Regulated sodium transport in the renal connecting tubule (CNT) via the epithelial sodium channel (ENaC)

The aldosterone-sensitive distal nephron (ASDN) includes the late distal convoluted tubule 2, the connecting tubule (CNT) and the collecting duct. The appropriate regulation of sodium (Na⁺) absorption in the ASDN is essential to precisely match urinary Na⁺ excretion to dietary Na⁺ intake whilst taking extra-renal Na⁺ losses into account. There is increasing evidence that Na⁺ transport in the CNT is of particular importance for the maintenance of body Na⁺ balance and for the long-term control of extra-cellular fluid volume and arterial blood pressure. Na⁺ transport in the CNT critically depends on the activity and abundance of the amiloride-sensitive epithelial sodium channel (ENaC) in the luminal membrane of the CNT cells. As a rate-limiting step for transepithelial Na⁺ transport, ENaC is the main target of hormones (e.g. aldosterone, angiotensin II, vasopressin and insulin/insulin-like growth factor 1) to adjust transepithelial Na⁺ transport in this tubular segment. In this review, we highlight the structural and functional properties of the CNT that contribute to the high Na⁺ transport capacity of this segment. Moreover, we discuss some aspects of the complex pathways and molecular mechanisms involved in ENaC regulation by hormones, kinases, proteases and associated proteins that control its function. Whilst cultured cells and heterologous expression systems have greatly advanced our knowledge about some of these regulatory mechanisms, future studies will have to determine the relative importance of the various pathways in the native tubule and in particular in the CNT.     

SGK1 increases Na,K-ATP cell-surface expression and function in Xenopus laevis oocytes

The Na⁺-retaining hormone aldosterone increases the cell-surface expression of the luminal epithelial sodium channel (ENaC) and the basolateral Na⁺ pump (Na,K-ATPase) in aldosterone-sensitive distal nephron cells in a coordinated fashion. To address the question of whether aldosterone-induced serum and glucocorticoid-regulated kinase-1 (SGK1) might be involved in mediating this regulation of Na,K-ATPase subcellular localization, similar to that of the epithelial Na⁺ channel (ENaC), we co-expressed the Na,K-ATPase (rat 1- and Xenopus laevis 1-subunits) and Xenopus SGK1 in Xenopus oocytes. Measurements of the Na⁺ pump current showed that wild-type SGK1 increases the function of exogenous Na,K-ATPase at the surface of Xenopus oocytes. This appeared to be secondary to an increase in Na,K-ATPase cell-surface expression as visualized by Western blotting of surface-biotinylated proteins. In contrast, the functional surface expression of two other exogenous transporters, the heterodimeric amino acid transporter LAT1-4F2hc and the Na⁺/phosphate cotransporter NaPi-IIa, was not increased by SGK1 co-expression. The total pool of exogenous Na,K-ATPase was increased by the co-expression of SGK1, and similarly also by ENaC co-expression. This latter effect depended on the [Na⁺] of the buffer and was not additive to that of SGK1. When the total Na,K-ATPase was increased by ENaC co-expression, SGK1 still increased Na,K-ATPase cell-surface expression. These observations in Xenopus oocytes suggest the possibility that SGK1 induction and/or activation could participate in the coordinated regulation of Na,K-ATPase and ENaC cell-surface expression in the aldosterone-sensitive distal nephron.     

Distal colonic Na+ absorption inhibited by luminal P2Y2 receptors

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y₂ and a luminal P2Y₄ receptor, stimulate K⁺ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na⁺ absorption in distal colonic mucosa of mice treated on a low Na⁺ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V _te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R _te): 24.1 ± 1.8 Ω cm², equivalent short circuit current (I _sc): −563.9 ± 63.8 μA/cm² (n = 21). Amiloride completely inhibited I _sc to −0.5 ± 8.5 μA/cm². Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I _sc by 160.7 ± 29.7 μA/cm² (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC₅₀ values of 10 μM and 3 μM respectively. In P2Y₂ knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na⁺ absorption was absent. In contrast, in P2Y₄ KO mice the inhibitory effect of luminal UTP on Na⁺ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na⁺ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y₂ and P2Y₄ receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption.     

