The thiazide-sensitive Na–Cl cotransporter is an aldosterone-induced protein

Although the collecting duct is regarded as the primary site at which mineralocorticoids regulate renal sodium transport in the kidney, recent evidence points to the distal convoluted tubule as a possible site of mineralocorticoid action. To investigate whether mineralocorticoids regulate the expression of the thiazide-sensitive Na–Cl cotransporter (TSC), the chief apical sodium entry pathway of distal convoluted tubule cells, we prepared an affinity-purified, peptide-directed antibody to TSC. On immunoblots, the antibody recognized a prominent 165-kDa band in membrane fractions from the renal cortex but not from the renal medulla. Immunofluorescence immunocytochemistry showed TSC labeling only in distal convoluted tubule cells. Semiquantitative immunoblotting studies demonstrated a large increase in TSC expression in the renal cortex of rats on a low-NaCl diet (207 ± 21% of control diet). Immunofluorescence localization in tissue sections confirmed the strong increase in TSC expression. Treatment of rats for 10 days with a continuous subcutaneous infusion of aldosterone also increased TSC expression (380 ± 58% of controls). Furthermore, 7-day treatment of rats with an orally administered mineralocorticoid, fludrocortisone, increased TSC expression (656 ± 114% of controls). We conclude that the distal convoluted tubule is an important site of action of the mineralocorticoid aldosterone, which strongly up-regulates the expression of TSC.     

Na,K-ATPase in isolated nephron segments in rats with experimental heart failure.

To characterize renal transport of Na+ in heart failure, urinary Na+ excretion (UNaV), aldosterone levels, and Na,K-ATPase activity in isolated nephron segments were determined in three groups: control rats, rats with heart failure and moderate sodium retention, and rats with heart failure and severe sodium retention. Heart failure was induced by a fistula between the aorta and vena cava. For the control group, UNaV was 0.66 +/- 0.04 (mean +/- SEM) mueq/min, and aldosterone was 18.4 +/- 3.5 ng%. Na,K-ATPase activity (in 10(-11) mol/mm/min) was 28.4 +/- 1.1 in the proximal convoluted tubule, 23.3 +/- 1.0 in the proximal straight tubule, 37.4 +/- 1.9 in the medullary thick ascending limb, 40.2 +/- 1.9 in the cortical thick ascending limb, 43.2 +/- 2.2 in the distal convoluted tubule, and 20.5 +/- 0.9 in the cortical collecting duct. For the group with moderate heart failure, UNaV was 0.35 +/- 0.02 (p less than 0.001 versus control), and aldosterone was 15.9 +/- 4.4 (p = NS versus control). Na,K-ATPase activity was unchanged in the proximal convoluted tubule, proximal straight tubule, medullary thick ascending limb, and cortical collecting duct, but it increased in the cortical thick ascending limb to 57.7 +/- 3.1 (p less than 0.001 versus control) and decreased in the distal convoluted tubule to 35.3 +/- 1.2 (p less than 0.005 versus control). For the group with severe heart failure, UNaV was 0.029 +/- 0.016 (p less than 0.001 versus control), and aldosterone was 186.0 +/- 14.8 (p less than 0.001 versus control).(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of Kininase Activity Along the Rabbit Nephron

The distribution of kininases along microdissected nephron segments was studied. Single nephrons from collagenase treated rabbit kidney were microdissected and divided into following 9 segments: 1) glomerulus; 2) early proximal tubule; 3) middle proximal tubule; 4) late proximal tubule; 5) cortical thick ascending limb; 6) distal convoluted tubule; 7) connecting tubule; 8) cortical collecting tubule; 9) medullary collecting tubule. Kininase activities in these nephron segments were measured with or without kininase II inhibitor. Kininase II and other peptidases were mainly localized in glomeruli and whole part of the proximal tubules.     

