Sam X. Cheng

WNK4 regulates activity of the epithelial Na+ channel in vitro and in vivo

Homeostasis of intravascular volume, Na+, Cl−, and K+ is interdependent and determined by the coordinated activities of structurally diverse mediators in the distal nephron and the distal colon. The behavior of these flux pathways is regulated by the renin–angiotensin–aldosterone system; however, the mechanisms that allow independent modulation of individual elements have been obscure. Previous work has shown that mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension with hyperkalemia, due to altered activity of specific Na-Cl cotransporters, K+ channels, and paracellular Cl− flux mediators of the distal nephron. By coexpression studies in Xenopus oocytes, we now demonstrate that WNK4 also inhibits the epithelial Na+ channel (ENaC), the major mediator of aldosterone-sensitive Na+ (re)absorption, via a mechanism that is independent of WNK4s kinase activity. This inhibition requires intact C termini in ENaC β- and γ-subunits, which contain PY motifs used to target ENaC for clearance from the plasma membrane. Importantly, PHAII-causing mutations eliminate WNK4s inhibition of ENaC, thereby paralleling other effects of PHAII to increase sodium balance. The relevance of these findings in vivo was studied in mice harboring PHAII-mutant WNK4. The colonic epithelium of these mice demonstrates markedly increased amiloride-sensitive Na+ flux compared with wild-type littermates. These studies identify ENaC as a previously unrecognized downstream target of WNK4 and demonstrate a functional role for WNK4 in the regulation of colonic Na+ absorption. These findings support a key role for WNK4 in coordinating the activities of diverse flux pathways to achieve integrated fluid and electrolyte homeostasis.

[Ca2+]i determines the effects of protein kinases A and C on activity of rat renal Na+,K+‐ATPase

1 It is well established that the activity of Na+,K+‐ATPase (NKA) is regulated by protein kinases A (PKA) and C (PKC), but results on their effects have been conflicting. The aim of this study was to examine if this is ascribed to the intracellular concentration of Ca2+ ([Ca2+]i). 2 Rat renal NKA was stably expressed in COS cells (green monkey kidney cells). Increases in [Ca2+]i were achieved with the Ca2+ ionophore A23187 and verified by direct measurements of [Ca2+]i using fura‐2 AM as an indicator. The activity of NKA was measured as ouabain‐sensitive 86Rb+ uptake and the state of phosphorylation of NKA was monitored with two site‐directed phosphorylation state‐specific antibodies. 3 Activation of PKA with forskolin decreased NKA activity by 45.5 ± 8.9 % at low [Ca2+]i (120 nM) and increased it by 40.5 ± 6.4 % at high [Ca2+]i (420 nM). The change in NKA activity by forskolin correlated with the level of increase in [Ca2+]i. 4 The effect of 1‐oleoyl‐2‐acetoyl‐sn‐glycerol (OAG), a specific PKC activator, on the activity of NKA was also Ca2+ dependent, being inhibitory when [Ca2+]i was low (29.3 ± 3.6 % decrease at 120 nM Ca2+) and stimulatory when [Ca2+]i was high (36.6 ± 10.1 % increase at 420 nM Ca2+). 5 The α subunit of NKA was phosphorylated under both low and high [Ca2+]i conditions upon PKA or PKC activation. PKA phosphorylates Ser943. PKC phosphorylates Ser23. 6 To see if the observed effects on NKA activity are secondary to changes in Na+ entry, we measured NKA hydrolytic activity using permeabilized membranes isolated from cells under controlled Na+ conditions. A decreased activity at low [Ca2+]i and no change in activity at high [Ca2+]i were observed following forskolin or OAG treatment. 7 Purified NKA from rat renal cortex was phosphorylated and inhibited by PKC. This phosphorylation‐associated inhibition of NKA was neither affected by Ca2+ nor by calmodulin, tested alone or together. 8 We conclude that effect of PKA/PKC on NKA activity is dependent on [Ca2+]i. This Ca2+ dependence may provide an explanation for the diversity of responses of NKA to activation of either PKA or PKC.