Effects of potassium bicarbonate on distal nephron Na−K-ATPase in adrenalectomized rabbits

Na–K-ATPase activity in the connecting tubule (CNT) and cortical collecting duct (CCD) has been shown to be influenced by KCl both in the presence and in the absence of aldosterone. To investigate if the aldosterone-independent effect of K⁺ on Na–K-ATPase can be produced by other K⁺ salts, we studied the effects of dietary KHCO₃ on Na–K-ATPase and ouabain-insensitive Mg-ATPase activities in four nephron segments of adrenalectomized (ADX) rabbits. The segments examined were: the distal convoluted tubule (DCT), CNT, CCD and medullary collecting duct (MCD). All diets were similar in composition except their KHCO₃ contents which were 100, 300, 500 and 700 meq/kg in groups 1 to 4 respectively. Increasing KHCO₃ in the diet increased K⁺ excretion (7×) and urine pH (6.6 to 8.3). Na–K-ATPase activity in the CCD increased >200% as dietary KHCO₃ was increased to 700 meq/kg. There was a linear relation between Na–K-ATPase activity in this segment and steady state plasma K⁺ as well as K⁺ excretion in the urine. However, Na–K-ATPase activity in the CCD was lower in KHCO₃-fed ADX rabbits than the KCl-fed animals studied previously under similar conditions. There were no significant differences in Na–K-ATPase activities in DCT, CNT and MCD among the four groups given different KHCO₃-diets. It is concluded that dietary intake of KHCO₃ can also influence Na–K-ATPase activity in the CCD independent of aldosterone.Abbreviations used ADX adrenalectomized - ATPase adenosine triphosphatase - CCD cortical collecting duct - CNT connecting tubule - DCT distal convoluted tubule - MCD medullary collecting duct     

Stimulation of the epithelial sodium channel (ENaC) by the serum- and glucocorticoid-inducible kinase (Sgk) involves the PY motifs of the channel but is independent of sodium feedback inhibition

The epithelial sodium channel (ENaC) is the major mediator of sodium transport across the apical membranes of the distal nephron, the distal colon, the respiratory tract and the ducts of exocrine glands. It is subject to feedback inhibition by increased intracellular Na⁺, a regulatory system wherein the ubiquitin protein ligases, Nedd4 and Nedd4-2, bind to conserved PY motifs in the C-termini of ENaC and inactivate the channel. It has been proposed recently that the kinase Sgk activates the channel as a consequence of phosphorylating Nedd4-2, thus preventing it from inhibiting the channels. This proposal predicts that Sgk should interfere with Na⁺ feedback regulation of ENaC. We have tested this prediction in Xenopus laevis oocytes and in mouse salivary duct cells and found that in neither system did increased activity of Sgk interrupt Na⁺ feedback inhibition of ENaC. We found, however, that Sgk stimulation was largely abolished in oocytes expressing ENaC channels with C-terminal truncations or mutated PY motifs. We were also unable to confirm that Sgk directly interacts with Nedd4-2 in vitro. We conclude that the stimulatory effect of Sgk on ENaC requires the presence of the channel’s PY motifs, but it is not due to the interruption of Na⁺ feedback regulation.Robert Rauh and Anuwat Dinudom cotributed equally to this work.     

Regulation of NaCl transport in the renal collecting duct: lessons from cultured cells

The fine control of NaCl absorption regulated by hormones takes place in the distal nephron of the kidney. In collecting duct principal cells, the epithelial sodium channel (ENaC) mediates the apical entry of Na⁺, which is extruded by the basolateral Na⁺,K⁺-ATPase. Simian virus 40-transformed and “transimmortalized” collecting duct cell lines, derived from transgenic mice carrying a constitutive, conditionally, or tissue-specific promoter-regulated large T antigen, have been proven to be valuable tools for studying the mechanisms controlling the cell surface expression and trafficking of ENaC and Na⁺,K⁺-ATPase. These cell lines have made it possible to identify sets of aldosterone- and vasopressin-stimulated proteins, and have provided new insights into the concerted mechanism of action of serum- and glucocorticoid-inducible kinase 1 (Sgk1), ubiquitin ligase Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), and 14-3-3 regulatory proteins in modulating ENaC-mediated Na⁺ currents. Epidermal growth factor and induced leucine zipper protein have also been shown to repress and stimulate ENaC-dependent Na⁺ absorption, respectively, by activating or repressing the mitogen-activated protein kinase externally regulated kinase_1/2. Overall, these findings have provided evidence suggesting that multiple pathways are involved in regulating NaCl absorption in the distal nephron.     