Integrated Control of Na Transport along the Nephron

The kidney filters vast quantities of Na at the glomerulus but excretes a very small fraction of this Na in the final urine. Although almost every nephron segment participates in the reabsorption of Na in the normal kidney, the proximal segments (from the glomerulus to the macula densa) and the distal segments (past the macula densa) play different roles. The proximal tubule and the thick ascending limb of the loop of Henle interact with the filtration apparatus to deliver Na to the distal nephron at a rather constant rate. This involves regulation of both filtration and reabsorption through the processes of glomerulotubular balance and tubuloglomerular feedback. The more distal segments, including the distal convoluted tubule (DCT), connecting tubule, and collecting duct, regulate Na reabsorption to match the excretion with dietary intake. The relative amounts of Na reabsorbed in the DCT, which mainly reabsorbs NaCl, and by more downstream segments that exchange Na for K are variable, allowing the simultaneous regulation of both Na and K excretion.     

The distal nephron of rat kidney: a target site for glucagon.

Glucagon-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity was measured in nine different portions of the rat nephron. Each sample contained a single piece of tubule isolated by microdissection from collagenase-treated kidney tissue. As compared to basal activity, 1 microM porcine glucagon stimulated adenylate cyclase 60-fold in the medullary portion and 40-fold in the cortical portion of the thick ascending limb, 23-fold in the early distal convoluted tubule, 11-rold in the cortical collecting tubule, and 8-fold in the medullary collecting tubule. No stimulation was observed in proximaly tubules and thin segments of the loop of Henle. Half-maximal stimulations were obtained with about 10 nM glucagon in the responsive nephron portions.     

Distribution of calcitonin-sensitive adenylate cyclase activity along the rabbit kidney tubule.

The adenylate cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] sensitivity to salmon calcitonin in 11 different segments of the rabbit nephron was investigated using a micromethod for enzyme activity measurements in samples, each containing a single piece of tubule. The required segments were isolated by microdissection from collagenase-treated rabbit kidneys. The results were expressed as femtomoles of adenosine 3':5'-cyclic monophosphate formed per mm of tubular length per 30 min of incubation time. In the presence of 0.1 mug/ml of synthetic salmon calcitonin, it was found that eight segments exhibited no hormonal sensitivity whereas maximal responses were induced in three segments, the "bright" portion of the distal convoluted tubule, the cortical and the medullary portions of the thick ascending limb of the loop of Henle (stimulated over control activity ratios were 32, 11, and27). The very high sensitivity to calcitonin of the adenylate cyclase contained in these three segments (0.01 ng/ml of salmon calcitonin inducing a 2-fold stimulation; half-miximal stimulation corresponding to about 0.3 ng/ml of salmon calcitonin) suggests that the distal convoluted tubule, as well as th cortical and medullary portions of the thick ascending limb of the loop of Henle represent physiological target structures of calcitonin action within the kidney.     

Evidence of a dynamic aldosterone-independent distal tubular control of renal sodium excretion in compensated liver cirrhosis

BACKGROUND AND AIM: In preascitic cirrhosis increased sodium retention occurs in kidney distal tubule in spite of normal aldosterone plasma levels. No clearance technique can dissect the respective contribution to sodium retention exerted by Henle's loop, distal convoluted tubule and collecting duct, so we evaluated proximal and distal tubular sodium handling in preascites during two manoeuvres that temporarily increase aldosterone secretion. METHODS: Ten patients with compensated cirrhosis and nine controls were studied in recumbency, during standing and after dopamine receptor blockade with metoclopramide through: 4 h renal clearances of sodium, potassium, lithium and creatinine; plasma levels of active renin and aldosterone. RESULTS: Whilst comparable in recumbency, aldosterone levels significantly rose during standing and after metoclopramide in both groups. In patients, dopaminergic blockade caused a fall of distal sodium delivery (P < 0.01) but urinary sodium excretion was unchanged because the reabsorbed fraction of distal sodium delivery also fell (P < 0.03). Cirrhotic patients showed the same findings in the passage from recumbency to standing. CONCLUSIONS: In preascitic cirrhosis, the distal tubular segments of the nephron are able to cope with decreases in tubular flow by reducing reabsorption at an aldosterone-independent site (possibly the loop of Henle).     

