Tight regulation of calcium levels is required for many critical biological functions. The Ca²⁺-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.

Three pregnancies with male offspring in one family were complicated by severe polyhydramnios and prematurity. One fetus died; the other two had transient massive salt-wasting and polyuria reminiscent of antenatal Bartter’s syndrome. To uncover the molecular cause of this possibly X-linked disease, we performed whole-exome sequencing of DNA from two members of the index family and targeted gene analysis of other members of this family and of six additional families with affected male fetuses. We also evaluated a series of women with idiopathic polyhydramnios who were pregnant with male fetuses. We performed immunohistochemical analysis, knockdown and overexpression experiments, and protein–protein interaction studies. We identified a mutation in MAGED2 in each of the 13 infants in our analysis who had transient antenatal Bartter’s syndrome. MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2) and maps to the X chromosome. We also identified two different MAGED2 mutations in two families with idiopathic polyhydramnios. Four patients died perinatally, and 11 survived. The initial presentation was more severe than in known types of antenatal Bartter’s syndrome, as reflected by an earlier onset of polyhydramnios and labor. All symptoms disappeared spontaneously during follow-up in the infants who survived. We showed that MAGE-D2 affects the expression and function of the sodium chloride cotransporters NKCC2 and NCC (key components of salt reabsorption in the distal renal tubule), possibly through adenylate cyclase and cyclic AMP signaling and a cytoplasmic heat-shock protein. We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartter’s syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.)

Activation of the Na⁺-K⁺-2Cl⁻-cotransporter (NKCC2) and the Na⁺-Cl⁻-cotransporter (NCC) by vasopressin includes their phosphorylation at defined, conserved N-terminal threonine and serine residues, but the kinase pathways that mediate this action of vasopressin are not well understood. Two homologous Ste20-like kinases, SPS-related proline/alanine-rich kinase (SPAK) and oxidative stress responsive kinase (OSR1), can phosphorylate the cotransporters directly. In this process, a full-length SPAK variant and OSR1 interact with a truncated SPAK variant, which has inhibitory effects. Here, we tested whether SPAK is an essential component of the vasopressin stimulatory pathway. We administered desmopressin, a V2 receptor–specific agonist, to wild-type mice, SPAK-deficient mice, and vasopressin-deficient rats. Desmopressin induced regulatory changes in SPAK variants, but not in OSR1 to the same degree, and activated NKCC2 and NCC. Furthermore, desmopressin modulated both the full-length and truncated SPAK variants to interact with and phosphorylate NKCC2, whereas only full-length SPAK promoted the activation of NCC. In summary, these results suggest that SPAK mediates the effect of vasopressin on sodium reabsorption along the distal nephron.

Sodium and its associated anions are the major determinant of extracellular fluid volume, and the reabsorption of Na⁺by the kidney plays a crucial role in long-term blood pressure control. The goal of this study was to investigate the extent to which inhibitors of transepithelial Na⁺transport (T_Na) along the nephron alter urinary solute excretion and T_Naefficiency and how those effects may vary along different nephron segments. To accomplish that goal, we used the multinephron model developed in the companion study (28). That model represents detailed transcellular and paracellular transport processes along the nephrons of a rat kidney. We simulated the inhibition of the Na⁺/H⁺exchanger (NHE3), the bumetanide-sensitive Na⁺-K⁺-2Cl⁻transporter (NKCC2), the Na⁺-Cl⁻cotransporter (NCC), and the amiloride-sensitive Na⁺channel (ENaC). Under baseline conditions, NHE3, NKCC2, NCC, and ENaC reabsorb 36, 22, 4, and 7%, respectively, of filtered Na⁺. The model predicted that inhibition of NHE3 substantially reduced proximal tubule T_Naand oxygen consumption (QO₂). Whole-kidney T_Naefficiency, as reflected by the number of moles of Na⁺reabsorbed per moles of O₂consumed (denoted by the ratio T_Na/QO₂), decreased by ∼20% with 80% inhibition of NHE3. NKCC2 inhibition simulations predicted a substantial reduction in thick ascending limb T_Naand QO₂; however, the effect on whole-kidney T_Na/QO₂was minor. Tubular K⁺transport was also substantially impaired, resulting in elevated urinary K⁺excretion. The most notable effect of NCC inhibition was to increase the excretion of Na⁺, K⁺, and Cl⁻; its impact on whole-kidney T_Naand its efficiency was minor. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na⁺(increased) and K⁺(decreased) and to have only a minor impact on whole-kidney T_Naand T_Na/QO₂. Overall, model predictions agree well with measured changes in Na⁺and K⁺excretion in response to diuretics and Na⁺transporter mutations.