Effect of low potassium-diet on Na−K-ATPase in rat nephron segments

Na–K-ATPase activity was determined in 10 segments of the rat nephron using a fluorometric microassay method [4]. The enzyme activity showed three peaks (>200 pmol ADP min^–1 mm^–1) along the nephron of normal rats. These peaks were in the S₁ portion of the proximal tubule, the medullary thick ascending limb from the inner stripe and the distal convoluted tubule. Feeding the rats a low potassium diet for 8 weeks produced a significant decrease in Na–K-ATPase activity in the cortical collecting duct, but no significant change in this enzyme in any other segment. The low potassium diet did not produce a significant change in Mg-ATPase in any nephron segments. We conclude that Na–K-ATPase activity along the rat nephron shows a pattern that is qualitatively similar to that seen in the rabbit nephron [4]. However, quantitatively the Na–K-ATPase activity in the rat nephron is greater than in the corresponding segments of the rabbit nephron. The results are consistent with the greater rate of glomerular filtration and Na⁺ reabsorption per rat nephron. Furthermore, our results suggest that the decrease in potassium excretion during potassium deficiency is modulated, at leat in part, by the level of Na–K-ATPase activity in the cortical collecting duct.     

The secretory K<Subscript>Ca</Subscript>1.1 channel localises to crypts of distal mouse colon: functional and molecular evidence

The colonic epithelium absorbs and secretes electrolytes and water. Ion and water absorption occurs primarily in surface cells, whereas crypt cells perform secretion. Ion transport in distal colon is regulated by aldosterone, which stimulates both Na⁺ absorption and K⁺ secretion. The electrogenic Na⁺ absorption is mediated by epithelial Na⁺ channel (ENaC) in surface cells. Previously, we identified the large conductance Ca²⁺-activated K⁺ channel, K_Ca1.1 or big potassium (BK) channel, as the only relevant K⁺ secretory pathway in mouse distal colon. The exact localisation of K_Ca1.1 channels along the crypt axis is, however, still controversial. The aim of this project was to further define the localisation of the K_Ca1.1 channel in mouse distal colonic epithelium. Through quantification of mRNA extracted from micro-dissected surface and crypt cells, we confirmed that Na⁺/K⁺/2Cl⁻ (NKCC1) is expressed primarily in the crypts and γ-ENaC primarily in the surface cells. The K_Ca1.1 α-subunit mRNA was like NKCC1, mainly expressed in the crypts. The crypt to surface expression pattern of the channels and transporters was not altered when plasma aldosterone was elevated. The mRNA levels for NKCC1, γ-ENaC and K_Ca1.1 α-subunit were, however, under these circumstances substantially augmented (K_Ca1.1 α-subunit, twofold; NKCC1, twofold and ENaC, tenfold). Functionally, we show that ENaC-mediated Na⁺ absorption and BK channel-mediated K⁺ secretion are two independent processes. These findings show that K_Ca1.1-mediated K⁺ secretion mainly occurs in the crypts of the murine distal colon. This is in agreement with the general model of ion secretion being preferentially located to the crypt and not surface enterocytes.     

Extracellular Na+ removal attenuates rundown of the epithelial Na+-channel (ENaC) by reducing the rate of channel retrieval

Regulation of the epithelial sodium channel (ENaC) is important for the long-term control of arterial blood pressure as evidenced by gain of function mutations of ENaC causing Liddles syndrome, a rare form of hereditary arterial hypertension. In Xenopus laevis oocytes expressing ENaC a spontaneous decline of ENaC currents over time, so-called rundown, is commonly observed. Mechanisms involved in rundown may be physiologically relevant and may be related to feedback regulation of ENaC by intra- or extracellular Na⁺. We tested the effect of extracellular Na⁺ removal on ENaC rundown. Spontaneous rundown of ENaC was largely prevented by extracellular Na⁺ removal and was partially prevented by primaquine suggesting that it is due to endocytic channel retrieval. Liddles syndrome mutation caused a reduced rate of rundown, and in oocytes expressing the mutated channel extracellular Na⁺ removal not only prevented rundown but even increased the ENaC currents (runup). Acute exposure to high extracellular Na⁺ drastically reduced whole-cell currents and surface expression of wild-type ENaC, while these effects were much smaller in ENaC with Liddles syndrome mutation consistent with a stabilization of the mutated channel in the plasma membrane. Interestingly, the apparent intracellular Na⁺ concentration [Na⁺]_i-app was high (>60 mM) in ENaC-expressing oocytes but rundown was not associated with a further increase in [Na⁺]_i-app. We conclude that the inhibitory effect of extracellular Na⁺ removal on rundown is due to an inhibition of endocytic ENaC retrieval.     