Calcium-sensing receptor inhibits secretagogue-induced electrolyte secretion by intestine via the enteric nervous system

Bacterial toxins such as cholera toxin induce diarrhea by both direct epithelial cell generation of cyclic nucleotides as well as stimulation of the enteric nervous system (ENS). Agonists of the extracellular calcium-sensing receptor (CaSR) can reduce toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Here we show that the CaSR is also highly expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, suggesting that CaSR agonists might also function through neuronal pathways. To test this hypothesis, rat colon segments containing intact ENS were isolated and mounted on Ussing chambers. Basal and cyclic nucleotide-stimulated electrolyte secretions were monitored by measuring changes in short-circuit current (I(sc)). CaSR was activated by R-568 and its effects were compared in the presence and absence of TTX. Consistent with active regulation of anion secretion by the ENS, a significant proportion of I(sc) in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. In the absence of TTX, activation of CaSR with R-568 significantly reduced basal I(sc) and cyclic AMP-stimulated I(sc); it also completely reversed the cAMP-stimulated secretory responses if the drug was applied after the forskolin stimulation. Such inhibitory effects of R-568 were either absent or significantly reduced when serosal TTX was present, suggesting that this agonist exerts its antisecretory effect on the intestine by inhibiting ENS. The present results suggest a new model for regulating intestinal fluid transport in which neuronal and nonneuronal secretagogue actions are modulated by the inhibitory effects of CaSR on the ENS. The ability of a CaSR agonist to reduce secretagogue-stimulated Cl(-) secretion might provide a new therapeutic approach for secretory and other ENS-mediated diarrheal conditions.

Epithelial CaSR deficiency alters intestinal integrity and promotes proinflammatory immune responses

The intestinal epithelium is equipped with sensing receptor mechanisms that interact with luminal microorganisms and nutrients to regulate barrier function and gut immune responses, thereby maintaining intestinal homeostasis. Herein, we clarify the role of the extracellular calcium‐sensing receptor (CaSR) using intestinal epithelium‐specific Casr −/− mice. Epithelial CaSR deficiency diminished intestinal barrier function, altered microbiota composition, and skewed immune responses towards proinflammatory. Consequently, Casr −/− mice were significantly more prone to chemically induced intestinal inflammation resulting in colitis. Accordingly, CaSR represents a potential therapeutic target for autoinflammatory disorders, including inflammatory bowel diseases.

Effects of okadaic acid, calyculin A, and PDBu on state of phosphorylation of rat renal Na+-K+-ATPase.

Several indirect lines of evidence suggest that protein kinases and phosphatases modulate the activity of renal Na+-K+-ATPase. The aim of this study was to examine whether such regulation may occur via modulation of the state of phosphorylation of Na+-K+-ATPase. Slices from rat renal cortex were prelabeled with [32P]orthophosphate and incubated with the inhibitors of protein phosphatase (PP)-1 and PP-2A, okadaic acid (OA) and calyculin A (CL-A), respectively, the protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), or the PP-2B inhibitor, FK-506. Phosphorylation of Na+-K+-ATPase α-subunit was evaluated by measuring the amount of [32P]phosphate incorporation into the immunoprecipitated protein. Incubation with either OA, CL-A, or PDBu caused four- to fivefold increases in the amount of [32P]phosphate incorporation into immunoprecipitated Na+-K+-ATPase α-subunit. OA and PDBu had a synergistic effect on the state of phosphorylation of Na+-K+-ATPase α-subunit. FK-506 did not affect Na+-K+-ATPase phosphorylation, neither alone nor in the presence of PDBu. Each of the drugs, OA, CL-A, and PDBu, inhibited the activity of Na+-K+-ATPase in microdissected proximal tubules. PDBu potentiated OA-induced inhibition of Na+-K+-ATPase activity. Inhibition of Na+-K+-ATPase required a lower dose of CL-A than of OA. On the basis of the inhibitory constant values of CL-A and OA for PP-1 and PP-2A, it is concluded that the tubular effect is mainly due to inhibition of PP-1. The PP-1 activity in rat renal cortex was ∼1.5 nmol Pi ⋅ mg protein-1 ⋅ min-1. Using a monoclonal anti-α antibody that fails to recognize the subunit when Ser23 is phosphorylated by PKC, we demonstrated that the dose response of PDBu inhibition of Na+-K+-ATPase correlated with the dose response of phosphorylation of the enzyme. The results suggest that the state of phosphorylation and activity of proximal tubular Na+-K+-ATPase are determined by the balance between the activities of protein kinases and phosphatases.

