Regulation of the water channel aquaporin-2 by cullin E3 ubiquitin ligases.

Aquaporin 2 (AQP2) is a vasopressin (VP)-regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play an essential role in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multisubunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we used an established cell model of the collecting duct (mpkCCD14 cells) to study the role of cullins in modulating AQP2. Western blotting identified Cul-1 to Cul-5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser261 phosphorylation, and attenuated VP-induced plasma membrane accumulation of AQP2 relative to the vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared with controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies.NEW & NOTEWORTHY Aquaporin 2 (AQP2) is essential for body water homeostasis and is regulated by the antidiuretic hormone vasopressin. The posttranslational modification ubiquitylation is a key regulator of AQP2 abundance and plasma membrane localization. Here we demonstrate that cullin-RING E3 ligases play a vital role in mediating some of the effects of vasopressin to increase AQP2 abundance and plasma membrane accumulation. The results suggest that manipulating cullin activity could be a novel strategy to alter kidney water handling.

Dissociation of sodium-chloride cotransporter expression and blood pressure during chronic high dietary potassium supplementation.

Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+-free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.

The E3 ubiquitin-protein ligase Nedd4-2 regulates the sodium chloride cotransporter NCC but is not required for a potassium-induced reduction of NCC expression.

Na+ and K+ balance is influenced by the activity of the sodium chloride cotransporter NCC in the distal convoluted tubule. NCC activity and abundance are reduced by high extracellular K+. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) has been proposed as a modulator of NCC abundance. Here, we examined the functional role of Nedd4-2 on NCC regulation and whether Nedd4-2 is important for the effects of high extracellular K+ on NCC. Total and plasma membrane levels of ubiquitylated NCC were lower in NCC-expressing MDCKI cells after Nedd4-2 deletion. NCC and phosphorylated NCC (pT58-NCC) levels were higher after Nedd4-2 deletion, and NCC levels on the plasma membrane were elevated. No significant changes were seen after Nedd4-2 knockdown in the levels of SPAK and phosphorylated SPAK (pS373-SPAK), the major NCC regulatory kinase. Nedd4-2 deficiency had no effect on the internalization rate of NCC from the plasma membrane, but NCC protein half-life was increased. In ex vivo experiments with kidney tubule suspensions from Nedd4-2 knockout (KO) mice, high K+ reduced total and pT58-NCC regardless of genotype. We conclude that Nedd4-2 is involved in ubiquitylation of NCC and modulating its plasma membrane levels and degradation. However, Nedd4-2 does not appear to be important for K+ induced reductions in NCC abundance.

The Hydrogen-Coupled Oligopeptide Membrane Cotransporter Pept2 is SUMOylated in Kidney Distal Convoluted Tubule Cells.

Protein post-translational modification by the Small Ubiquitin-like MOdifier (SUMO) on lysine residues is a reversible process highly important for transcription and protein stability. In the kidney, SUMOylation appears to be important for the cellular response to aldosterone. Therefore, in this study, we generated a SUMOylation profile of the aldosterone-sensitive kidney distal convoluted tubule (DCT) as a basis for understanding SUMOylation events in this cell type. Using mass spectrometry-based proteomics, 1037 SUMO1 and 552 SUMO2 sites, corresponding to 546 SUMO1 and 356 SUMO2 proteins, were identified from a modified mouse kidney DCT cell line (mpkDCT). SUMOylation of the renal hydrogen-coupled oligopeptide and drug co-transporter (Pept2) at one site (K139) was found to be highly regulated by aldosterone. Using immunolabelling of mouse kidney sections Pept2 was localized to DCT cells in vivo. Aldosterone stimulation of mpkDCT cell lines expressing wild-type Pept2 or mutant K139R-Pept2, post-transcriptionally increased Pept2 expression up to four-fold. Aldosterone decreased wild-type Pept2 abundance in the apical membrane domain of mpkDCT cells, but this response was absent in K139R-Pept2 expressing cells. In summary, we have generated a SUMOylation landscape of the mouse DCT and determined that SUMOylation plays an important role in the physiological regulation of Pept2 trafficking by aldosterone.

