Steven C. Hebert

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: I. ADH increases transcellular conductance pathways.

SummaryThis paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfusedin vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (Gcmin) of the transcellular conductance, estimated from Ba++ blockade of apical membrane K+ channels, indicated thatGcmin was approximately 30–40% of the measured transepithelial conductance. In apical membranes, K+ was the major conductive species; and ADH increased the magnitude of a Ba++-sensitive K+ conductance under conditions where net Cl− absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl− conductance; this ADH-dependent increase in basal Cl− conductance depended on a simultaneous hormone-dependent increase in the rate of net Cl− absorption. Cl− removal from luminal solutions had no detectable effect onGe, and net Cl− absorption was reduced at luminal K+ concentrations less than 5mm; thus apical Cl− entry may have been a Na+,K+,2Cl− cotransport process having a negligible conductance. The net rate of K+ secretion was approximately 10% of the net rate of Cl− absorption, while the chemical rate of net Cl− absorption was virtually equal to the equivalent short-circuit current. Thus net Cl− absorption was rheogenic; and approximately half of net Na+ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K+ was the principal conductive species in apical plasma membranes, support the view that the majority of K+ efflux from cell to lumen through the Ba++-sensitive apical K+ conductance pathway was recycled into cells by Na+,K+,2Cl− cotransport.

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl− absorption

SummaryCellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,Ve, which attends net Cl− absorption,JClnet, and to assess how ADH might augment bothJClmet andVe in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba++ for 5mm K+ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,Rc/Rbl, and increasing luminal K+ from 5 to 50mm in the presence of luminal 10−4m furosemide resulted in a 53-mV depolarization of apical membrane voltage,Va. Thus K+ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10−4m luminal furosemide reducedVe andJClnet to near zero values and hyperpolarized bothVa andVbl, the voltage across basolateral membranes; however, the depolarization ofVbl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofVa, Thus ADH-dependent increases inVb were referable to greater depolarizations ofVbl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofVa was referable to a decrease in the K+ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofVbl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl− current. Thus an increase in intracellular Cl− activity may have contributed to the depolarization ofVbl during net Cl− absorption, and the intracellular Cl− activity was likely greater with ADH than without hormone. Since ADH increases apical K+ conductance and since the chemical driving force for electroneutral Na+,K+,2Cl− cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba++-sensitive conductance K+ channels and electroneutral Na+,K+,2Cl− cotransport units in apical plasma membranes.

N-Glycosylation at Two Sites Critically Alters Thiazide Binding and Activity of the Rat Thiazide-sensitive Na+:Cl− Cotransporter

The rat thiazide-sensitive Na-Cl cotransporter (rNCC) is expressed in the renal distal convoluted tubule and is the site of action of an important class of antihypertensive agents, the thiazide diuretics. The amino acid sequence contains two potential N-linked glycosylation consensus sites, N404 and N424. Either enzymatic deglycosylation or tunicamycin reduced the cotransporter to its core molecular weight (113 kD). Glycosylation site single mutants expressed in oocytes ran as thick bands at 115 kD, consistent with the high-mannose glycoprotein. The double mutant produced the single thin 113-kD band seen in the deglycosylated cotransporter. Functional expression of cotransporters in Xenopus laevis oocytes revealed that the mutants displayed drastically decreased thiazide-sensitive (22)Na(+) uptake compared with wild-type NCC. Analysis of enhanced green fluorescence protein (EGFP)-tagged cotransporters demonstrated that this decrease in function is predominantly secondary to decreased surface expression. The elimination of glycosylation in the double mutant increased thiazide sensitivity by more than two orders of magnitude and also increased Cl(-) affinity. Thus, we have demonstrated that rNCC is N-glycosylated in vivo at two sites, that glycosylation is essential for efficient function and surface expression of the cotransporter, and that the elimination of glycosylation allows much greater access of thiazide diuretics to their binding site.

Electroneutral Na-coupled cotransporter expression in the kidney during variations of NaCl and water metabolism.