Mechanisms of actions of guanylin peptides in the kidney

After a salty meal, stimulation of salt excretion via the kidney is a possible mechanism to prevent hypernatremia and hypervolemia. Besides the well known hormonal regulators of salt and water excretion in the distal nephron, arginine vasopressin and aldosterone, guanylin (GN) peptides produced in the intestine were proposed to be intestinal natriuretic peptides. These peptides inhibit Na⁺ absorption in the intestine and induce natriuresis, kaliuresis and diuresis in the kidney. The signaling pathway of GN peptides in the intestine is well known. They activate enterocytes via guanylate cyclase C (GC-C) and increase the cellular concentration of cGMP which leads to secretion of Cl^–, HCO₃^– and water into the intestinal lumen and to inhibition of Na⁺ absorption. Guanylin peptides are filtered in the glomerulus, and additionally synthesized and excreted by tubular cells. They activate receptors located in the luminal membrane of the tubular cells along the nephron. In GC-C deficient mice renal effects of GN peptides are retained. In human, rat, and opossum proximal tubule cells, a cGMP-dependent signaling was demonstrated, but in addition GN peptides apparently also activate a PT-sensitive G-protein coupled receptor. A similar dual signaling pathway is also known for other natriuretic peptides like atrial natriuretic peptide. A cGMP-independent signaling pathway of GN peptides is also shown for principal cells of the human cortical collecting duct where the final hormonal regulation of electrolyte homeostasis takes place. This review will focus on the current knowledge on renal actions of GN peptides and specifically address novel GC-C- and cGMP-independent signaling mechanisms.     

Neuropeptide Y regulates rat renal tubular Na,K-ATPase through several signalling pathways

Neuropeptide Y (NPY) has at least three receptors (Y₁, Y₂ and Y₃) through which it influences different mechanisms in many cell types. Previous data suggest that the Y₂ receptor may be divided into prejunctional and postjunctional subgroups. We have examined the intracellular signalling pathways of the postjunctional Y₂ receptor in rat renal proximal tubules. The results indicate that NPY regulates Na⁺,K⁺-ATPase through several signalling pathways: (1) In proximal tubule (PT) cells NPY increased intracellular calcium. The response was blocked by removing extracellular calcium and was also blocked by using nifedipine. This suggests that calcium was increased by influx from the extracellular space through L -type calcium channels. (2) NPY increased Na⁺,K⁺-ATPase activity in PT segments and this effect was also blocked by nifedipine. CaMKII-Ala²⁸⁶[281–302] a blocker of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibited the NPY-stimulated Na⁺,K⁺-ATPase activity. This implies that increased intracellular calcium activates CaMKII which subsequently increases Na⁺,K⁺-ATPase activity. CaMKII thus appear to act similar to what has been proposed for protein phosphatase 2B. (3) Calphostin C, an inhibitor of protein kinase C (PKC), did not inhibit NPY-stimulated Na⁺,K⁺-ATPase activity. PKC is, therefore, unlikely to be involved. (4) Y₂ receptors are negatively coupled to the cAMP pathway. NPY attenuated forskolin-stimulated cAMP production in renal tubules and exogenous cAMP counteracted the NPY-stimulated Na⁺,K⁺-ATPase activity. This illustrated the importance of NPY for the regulation of renal sodium handling. We also propose that the renal tubule cell is a good model for studying the function and mechanisms of postjunctional Y₂ receptors.     

Dual effect of temperature on the human epithelial Na+ channel

The amiloride-sensitive epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption in the distal segments of the nephron, in the colon and in the airways. Its activity is regulated by intracellular and extracellular factors but the mechanisms of this regulation are not yet completely understood. Recently, we have shown that the fast regulation of ENaC by the extracellular [Na⁺], a phenomenon termed self-inhibition, is temperature dependent. In the present study we examined the effects of temperature on the single-channel properties of ENaC. Single-channel recordings from excised patches showed that the channel open probability (P _o, estimated from the number of open channels N·P _o, where N is the total number of channels) increased on average two- to threefold while the single-channel conductance decreased by about half when the temperature of the perfusion solution was lowered from ~30 to ~15 °C. The effects of temperature on the single-channel conductance and P _o explain the changes of the macroscopic current that can be observed upon temperature changes and, in particular, the paradoxical effect of temperature on the current carried by ENaC.     