Large-Scale Proteomic Assessment of Urinary Extracellular Vesicles Highlights Their Reliability in Reflecting Protein Changes in the Kidney.

BACKGROUND: Urinary extracellular vesicles (uEVs) are secreted into urine by cells from the kidneys and urinary tract. Although changes in uEV proteins are used for quantitative assessment of protein levels in the kidney or biomarker discovery, whether they faithfully reflect (patho)physiologic changes in the kidney is a matter of debate. METHODS: Mass spectrometry was used to compare in an unbiased manner the correlations between protein levels in uEVs and kidney tissue from the same animal. Studies were performed on rats fed a normal or high K+ diet. RESULTS: Absolute quantification determined a positive correlation (Pearson R=0.46 or 0.45, control or high K+ respectively, P<0.0001) between the approximately 1000 proteins identified in uEVs and corresponding kidney tissue. Transmembrane proteins had greater positive correlations relative to cytoplasmic proteins. Proteins with high correlations (R>0.9), included exosome markers Tsg101 and Alix. Relative quantification highlighted a monotonic relationship between altered transporter/channel abundances in uEVs and the kidney after dietary K+ manipulation. Analysis of genetic mouse models also revealed correlations between uEVs and kidney. CONCLUSION: This large-scale unbiased analysis identifies uEV proteins that track the abundance of the parent proteins in the kidney. The data form a novel resource for the kidney community and support the reliability of using uEV protein changes to monitor specific physiologic responses and disease mechanisms.

High dietary potassium causes ubiquitin-dependent degradation of the kidney sodium-chloride cotransporter.

The thiazide-sensitive sodium-chloride cotransporter (NCC) in the renal distal convoluted tubule (DCT) plays a critical role in regulating blood pressure (BP) and K+ homeostasis. During hyperkalemia, reduced NCC phosphorylation and total NCC abundance facilitate downstream electrogenic K+ secretion and BP reduction. However, the mechanism for the K+-dependent reduction in total NCC levels is unknown. Here, we show that NCC levels were reduced in ex vivo renal tubules incubated in a high-K+ medium for 24-48 h. This reduction was independent of NCC transcription, but was prevented using inhibitors of the proteasome (MG132) or lysosome (chloroquine). Ex vivo, high K+ increased NCC ubiquitylation, but inhibition of the ubiquitin conjugation pathway prevented the high K+-mediated reduction in NCC protein. In tubules incubated in high K+ media ex vivo or in the renal cortex of mice fed a high K+ diet for 4 days, the abundance and phosphorylation of heat shock protein 70 (Hsp70), a key regulator of ubiquitin-dependent protein degradation and protein folding, were decreased. Conversely, in similar samples the expression of PP1α, known to dephosphorylate Hsp70, was also increased. NCC coimmunoprecipitated with Hsp70 and PP1α, and inhibiting their actions prevented the high K+-mediated reduction in total NCC levels. In conclusion, we show that hyperkalemia drives NCC ubiquitylation and degradation via a PP1α-dependent process facilitated by Hsp70. This mechanism facilitates K+-dependent reductions in NCC to protect plasma K+ homeostasis and potentially reduces BP.

Activation of the kidney sodium chloride cotransporter by the ß2-adrenergic receptor agonist salbutamol increases blood pressure.

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the ß2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to ß1-adrenergic receptors, the ß2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In ex vivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 µM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, ß2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3-Ubiquitin Ligases.

The thiazide-sensitive sodium chloride cotransporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which is controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of cullins (Cul1, 3, 4, and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/Δ9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect cullin neddylation levels in ex vivo renal tubules. In the short term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan-cullin inhibitor), but the response to low extracellular K+ was absent. In the long term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation, and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

Advances in Aquaporin-2 trafficking mechanisms and their implications for treatment of water balance disorders.