Decreases in blood pH activate NHE3, the proximal tubular apical membrane Na/H antiporter. In cultured renal epithelial cells, activation of the endothelin-B (ET_B) receptor increases NHE3 activity. To examine the role of the ET_B receptor in the response to acidosis in vivo, the present studies examined ET_B receptor–deficient mice, rescued from neonatal lethality by expression of a dopamine β-hydroxylase promoter/ET_B receptor transgene (Tg/Tg:ET_B^–/– mice). In proximal tubule suspensions from Tg/Tg:ET_B^+/– mice, 10^–8 M endothelin-1 (ET-1) increased NHE3 activity, but this treatment had no effect on tubules from Tg/Tg:ET_B^–/– mice. Acid ingestion for 7 days caused a greater decrease in blood HCO₃^– concentration in Tg/Tg:ET_B^–/– mice compared with Tg/Tg:ET_B^+/+ and Tg/Tg:ET_B^+/– mice. Whereas acid ingestion increased apical membrane NHE3 by 42–46% in Tg/Tg:ET_B^+/+ and Tg/Tg:ET_B^+/– mice, it had no effect on NHE3 in Tg/Tg:ET_B^–/– mice. In C57BL/6 mice, excess acid ingestion increased renal cortical preproET-1 mRNA expression 2.4-fold and decreased preproET-3 mRNA expression by 37%. On a control diet, Tg/Tg:ET_B^–/– mice had low rates of ammonium excretion, which could not be attributed to an inability to acidify the urine, as well as hypercitraturia, with increased titratable acid excretion. Acid ingestion increased ammonium excretion, citrate absorption, and titratable acid excretion to the same levels in Tg/Tg:ET_B^–/– and Tg/Tg:ET_B^+/+ mice. In conclusion, metabolic acidosis increases ET-1 expression, which increases NHE3 activity via the ET_B receptor.

Chronic metabolic acidosis (CMA) is associated with an adaptive increase in the bicarbonate absorptive capacity of the rat medullary thick ascending limb (MTAL). To specify whether NHE-3, the apical MTAL Na/H exchanger, is involved in this adaptation, NHE-3 mRNA was quantified by a competitive RT-PCR using an internal standard which differed from the wild-type NHE-3 mRNA by an 80-bp deletion. CMA increased NHE-3 mRNA from 0.025+/-0.003 to 0.042+/-0.009 amol/ng total RNA (P < 0.005). NHE-3 transport activity was measured as the initial proton flux rate calculated from the Na-dependent cell pH recovery of Na-depleted acidified MTAL cells in the presence of 50 microM HOE694 which specifically blocks NHE-1, the basolateral MTAL NHE isoform. CMA caused a 68% increase in NHE-3 transport activity (P < 0.001). In addition, CMA was associated with a 71% increase in NHE-3 protein abundance (P < 0.05) as determined by Western blot analysis on MTAL membranes using a polyclonal antiserum directed against a cytoplasmic epitope of rat NHE-3. Thus, NHE-3 adapts to CMA in the rat MTAL via an increase in the mRNA transcript that enhances NHE-3 protein abundance and transport activity.