Calcium-sensing receptor stimulates Cl(-)- and SCFA-dependent but inhibits cAMP-dependent HCO3(-) secretion in colon.

Colonic bicarbonate (HCO3(-)) secretion is a well-established physiological process that is closely linked to overall fluid and electrolyte movement in the mammalian colon. These present studies show that extracellular calcium-sensing receptor (CaSR), a fundamental mechanism for sensing and regulating ionic and nutrient compositions of extracellular milieu in the small and large intestine, regulates HCO3(-) secretion. Basal and induced HCO3(-) secretory responses to CaSR agonists were determined by pH stat techniques used in conjunction with short-circuit current measurements in mucosa from rat distal colon mounted in Ussing chambers. R568, a specific CaSR activator, stimulated lumen Cl(-)- and short-chain fatty acid (SCFA)-dependent HCO3(-) secretion but inhibited cyclic nucleotide-activated HCO3(-) secretion. Consequently, at physiological conditions (either at basal or during lumen acid challenge) when electroneutral Cl(-)/HCO3(-) and SCFA/HCO3(-) exchangers dominate, CaSR stimulates HCO3(-) secretion; in contrast, in experimental conditions that stimulate fluid and HCO3(-) secretion, e.g., when forskolin activates electrogenic cystic fibrosis transmembrane conductance regulator-mediated HCO3(-) conductance, CaSR activation inhibits HCO3(-) secretion. Corresponding changes in JHCO3 (μeq·h(-1)·cm(-2), absence vs. presence of R568) were 0.18 ± 0.03 vs. 0.31 ± 0.08 under basal nonstimulated conditions and 1.85 ± 0.23 vs. 0.45 ± 0.06 under forskolin-stimulated conditions. Similarly, activation of CaSR by R568 stimulated Cl(-)- and SCFA-dependent HCO3(-) secretion and inhibited cAMP-dependent HCO3(-) secretion in colon mucosa of wild-type mice; such effects were abolished in CaSR-null mice. These results suggest a new paradigm for regulation of intestinal ion transport in which HCO3(-) secretion may be fine-tuned by CaSR in accordance with nutrient availability and state of digestion and absorption. The ability of CaSR agonists to inhibit secretagogue-induced intestinal HCO3(-) secretion suggests that modulation of CaSR activity may provide a new therapeutic approach to correct HCO3(-) deficit and metabolic acidosis, a primary cause of morbidity and mortality in acute infectious diarrheal illnesses.

Corrigendum: The Extracellular Calcium-Sensing Receptor in the Intestine: Evidence for Regulation of Colonic Absorption, Secretion, Motility, and Immunity