In mammals, conservation of body water is critical for survival and is dependent on the kidneys ability to minimize water loss in the urine during periods of water deprivation. The collecting duct water channel aquaporin-2 (AQP2) plays an essential role in this homeostatic response by facilitating water reabsorption along osmotic gradients. The ability to increase the levels of AQP2 in the apical plasma membrane following an increase in plasma osmolality is a rate-limiting step in water reabsorption, a process that is tightly regulated by the antidiuretic hormone arginine vasopressin (AVP). In this review, the focus is on the role of the carboxyl-terminus of AQP2 as a key regulatory point for AQP2 trafficking. We provide an overview of AQP2 structure, disease-causing mutations in the AQP2 carboxyl-terminus, the role of post-translational modifications such as phosphorylation and ubiquitylation in the tail domain, and their implications for balanced trafficking of AQP2. Finally, we discuss how various modifications of the AQP2 tail facilitate selective protein:protein interactions that modulate the AQP2 trafficking mechanism.

The Deubiquitylase USP4 Interacts with the Water Channel AQP2 to Modulate Its Apical Membrane Accumulation and Cellular Abundance.

Aquaporin 2 (AQP2) mediates the osmotic water permeability of the kidney collecting duct in response to arginine vasopressin (VP) and is essential for body water homeostasis. VP effects on AQP2 occur via long-term alterations in AQP2 abundance and short-term changes in AQP2 localization. Several of the effects of VP on AQP2 are dependent on AQP2 phosphorylation and ubiquitylation; post-translational modifications (PTM) that modulate AQP2 subcellular distribution and function. Although several protein kinases, phosphatases, and ubiquitin E3 ligases have been implicated in AQP2 PTM, how AQP2 is deubiquitylated or the role of deubiquitylases (DUBS) in AQP2 function is unknown. Here, we report a novel role of the ubiquitin-specific protease USP4 in modulating AQP2 function. USP4 co-localized with AQP2 in the mouse kidney, and in mpkCCD14 cells USP4 and AQP2 abundance are increased by VP. AQP2 and USP4 co-immunoprecipitated from mpkCCD14 cells and mouse kidney, and in vitro, USP4 can deubiquitylate AQP2. In mpkCCD14 cells, shRNA mediated knockdown of USP4 decreased AQP2 protein abundance, whereas no changes in AQP2 mRNA levels or VP-induced cAMP production were detected. VP-induced AQP2 membrane accumulation in knockdown cells was significantly reduced, which was associated with higher levels of ubiquitylated AQP2. AQP2 protein half-life was also significantly reduced in USP4 knockdown cells. Taken together, the data suggest that USP4 is a key regulator of AQP2 deubiquitylation and that loss of USP4 leads to increased AQP2 ubiquitylation, decreased AQP2 levels, and decreased cell surface AQP2 accumulation upon VP treatment. These studies have implications for understanding body water homeostasis.

Adenylyl Cyclase 6 Expression Is Essential for Cholera Toxin-Induced Diarrhea.

BACKGROUND: Cholera toxin (CT)-induced diarrhea is mediated by cyclic adenosine monophosphate (cAMP)-mediated active Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). Although the constitutive activation of adenylyl cyclase (AC) in response to CT is due to adenosine diphosphate ribosylation of the small G protein α-subunit activating CFTR with consequent secretory diarrhea, the AC isoform(s) involved remain unknown. METHODS: We generated intestinal epithelial cell-specific adenylyl cyclase 6 (AC6) knockout mice to study its role in CT-induced diarrhea. RESULTS: AC6 messenger RNA levels were the highest of all 9 membrane-bound AC isoforms in mouse intestinal epithelial cells. Intestinal epithelial-specific AC6 knockout mice (AC6loxloxVillinCre) had undetectable AC6 levels in small intestinal and colonic epithelial cells. No significant differences in fluid and food intake, plasma electrolytes, intestinal/colon anatomy and morphology, or fecal water content were observed between genotypes. Nevertheless, CT-induced fluid accumulation in vivo was completely absent in AC6loxloxVillinCre mice, associated with a lack of forskolin- and CT-induced changes in the short-circuit current (ISC) of the intestinal mucosa, impaired cAMP generation in acutely isolated small intestinal epithelial cells, and significantly impaired apical CFTR levels in response to forskolin. CONCLUSIONS: AC6 is a novel target for the treatment of CT-induced diarrhea.