Influence of head-down bed rest on the circadian rhythms of hormones and electrolytes involved in hydroelectrolytic regulation

We investigated in six men the impact of a 17-day head-down bed rest (HDBR) on the circadian rhythms of the hormones and electrolytes involved in hydroelectrolytic regulation. This HDBR study was designed to mimic an actual spaceflight. Urine samples were collected at each voiding before, during and after HDBR. Urinary excretion of aldosterone, arginine vasopressin (AVP), cyclic guanosine monophosphate (cGMP), cortisol, electrolytes (Na⁺ and K⁺) and creatinine were determined. HDBR resulted in a significant reduction of body mass (P<0.01) and of caloric intake [mean (SEM) 2,778 (37) kcal·24 h^–1 to 2,450 (36) kcal·24 h^–1, where 1 kcal·h^–1=1.163 J·s^–1; P<0.01]. There was a significant increase in diastolic blood pressure [71.8 (0.7) mmHg vs 75.6 (0.91) mmHg], with no significant changes in either systolic blood pressure or heart rate. The nocturnal hormonal decrease of aldosterone was clearly evident only before and after HDBR, but the day/night difference did not appear during HDBR. The rhythm of K⁺ excretion was unchanged during HDBR, whereas for Na⁺ excretion, a large decrease was shown during the night as compared to the day. The circadian rhythm of cortisol persisted. These data suggest that exposure to a 17-day HDBR could induce an exaggeration of the amplitude of the Na⁺ rhythm and abolition of the aldosterone rhythm. Electronic Publication     

Regulation of the epithelial Na+ channel and airway surface liquid volume by serine proteases

Mammalian airways are protected from infection by a thin film of airway surface liquid (ASL) which covers airway epithelial surfaces and acts as a lubricant to keep mucus from adhering to the epithelial surface. Precise regulation of ASL volume is essential for efficient mucus clearance and too great a reduction in ASL volume causes mucus dehydration and mucus stasis which contributes to chronic airway infection. The epithelial Na⁺ channel (ENaC) is the rate-limiting step that governs Na⁺ absorption in the airways. Recent in vitro and in vivo data have demonstrated that ENaC is a critical determinant of ASL volume and hence mucus clearance. ENaC must be cleaved by either intracellular furin-type proteases or extracellular serine proteases to be active and conduct Na⁺, and this process can be inhibited by protease inhibitors. ENaC can be regulated by multiple pathways, and once proteolytically cleaved ENaC may then be inhibited by intracellular second messengers such as cAMP and PIP₂. In the airways, however, regulation of ENaC by proteases seems to be the predominant mode of regulation since knockdown of either endogenous serine proteases such as prostasin, or inhibitors of ENaC proteolysis such as SPLUNC1, has large effects on ENaC activity in airway epithelia. In this review, we shall discuss how ENaC is proteolytically cleaved, how this process can regulate ASL volume, and how its failure to operate correctly may contribute to chronic airway disease.     

Sodium transport-related proteins in the mammalian distal nephron – distribution, ontogeny and functional aspects

The mammalian distal nephron plays a pivotal role in adjusting urinary sodium excretion. Successive portions of the renal tubule are formed to adapt to this function, and an axial heterogeneity of the distal segments has been defined. The specific transport properties of these epithelia are accomplished by the expression of proteins (cotransporters, exchangers, channels) governing the movement of ions on either cell side. Molecular cloning of these proteins has had a marked impact on the study of their localization and function in the healthy and diseased kidney. Electroneutral cation-chloride cotransporters [Na(K)CC] have been localized to the thick ascending limb and the distal convoluted tubule using specific probes. Proteins implicated in the function of aldosterone target cells, such as the epithelial Na⁺ channel (ENaC), the mineralocorticoid receptor (MR) and 11β-hydroxysteroid dehydrogenase type 2 (11HSD2), an enzyme that confers mineralocorticoid specificity, have been found in the terminal portion of the nephron and the collecting duct. A mineralocorticoid-sensitive component of thiazide-sensitive NaCl transport has been identified in the distal convoluted tubule. Analysis of the ontogeny of these proteins in the maturing kidney has provided a detailed picture of epithelial differentiation and morphological specialization of the renal tubule. The study of mutations of the proteins related with NaCl transport has led to the identification of the molecular causes of inherited human diseases associated with hypo- or hypertension, and the respective sites of an impaired ion transport could be mapped to the renal tubule. Accepted: 13 April 1999     

