemic pH by increasing H(+)-ATPase in the plasma membrane. Therefore, defects in NBCe2 can cause dRTA, and NBCe2 has an important role to regulate urinary acidification and the transport of K(+) and Ca(2+) in the distal nephron.

Low Na, high K diet and the role of aldosterone in BK-mediated K excretion.

A low Na, high K diet (LNaHK) is associated with a low rate of cardiovascular (CV) disease in many societies. Part of the benefit of LNaHK relies on its diuretic effects; however, the role of aldosterone (aldo) in the diuresis is not understood. LNaHK mice exhibit an increase in renal K secretion that is dependent on the large, Ca-activated K channel, (BK-α with accessory BK-ß4; BK-α/ß4). We hypothesized that aldo causes an osmotic diuresis by increasing BK-α/ß4-mediated K secretion in LNaHK mice. We found that the plasma aldo concentration (P[aldo]) was elevated by 10-fold in LNaHK mice compared with control diet (Con) mice. We subjected LNaHK mice to either sham surgery (sham), adrenalectomy (ADX) with low aldo replacement (ADX-LA), or ADX with high aldo replacement (ADX-HA). Compared to sham, the urinary flow, K excretion rate, transtubular K gradient (TTKG), and BK-α and BK-ß4 expressions, were decreased in ADX-LA, but not different in ADX-HA. BK-ß4 knockout (ß4KO) and WT mice exhibited similar K clearance and TTKG in the ADX-LA groups; however, in sham and ADX-HA, the K clearance and TTKG of ß4KO were less than WT. In response to amiloride treatment, the osmolar clearance was increased in WT Con, decreased in WT LNaHK, and unchanged in ß4KO LNaHK. These data show that the high P[aldo] of LNaHK mice is necessary to generate a high rate of BK-α/ß4-mediated K secretion, which creates an osmotic diuresis that may contribute to a reduction in CV disease.

Coupled ATP and potassium efflux from intercalated cells.

Increased flow in the distal nephron induces K secretion through the large-conductance, calcium-activated K channel (BK), which is primarily expressed in intercalated cells (IC). Since flow also increases ATP release from IC, we hypothesized that purinergic signaling has a role in shear stress (τ; 10 dynes/cm(2)) -induced, BK-dependent, K efflux. We found that 10 µM ATP led to increased IC Ca concentration, which was significantly reduced in the presence of the P(2) receptor blocker suramin or calcium-free buffer. ATP also produced BK-dependent K efflux, and IC volume decrease. Suramin inhibited τ-induced K efflux, suggesting that K efflux is at least partially dependent on purinergic signaling. BK-ß4 small interfering (si) RNA, but not nontarget siRNA, decreased ATP secretion and both ATP-dependent and τ-induced K efflux. Similarly, carbenoxolone (25 µM), which blocks connexins, putative ATP pathways, blocked τ-induced K efflux and ATP secretion. Compared with BK-ß4(-/-) mice, wild-type mice with high distal flows exhibited significantly more urinary ATP excretion. These data demonstrate coupled electrochemical efflux between K and ATP as part of the mechanism for τ-induced ATP release in IC.