Collecting Duct Intercalated Cell Function and Regulation

Intercalated cells are kidney tubule epithelial cells with important roles in the regulation of acid-base homeostasis. However, in recent years the understanding of the function of the intercalated cell has become greatly enhanced and has shaped a new model for how the distal segments of the kidney tubule integrate salt and water reabsorption, potassium homeostasis, and acid-base status. These cells appear in the late distal convoluted tubule or in the connecting segment, depending on the species. They are most abundant in the collecting duct, where they can be detected all the way from the cortex to the initial part of the inner medulla. Intercalated cells are interspersed among the more numerous segment-specific principal cells. There are three types of intercalated cells, each having distinct structures and expressing different ensembles of transport proteins that translate into very different functions in the processing of the urine. This review includes recent findings on how intercalated cells regulate their intracellular milieu and contribute to acid-base regulation and sodium, chloride, and potassium homeostasis, thus highlighting their potential role as targets for the treatment of hypertension. Their novel regulation by paracrine signals in the collecting duct is also discussed. Finally, this article addresses their role as part of the innate immune system of the kidney tubule.     

Decreased ENaC expression compensates the increased NCC activity following inactivation of the kidney-specific isoform of WNK1 and prevents hypertension

Mutations in WNK1 and WNK4 lead to familial hyperkalemic hypertension (FHHt). Because FHHt associates net positive Na(+) balance together with K(+) and H(+) renal retention, the identification of WNK1 and WNK4 led to a new paradigm to explain how aldosterone can promote either Na(+) reabsorption or K(+) secretion in a hypovolemic or hyperkalemic state, respectively. WNK1 gives rise to L-WNK1, an ubiquitous kinase, and KS-WNK1, a kinase-defective isoform expressed in the distal convoluted tubule. By inactivating KS-WNK1 in mice, we show here that this isoform is an important regulator of sodium transport. KS-WNK1(-/-) mice display an increased activity of the Na-Cl cotransporter NCC, expressed specifically in the distal convoluted tubule, where it participates in the fine tuning of sodium reabsorption. Moreover, the expression of the ROMK and BKCa potassium channels was modified in KS-WNK1(-/-) mice, indicating that KS-WNK1 is also a regulator of potassium transport in the distal nephron. Finally, we provide an alternative model for FHHt. Previous studies suggested that the activation of NCC plays a central role in the development of hypertension and hyperkalemia. Even though the increase in NCC activity in KS-WNK1(-/-) mice was less pronounced than in mice overexpressing a mutant form of WNK4, our study suggests that the activation of Na-Cl cotransporter is not sufficient by itself to induce a hyperkalemic hypertension and that the deregulation of other channels, such as the Epithelial Na(+) channel (ENaC), is probably required.     

Kidney function and Na+-K+-ATPase and succinate dehydrogenase activity in the cells of the renal tubules of rats with constriction of the thoracic portion of the vena cava inferior]

Circulatory insufficiency in rats was induced by constriction of the thoracic portion of vena cava inferior. Two-three days after the operation renal function was studied and the activity of succinate dehydrogenase and Na+K+-ATPase in the cells of the renal tubules was determined cytochemically. On infusion of 0.9% NaCl solution into the stomach, the filtration in the "caval" rats did not change whereas the excretion of sodium and its concentration in the urine were much lower than in the sham operated rats. At the same time, the activity of Na+K+-ATPase and succinate dehydrogenase in the "caval" rats increased in the cells of the proximal tubule, diminished in Henle's loop, and remained unchanged in the cells of the distal convoluted tubule. Comparison of the results of functional and cytochemical study of the kidneys in caval rats allows the conclusion that intensified proximal reabsorption may be the main cause of antinatriuresis in these animals. The lesser load experienced by the distal segment promotes fuller reabsorption of sodium in this part of the nephron too.     