The purpose of the present study was to analyze the long-term regulation of renal bumetanide-sensitive Na+-K+-2Cl- cotransporter and thiazide-sensitive Na+-Cl- cotransporter gene expression during changes in NaCl and water metabolism. Male Wistar rats exposed to high or low NaCl intake, saline loading, dehydration, water loading, and furosemide administration during 7 days were studied. Control groups had access to regular food and tap water. Rats were kept in metabolic cages for 4 days before and during the experiment to determine daily urinary electrolyte excretion and osmolarity. At the end of the experiment, creatinine clearance and serum electrolyte levels were also measured. Kidneys were excised and macroscopically subdivided into cortex and outer and inner medulla. Total RNA was extracted from each individual cortex or outer medulla by use of the guanidine/cesium chloride method. The Na+-K+-2Cl- cotransporter expression in outer medulla total RNA was assessed by nonradioactive Northern blot analysis and the Na+-Cl- cotransporter expression in renal cortex total RNA was assessed by semiquantitative polymerase chain reaction. Experimental maneuvers were adequately tolerated, and all groups developed the appropriate renal response to each challenge. However, the level of expression of both cotransporters did not change in any model, except for a 2.8-fold increase in the Na+-Cl- cotransporter expression during dehydration. We conclude that nephron adaptation to 7-day modifications in NaCl and water metabolism does not include changes in the amount of electroneutral sodium-coupled cotransporter gene expression at the mRNA level.

The effects of antidiuretic hormone (ADH) on solute and water transport in the mammalian nephron

The biochemical and physiological events responsible for osmotic homeostasis range from vasopressin (antidiuretic hormone, ADH) synthesis and release from the central nervous system to the action of ADH on specific renal tubular cells, the latter being ultimately responsible for water conservation by the kidney. Although a general review of this subject would appear timely, the explosion of knowledge in this field over the last decade has been great; thus covering all recent events would permit only a superficial analysis. Consequently, this review has been restricted to an examination of the effects of antidiuretic hormone on solute and volume flows in the mammalian nephron. Although such a choice might appear myopic, we believe it to be justifiable for several reasons. First, an understanding of the physiological effects of ADH on renal tubular epithelia forms a basis for analyzing the pathophysiology of clinical disorders of renal water transport. Second, recent experimental observations on the temperature-dependence of tracer water diffusion and net volume flow in cortical collecting tubules, both in the presence and absence of ADH, provide a way of assigning specific characteristics to the water permeation sites in apical membranes of these tubules, and to the way ADH affects these regions. In addition, this type of analysis demonstrates the importance of explicit knowledge of the major barriers which impede transport across epithelia when interpreting apparent breaks in Arrhenius activation energy relations, either for water transport or for solute transport. Third, the discovery of an ADH-induced NaC1 absorptive mechanism (Hall, 1979; Hall & Varney, 1979; Hebert etal., 1980b) in the mouse medullary thick ascending limb of Henles loop provides a way of investigating the

Inherited and Acquired Disorders of the Extracellular CA2+0-Sensing Receptor

Publisher Summary This chapter discusses the inherited and acquired disorders of the extracellular CA 2+ O -sensing receptor. Calcium (Ca 2+) ions are of critical importance for a wide variety of vital bodily functions within both the extracellular and intracellular compartments. The application of molecular techniques has enabled cloning of an extracellular Ca 2+ o -sensing receptor (CaR), initially from parathyroid. The inherited disorders of Ca 2+ o -sensing are familial hypocalciuric hypercalcemia (FHH); neonatal severe primary hyperparathyroidism (NSHPT); mice with targeted disruption of the CaR gene: an animal model for FHH and NSHPT; and autosomal dominant hypocalcemia (ADH). Detection of mutations in the CaR in patients with sporadic, asymptomatic hypercalcemia could clearly be helpful in diagnosing de novo cases of FHH, although the large size of the CaRs coding sequence makes this an arduous undertaking if direct sequencing is carried out. More rapid procedures for detecting point mutations (that is, the use of denaturing gradient gel electrophoresis) could facilitate mutational analysis in such cases. The recognition that parathyroid cells recognize and respond to changes in Ca 2+ o through a cell surface, G protein-linked receptor led to attempts to develop drugs targeted at the receptor. Calcimimetics, drugs that mimic the effects of high Ca 2+ o on the receptor, for instance, is developed recently. One such agent, NPS R-568, is currently in the process of undergoing clinical trials for the treatment of primary and secondary hyperparathyroidism.

Rapid Cell Volume Regulation by the Mouse Medullary Thick Ascending Limb of Henle

In this paper, we summarize our current knowledge regarding cell volume regulation in mouse medullary thick ascending loop of Henle (MTAL) cells. It has become apparent that arginine vasopressin (ADH) plays a central role in this process (at least in certain species). During antidiuresis ADH increases the rate of NaCl absorption by the MTAL, thereby enhancing the single effect of countercurrent multiplication. In addition, ADH is required for MTAL cells to regulate their volume in the more hypertonic environment. ADH appears to activate normally quiescent basolateral Na+:H+ exchangers which mediate Na+ uptake into MTAL cells during volume regulatory increase (VRI). This action of ADH may be mediated via an increase in cytosolic calcium. The trade off for this effect of ADH appears to be that the MTAL cells are no longer able to regulate their cell volume completely following reductions in interstitial osmolality. This is a direct result of an inverse relationship between the rates of salt absorption and volume regulatory decrease (VRD). This may not present a problem for the MTAL in vivo for two reasons. First, when interstitial osmolality is increased, NaCl absorption is reduced (see [1–3]). Second, when interstitial osmolality decreases during the transition from an antidiuretic to a water diuretic state the circulating level of ADH falls, and consequently, the rapid VRD response would be restored.