Different from other epithelia, the intestinal epithelium has the complex task of providing a barrier impeding the entry of toxins, food antigens, and microbes, while at the same time allowing for the transfer of nutrients, electrolytes, water, and microbial metabolites. These molecules/organisms are transported either transcellularly, crossing the apical and basolateral membranes of enterocytes, or paracellularly, passing through the space between enterocytes. Accordingly, the intestinal epithelium can affect energy metabolism, fluid balance, as well as immune response and tolerance. To help accomplish these complex tasks, the intestinal epithelium has evolved many sensing receptor mechanisms. Yet, their roles and functions are only now beginning to be elucidated. This article explores one such sensing receptor mechanism, carried out by the extracellular calcium-sensing receptor (CaSR). In addition to its established function as a nutrient sensor, coordinating food digestion, nutrient absorption, and regulating energy metabolism, we present evidence for the emerging role of CaSR in the control of intestinal fluid homeostasis and immune balance. An additional role in the modulation of the enteric nerve activity and motility is also discussed. Clearly, CaSR has profound effects on many aspects of intestinal function. Nevertheless, more work is needed to fully understand all functions of CaSR in the intestine, including detailed mechanisms of action and specific pathways involved. Considering the essential roles CaSR plays in gastrointestinal physiology and immunology, research may lead to a translational opportunity for the development of novel therapies that are based on CaSRs unique property of using simple nutrients such as calcium, polyamines, and certain amino acids/oligopeptides as activators. It is possible that, through targeting of intestinal CaSR with a combination of specific nutrients, oral solutions that are both inexpensive and practical may be developed to help in conditioning the gut microenvironment and in maintaining digestive health.

Colonic Immune Suppression, Barrier Dysfunction, and Dysbiosis by Gastrointestinal Bacillus anthracis Infection

Gastrointestinal (GI) anthrax results from the ingestion of Bacillus anthracis. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated B. anthracis Sterne strain (pXO1+ pXO2−) spores that resulted in rapid animal death. B. anthracis Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only B. anthracis, but also of commensals. Disease progression significantly correlated with the deterioration of innate and T cell functions. Our studies provide critical immunologic and physiologic insights into the pathogenesis of GI anthrax infection, whereupon cleavage of mitogen-activated protein kinases (MAPKs) in immune cells may play a central role in promoting dysfunctional immune responses against this deadly pathogen.

The role of the calcium-sensing receptor in gastrointestinal inflammation.

The gastrointestinal (GI) tract must balance the extraction of energy and metabolic end-products from ingested nutrition and resident gut microbes and the maintenance of a symbiotic relationship with this microbiota, with the ability to mount functional immune responses to pathogenic organisms to maintain GI health. The gut epithelium is equipped with bacteria-sensing mechanisms that discriminate between pathogenic and commensal microorganisms and regulate host responses between immunity and tolerance. The epithelium also expresses numerous nutrient-sensing receptors, but their importance in the preservation of the gut microbiota and immune homeostasis remains largely unexplored. Observations that a deficiency in the extracellular calcium-sensing receptor (CaSR) using intestinal epithelium-specific receptor knockout mice resulted in diminished intestinal barrier integrity, altered composition of the gut microbiota, modified expression of intestinal pattern recognition receptors, and a skewing of local and systemic innate responses from regulatory to stimulatory, may change the way that this receptor is considered as a potential immunotherapeutic target in gut homeostasis. These findings suggest that pharmacologic CaSR activators and CaSR-based nutrients such as calcium, polyamines, phenylalanine, tryptophan, and oligo-peptides might be useful in conditioning the gut microenvironment, and thus, in the prevention and treatment of disorders such as inflammatory bowel disease (IBD), infectious enterocolitis, and other inflammatory and secretory diarrheal diseases. Here, we review the emerging roles of the CaSR in intestinal homeostasis and its therapeutic potential for gut pathology.

Calcium ameliorates diarrhea in immunocompromised children.

ABSTRACT Treatment of infectious diarrheas remains a challenge, particularly in immunocompromised patients in whom infections usually persist and resultant diarrhea is often severe and protracted. Children with infectious diarrhea who become dehydrated are normally treated with oral or intravenous rehydration therapy. Although rehydration therapy can replace the loss of fluid, it does not ameliorate diarrhea. Thus, during the last decades, there has been continuous effort to search for ways to safely stop diarrhea. Herein, we report 3 immunocompromised children who developed severe and/or protracted infectious diarrhea. Their diarrheas were successfully “halted” within 1 to 2 days following the administration of calcium.