Pharmacodynamic and kinetic considerations on diuretics as a basis for differential therapy

Summary Diuretics are classified according to their site of action in the nephron: loop diuretics, thiazides, and antikaliuretics. During peak diuresis the pattern of electrolyte excretion is constant and characteristic for a class of diuretics. The ratio of diuretic-induced excretion of K⁺ to Na⁺ is 0.12 for loop diuretics, 0.20 for thiazides, and –0.21 for antikaliuretics. The ratio of Ca²⁺ to Na⁺ is 0.02 for loop diuretics and 0.003 for thiazides. Mg²⁺ excretion follows K⁺ excretion in a ratio of 0.15. The natriuretic effect of a diuretic directly depends on the renal clearance of the drug and is proportionate to the number of intact nephrons. Not only loop diuretics but also thiazides and antikaliuretics were demonstrated to be effective natriuretic drugs down to end-stage renal disease. In renal failure FE_Na is doubled with every halfening of GFR. Loop diuretics increase FE_Na to a maximum of 24%, thiazides to 10–15%, and FE_Na is doubled by antikaliuretics. Comedication of loop diuretics with thiazides in renal failure may therefore be more effective than increasing monotherapy. In liver disease, nonrenal drug clearance is reduced the more the patient's direct bilirubin rises thus causing an increase in AUC and urinary excretion of parent drug and metabolites. Despite increased A_e, the cirrhotic patient may become resistant to diuretics as may patients with congestive heart failure or nephrotic syndrome. This is considered to be due to reduced Na⁺ load available at the diuretic's site of action following avid proximal Na⁺ reabsorption. In reduced EABV a short-term comedication of loop diuretics with carboanhydratase inhibitors is considered a more effective diuretic strategy than vigorously increasing monotherapy.Abbreviations GFR glomerular filtration rate (ml/min) - A_e amount of drug excreted into the urine (% of given dose) - AUC area under the plasma level time curve (g·h/ml) - Cl_pl total plasma clearance=Dose_i.v./AUC (ml/min) - Cl_r renal clearance=A_e/AUC (ml/min) - Cl_nr nonrenal clearance=Cl_pl-Cl_r (ml/min) - FE_Na fractional sodium excretion (%)=Na excreted x 100/Na filtered=urine sodium × urine volume per minute x 100/plasma sodium x GFR - CHF congestive heart failure - EABV effective arterial blood volume - RAA Renin angiotensin aldosterone system - NE Norepinephrin - NSAIDs Nonsteroidal anti-inflammatory drugs Gratefully dedicated to Prof. Dr. K.J. Ullrich     

Renal effects of neuropeptide Y

 Neuropeptide Y (NPY) is a co-transmitter of the sympathetic nervous system including the renal nerves. The kidney expresses NPY receptors, which can also be activated by peptide YY (PYY), a circulating hormone released from gastrointestinal cells. Five subtypes of NPY receptors have been cloned, among which Y₁, Y₂ and Y₅ appear to be involved in the regulation of renal function. NPY produces potent renal vasoconstriction in vitro in isolated interlobar arteries and in the isolated perfused kidney and in vivo upon intrarenal or systemic administration via a Y₁ receptor. Nevertheless glomerular filtration rate is altered only little if at all by NPY, indicating a greater effect on the vas efferens than the vas afferens. NPY can inhibit renin release via Y₁-like receptors. NPY can stimulate Na⁺/K⁺ adenosine triphosphatase (Na⁺/K⁺-ATPase) in proximal tubules via Y₂ receptors and can antagonize the effects of vasopressin on isolated collecting ducts. It can also act prejunctionally to inhibit noradrenaline release via Y₂ receptors. Despite the profound reductions of renal blood flow, systemic NPY infusion can cause diuresis and natriuresis; this is largely independent of pressure natriuresis mechanisms and is possibly mediated by an extrarenal Y₅ receptor. Studies with the converting enzyme inhibitor ramiprilat and the bradykinin receptor antagonist icatibant indicate that bradykinin mediates, at least partly, diuretic NPY effects. NPY antagonists enhance basal renal blood flow but do not alter basal diuresis or natriuresis indicating that renovascular, but not tubular, NPY receptors may be tonically activated by endogenous NPY.     