Gitelman's syndrome: an overlooked cause of chronic hypokalemia and hypomagnesemia in adults

In 1966, Gitelman described a benign variant of classical Bartter's syndrome in adults characterized by consistent hypomagnesemia and hypocalciuria, hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism with normal blood pressure. A specific gene has been found responsible for this disorder, encoding the thiazide-sensitve Na-Cl coporter (TSC) in the distal convoluted tubule. Mutant alleles result in loss of normal TSC function and the phenotype is identical to patients with chronic use of thiazide diuretics. Gitelman's syndrome is a more common cause of chronic hypokalemia than Bartter's syndrome, with which it is often confused. The distinguishing features between both syndromes are discussed.     

Disorders of distal nephron function

In this review, the distal nephron is considered to be that portion of the renal tubule commencing with the thick ascending limb of the loop of Henle and ending with the papillary collecting duct. The collecting duct, including its subdivisions in the cortex and medulla, originates from a different embryologic anlage than more proximal nephron segments, which may explain its morphologic and functional dissimilarities from the thick ascending limb and the distal convoluted tubule. This review summarizes selected aspects of the physiology of the distal nephron, with particular emphasis on the physiology of distal nephron transport of sodium, potassium, chloride and hydrogen ion. The pathophysiologic features of the following disorders of distal nephron function are reviewed: (1) pseudohypoaldosteronism, a heterogenous group of disorders in which the signs and symptoms are suggestive of aldosterone deficiency, but in which aldosterone levels are supernormal and administration of exogenous mineralocorticoid is not ameliorative; (2) pseudohyperaldosteronism (Liddle syndrome), a familial disorder in which the clinical manifestations closely resemble those resulting from an aldosterone-producing adenoma of the adrenal gland (primary aldosteronism), but in which the measured rate of aldosterone secretion and excretion is greatly subnormal; (3) Bartter syndrome and related syndromes of renal potassium wasting; (4) type 1 renal tubular acidosis (classic, distal); (5) type 4 renal tubular acidosis (hyperkalemic). Reference citations are generally to articles reporting recent advances in these areas and to review articles that contain comprehensive bibliographies.     

Epidermal growth factor gene expression is regulated differently in mouse kidney and submandibular gland

The concentration of the peptide mitogen epidermal growth factor (EGF) is hormonally and developmentally regulated in the granular convoluted tubule cells of the mouse submandibular gland. Using a labeled EGF nucleic acid probe, we have demonstrated that submandibular gland EGF mRNA concentrations increase during postnatal development of the gland and after the administration of testosterone or thyroid hormone. Recently, it was reported that EGF mRNA is present in kidney as well as a number of other mouse tissues. A comparison of EGF gene regulation in submandibular gland and kidney revealed that kidney EGF mRNA levels also increase during the postnatal period. Opposite sex differences were observed, with submandibular gland levels being about 16-fold higher in the male than in the female and kidney levels being 2- to 4-fold higher in the female than in the male. Renal EGF mRNA concentrations are less responsive to hormones than those in the submandibular gland. Renal EGF was localized immunocytochemically to the cells of distal convoluted tubules.     

Sodium transporters in the distal nephron and disease implications

The post-macula densa segments of the renal tubule—that is, the distal convoluted tubule, connecting tubule, and collecting duct—play a central role in determining final urine sodium excretion. The major regulated sodium transporters and channels in these cell types include the thiazide-sensitive (Na-Cl) cotransporter (NCC), the epithelial sodium channel (ENaC), and Na-K-ATPase. Furthermore, although not involved in sodium reabsorption, the anion exchanger, pendrin, and the basolateral bumetanide-sensitive Na-K-2Cl cotransporter (NKCC1 or BSC2) have roles in blood-volume maintenance. Mutations in several of these major sodium transporters, channel subunits, and their regulatory proteins have been linked to human diseases such as Liddle’s syndrome, Gitelman’s syndrome, and Gordon’s syndrome, emphasizing the need for appropriate regulation of sodium at these sites for maintenance of sodium balance and normotension.     

Two highly homologous members of the ClC chloride channel family in both rat and human kidney.