Klotho Lacks an FGF23-Independent Role in Mineral Homeostasis.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone regulating vitamin D hormone production and renal handling of minerals by signaling through an FGF receptor/αKlotho (Klotho) receptor complex. Whether Klotho has FGF23-independent effects on mineral homeostasis is a controversial issue. Here, we aimed to shed more light on this controversy by comparing male and female triple knockout mice with simultaneous deficiency in Fgf23 and Klotho and a nonfunctioning vitamin D receptor (VDR) (Fgf23/Klotho/VDR) with double (Fgf23/VDR, Klotho/VDR, and Fgf23/Klotho) and single Fgf23, Klotho, and VDR mutants. As expected, 4-week-old Fgf23, Klotho, and Fgf23/Klotho knockout mice were hypercalcemic and hyperphosphatemic, whereas VDR, Fgf23/VDR, and Klotho/VDR mice on rescue diet were normocalcemic and normophosphatemic. Serum levels of calcium, phosphate, and sodium did not differ between 4-week-old triple Fgf23/Klotho/VDR and double Fgf23/VDR or Klotho/VDR knockout mice. Notably, 3-month-old Fgf23/Klotho/VDR triple knockout mice were indistinguishable from double Fgf23/VDR and Klotho/VDR compound mutants in terms of serum calcium, serum phosphate, serum sodium, and serum PTH, as well as urinary calcium and sodium excretion. Protein expression analysis revealed increased membrane abundance of sodium-phosphate co-transporter 2a (NaPi-2a), and decreased expression of sodium-chloride co-transporter (NCC) and transient receptor potential cation channel subfamily V member 5 (TRPV5) in Fgf23/Klotho/VDR, Fgf23/VDR, and Klotho/VDR mice, relative to wild-type and VDR mice, but no differences between triple and double knockouts. Further, ex vivo treatment of live kidney slices isolated from wild-type and Klotho/VDR mice with soluble Klotho did not induce changes in intracellular phosphate, calcium or sodium accumulation assessed by two-photon microscopy. In conclusion, our data suggest that the main physiological function of Klotho for mineral homeostasis in vivo is its role as co-receptor mediating Fgf23 action. © 2017 American Society for Bone and Mineral Research.

Excessive Osteocytic Fgf23 Secretion Contributes to Pyrophosphate Accumulation and Mineralization Defect in Hyp Mice.

X-linked hypophosphatemia (XLH) is the most frequent form of inherited rickets in humans caused by mutations in the phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX). Hyp mice, a murine homologue of XLH, are characterized by hypophosphatemia, inappropriately low serum vitamin D levels, increased serum fibroblast growth factor-23 (Fgf23), and osteomalacia. Although Fgf23 is known to be responsible for hypophosphatemia and reduced vitamin D hormone levels in Hyp mice, its putative role as an auto-/paracrine osteomalacia-causing factor has not been explored. We recently reported that Fgf23 is a suppressor of tissue nonspecific alkaline phosphatase (Tnap) transcription via FGF receptor-3 (FGFR3) signaling, leading to inhibition of mineralization through accumulation of the TNAP substrate pyrophosphate. Here, we report that the pyrophosphate concentration is increased in Hyp bones, and that Tnap expression is decreased in Hyp-derived osteocyte-like cells but not in Hyp-derived osteoblasts ex vivo and in vitro. In situ mRNA expression profiling in bone cryosections revealed a ~70-fold up-regulation of Fgfr3 mRNA in osteocytes versus osteoblasts of Hyp mice. In addition, we show that blocking of increased Fgf23-FGFR3 signaling with anti-Fgf23 antibodies or an FGFR3 inhibitor partially restored the suppression of Tnap expression, phosphate production, and mineralization, and decreased pyrophosphate concentration in Hyp-derived osteocyte-like cells in vitro. In vivo, bone-specific deletion of Fgf23 in Hyp mice rescued the suppressed TNAP activity in osteocytes of Hyp mice. Moreover, treatment of wild-type osteoblasts or mice with recombinant FGF23 suppressed Tnap mRNA expression and increased pyrophosphate concentrations in the culture medium and in bone, respectively. In conclusion, we found that the cell autonomous increase in Fgf23 secretion in Hyp osteocytes drives the accumulation of pyrophosphate through auto-/paracrine suppression of TNAP. Hence, we have identified a novel mechanism contributing to the mineralization defect in Hyp mice.