Evidence for a subgroup of essential hypertensives with non-suppressible excretion of aldosterone during sodium loading

Summary In patients with grade I and II essential hypertension studied during sodium loading (Na⁺ excretion above 175 meq·d^–1) we found a bimodal behaviour of aldosterone excretion and could distinguish two groups of patients: In the major part of essential hypertensives sodium loading led to a suppression of aldosterone excretion below 6 µg·d^–1, which is the highest control value during sodium loading, with an average of 2.7±1.4 (SD) µg·d^–1. Aldosterone excretion in a second group of patients was not suppressible below 6 µg·d^–1 despite forced sodium loading; it resulted in an average value of 10.0±3.0 (SD) µg·d^–1. During sodium deprivation or free sodium intake, aldosterone excretion in the first group of patients followed exactly the behaviour of normotensive controls, while in the second group of essential hypertensives the correlation of aldosterone excretion and log. Na excretion or log. Na⁺/K⁺ ratio in 24 h urine (r=–0.59) was far below the control value ofr=–0.87. Serum potassium concentration during sodium loading was significantly (p<0.001) lower (3.81±0.44 meq·l^–1) in the essential hypertensives with non-suppressible aldosterone excretion compared to those with suppressible aldosterone excretion (4.26±0.37 meq·l^–1). The blood pressure response to treatment with 200 mg spironolactone·d^–1 was better (p<0.05) in patients with non-suppressible aldosterone excretion compared to the essential hypertensives with normal aldosterone regulation. The plasma renin activity of both groups of patients was not significantly different, however, a tendency prevailed towards lower PRA-values in the patient group with non-suppressible aldosterone excretion during sodium loading.With the technical help of Mrs. R. Schendschilorz and Mrs. G. Suckau     

Downregulation of the renal outer medullary K+ channel ROMK by the AMP-activated protein kinase

The 5′-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca²⁺ activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na⁺ channel ENaC and the voltage-gated K⁺ channel KCNE1/KCNQ1. It is partially effective by decreasing PIP₂ formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K⁺ channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK^γR70Q (AMPK^α1-HA+AMPK^β1-Flag+AMPKγ1^R70Q), or of inactive AMPK^αK45R (AMPK^α1K45R+AMPK^β1-Flag+AMPK^γ1-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence in Xenopus oocytes and western blot in whole kidney tissue. Moreover, renal Na⁺ and K⁺ excretion were determined in AMPK^α1-deficient mice (ampk ^?/? ) and wild-type mice (ampk ^+/+ ) prior to and following an acute K⁺ load (111 mM KCl, 30 mM NaHCO₃, 4.7 mM NaCl, and 2.25 g/dl BSA) at a rate of 500 μl/h. As a result, coexpression of AMPK^γR70Q but not of AMPK^αK45R significantly decreased the current in ROMK1-expressing Xenopus oocytes. Injection of phosphatidylinositol PI_(4,5)P₂ significantly increased the current in ROMK1-expressing Xenopus oocytes, an effect reversed in the presence of AMPK^γR70Q. Under control conditions, no significant differences between ampk ^?/? and ampk ^+/+ mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na⁺, and K⁺ concentrations as well as absolute and fractional Na⁺ and K⁺ excretion. Following an acute K⁺ load, GFR, urinary flow rate, serum aldosterone, plasma Na⁺, and K⁺ concentration were again similar in both genotypes, but renal absolute and fractional Na⁺ and K⁺ excretion were higher in ampk ^?/? than in ampk ^+/+ mice. According to micropuncture following a K⁺ load, delivery of Na⁺ to the early distal tubule but not delivery of K⁺ to late proximal and early distal tubules was increased in ampk ^?/? mice. The upregulation of renal ROMK1 protein expression by acute K⁺ load was more pronounced in ampk ^?/? than in ampk ^+/+ mice. In conclusion, AMPK downregulates ROMK, an effect compromising the ability of the kidney to excrete K⁺ following an acute K⁺ load.