Inherited Diseases of the Calcium-Sensing Receptor: Impact on Parathyroid and Renal Function

Publisher Summary This chapter gives an overview on inherited diseases of the calcium-sensing receptor (CaR or CaSR) that have significant impact on parathyroid and renal function. One of the diseases, neonatal severe primary hyperparathyroidism (NSHPT), typically presents within the first six months of life. Affected infants have severe, symptomatic, parathyroid hormone (PTH)-dependent hypercalcemia as well as the bony changes of severe hyperparathyroidism. Infants with NSHPT can also manifest polyuria, dehydration, hypotonia, and failure to thrive. A prominent component of the disorder is the hyperparathyroid bone disease, which can lead to multiple fractures of the long bones and other skeletal sites. Patients with autosomal dominant form of hypocalcemia/hypoparathyroidism are commonly asymptomatic, similar to the majority of patients with familial hypocalciuric hypercalcemia (FHH). Some exhibit neuromuscular irritability, seizures, and calcification of the basal ganglia, complications commonly seen in other forms of hypoparathyroidism in which the CaR gene is normal. During febrile episodes, patients with autosomal dominant hypoparathyroidism (ADH), particularly children, can exhibit symptomatic hypocalcemia and, in some cases, develop seizures. Patients with ADH appear particularly susceptible to complications during treatment with Ca2+ and vitamin D analogs aimed at increasing their serum Ca2+ concentrations toward normal. They are especially prone to developing renal complications, including nephrocalcinosis, nephrolithiasis, and renal impairment during treatment of ADH patients with Ca2+and vitamin D. The renal complications observed during treatment of ADH generally occur in a setting in which the clinician has tried to correct the serum Ca2+ concentration to or close to the normal range. Treatment with Ca2+ supplements and vitamin D metabolites should be reserved for those patients with symptomatic ADH.

Roderick MacKinnon, MD, honored by 2003 nobel prize in chemistry for work on elucidating the structure of ion channels

This past year, Rod Mackinnon shared the Nobel Prize in Chemistry for his groundbreaking discoveries in defining the structures of potassium and chloride channels. This work sheds new light on the mechanisms by which channels determine which ion to permit passage (called selectivity) and open or

Inherited Diseases of the Calcium-Sensing Receptor

Publisher Summary This chapter gives an overview on inherited diseases of the calcium-sensing receptor (CaR or CaSR) that have significant impact on parathyroid and renal function. One of the diseases, neonatal severe primary hyperparathyroidism (NSHPT), typically presents within the first six months of life. Affected infants have severe, symptomatic, parathyroid hormone (PTH)-dependent hypercalcemia as well as the bony changes of severe hyperparathyroidism. Infants with NSHPT can also manifest polyuria, dehydration, hypotonia, and failure to thrive. A prominent component of the disorder is the hyperparathyroid bone disease, which can lead to multiple fractures of the long bones and other skeletal sites. Patients with autosomal dominant form of hypocalcemia/hypoparathyroidism are commonly asymptomatic, similar to the majority of patients with familial hypocalciuric hypercalcemia (FHH). Some exhibit neuromuscular irritability, seizures, and calcification of the basal ganglia, complications commonly seen in other forms of hypoparathyroidism in which the CaR gene is normal. During febrile episodes, patients with autosomal dominant hypoparathyroidism (ADH), particularly children, can exhibit symptomatic hypocalcemia and, in some cases, develop seizures. Patients with ADH appear particularly susceptible to complications during treatment with Ca2+ and vitamin D analogs aimed at increasing their serum Ca2+ concentrations toward normal. They are especially prone to developing renal complications, including nephrocalcinosis, nephrolithiasis, and renal impairment during treatment of ADH patients with Ca2+and vitamin D. The renal complications observed during treatment of ADH generally occur in a setting in which the clinician has tried to correct the serum Ca2+ concentration to or close to the normal range. Treatment with Ca2+ supplements and vitamin D metabolites should be reserved for those patients with symptomatic ADH.