We have cloned two closely related putative Cl- channels from both rat kidney (designated rClC-K1 and rClC-K2) and human kidney (hClC-Ka and hClC-Kb) by sequence homology to the ClC family of voltage-gated Cl- channels. While rClC-K1 is nearly identical to ClC-K1, a channel recently isolated by a similar strategy, rClC-K2 is 80% identical to rClC-K1 and is encoded by a different gene. hClC-Ka and hClC-Kb show approximately 90% identity, while being approximately 80% identical to the rat proteins. All ClC-K gene products are expressed predominantly in the kidney. While rClC-K1 is expressed strongly in the cortical thick ascending limb and the distal convoluted tubule, with minor expression in the S3 segment of the proximal tubule and the cortical collecting tubule, rClC-K2 is expressed in all segments of the nephron examined, including the glomerulus. Since they are related more closely to each other than to the rat proteins, hClC-Ka and hClC-Kb cannot be regarded as strict homologs of rClC-K1 or rClC-K2. After injection of ClC-K cRNAs into oocytes, corresponding proteins were made and glycosylated, though no additional Cl- currents were detectable. Glycosylation occurs between domains D8 and D9, leading to a revision of the transmembrane topology model for ClC channels.     

Calbindin in vertebrate classes: immunohistochemical localization and Western blot analysis

Calbindin immunoreactivity was investigated in various vertebrates. Positive labeling was observed in the absorptive cells of the duodenum of all birds and reptiles but not in mammals, amphibia, or fish. Staining was present in the kidney distal convoluted tubule from amphibia and higher vertebrates. Fish kidney was negative. In the central nervous system of all species investigated, cellular bodies and fibers were Calbindin positive. Their distribution was quite broad and correlates well with the previously reported mapping for chick and rat. Western blot analysis revealed two Calbindins in brain from mammals, birds, reptiles, and amphibia (27,000 and 29,000 Da). Only one band was detected in fish. We conclude that Calbindin from the evolutionary point of view is primarily a neuronal protein, with a highly conservative character.     

Deletion of WNK1 first intron results in misregulation of both isoforms in renal and extrarenal tissues

Large deletions in intron 1 of the with-no-lysine kinase type 1 (WNK1) gene cause familial hyperkalemic hypertension. Alternative promoters generate functionally different isoforms: long ubiquitous isoforms (L-WNK1) and a kidney-specific isoform (KS-WNK1) lacking kinase activity. It remains unclear whether the disease-causing mutations selectively modify the synthesis of 1 or both types of isoforms. Using a transgenic mouse model, we found that intron 1 deletion resulted in the overexpression of L- and KS-WNK1 in the distal convoluted tubule and ubiquitous ectopic KS-WNK1 expression. Phylogenetic and functional analysis of the minimal 22-kb intron 1 deletion identified 1 repressor and 1 insulator, potentially preventing interactions between the regulatory elements of L-WNK1 and KS-WNK1. These results provide the first insight into the molecular mechanisms of WNK1-induced familial hyperkalemic hypertension.     

Evaluation of urinary Tamm-Horsfall protein in post-menopausal diabetic women

Tamm-Horsfall protein (THP), a glycoprotein produced in the thick ascending limb (TAL) and the early distal convoluted tubule (DCT), is normally excreted in large amounts in urine. Urinary THP may be a useful marker for renal damage. The goal of this research project was to determine the THP excretion in control and diabetic post-menopausal women. Twenty-four hour urine samples were collected from 19 controls and 19 diabetic patients (11 non-insulin dependent diabetic mellitus (non-IDDM) patients, and 8 insulin dependent diabetic mellitus (IDDM) patients). Polyacrylamide gel electrophoresis (PAGE), Western blotting, and enzyme linked immunosorbent assay (ELISA) methods were used. It was determined that urinary THP concentrations were significantly decreased in patients with IDDM compared to patients with non-IDDM and controls. In conclusion, laboratory quantitation of urinary THP may be a useful indicator of cellular abnormalities such as reduced protein (THP) synthesis of the cells of the TAL and early DCT in some IDDM patients.