Renal potassium homeostasis: a short historical perspective.

During the past century, investigators have increased our understanding of renal potassium excretion significantly using many techniques. Notable among these were renal clearance experiments, renal micropuncture, isolated tubule microperfusion, and electrophysiological and patch clamp analysis. These experiments have been made possible by technical advances that have allowed the measurement of potassium in progressively smaller quantities. Initially, the kidney was viewed as controlling potassium excretion by the regulated absorption of potassium from the glomerular filtrate, predominantly in the proximal tubule. This concept was supplanted when clearance experiments deduced and subsequent micropuncture studies directly identified the importance of the distal nephron and collecting duct as the principal site responsible for the regulation of potassium excretion. Additional micropuncture and microperfusion studies showed that a component of potassium secreted by the distal cortical nephron and cortical collecting duct is reabsorbed in the medullary collecting duct, which results in renal medullary potassium recycling. Studies have defined the cellular and molecular mechanisms responsible for potassium secretion and potassium reabsorption in the collecting duct. Further understanding of renal potassium handling will require integrated investigation of the renal and extrarenal signaling systems that control these transport mechanisms.

Angiotensin II stimulates H⁺-ATPase activity in intercalated cells from isolated mouse connecting tubules and cortical collecting ducts.

Intercalated cells in the collecting duct system express V-type H(+)-ATPases which participate in acid extrusion, bicarbonate secretion, and chloride absorption depending on the specific subtype. The activity of H(+)-ATPases is regulated by acid-base status and several hormones, including angiotensin II and aldosterone. Angiotensin II stimulates chloride absorption mediated by pendrin in type B intercalated cells and this process is energized by the activity of H(+)-ATPases. Moreover, angiotensin II stimulates bicarbonate secretion by the connecting tubule (CNT) and early cortical collecting duct (CCD). In the present study we examined the effect of angiotensin II (10 nM) on H(+)-ATPase activity and localization in isolated mouse connecting tubules and cortical collecting ducts. Angiotensin II stimulated Na(+)-independent intracellular pH recovery about 2-3 fold, and this was abolished by the specific H(+)-ATPase inhibitor concanamycin. The effect of angiotensin II was mediated through type 1 angiotensin II receptors (AT(1)-receptors) because it could be blocked by saralasin. Stimulation of H(+)-ATPase activity required an intact microtubular network--it was completely inhibited by colchicine. Immunocytochemistry of isolated CNT/CCDs incubated in vitro with angiotensin II suggests enhanced membrane associated staining of H(+)-ATPases in pendrin expressing intercalated cells. In summary, angiotensin II stimulates H(+)-ATPases in CNT/CCD intercalated cells, and may contribute to the regulation of chloride absorption and bicarbonate secretion in this nephron segment.

A long affair with renal tubules.

This essay provides a summary of my professional activities. My interest in renal physiology started as a medical student in Vienna, when I became acquainted with Homer Smith's essays on kidney function. After moving to the United States in 1951, I was fortunate to be mentored by Robert Pitts, in whose Department of Physiology at Cornell Medical College in New York I was given early independence, intellectual stimulation, and the opportunity to pursue experiments on single renal tubules. The problem of how the nephron manages its myriad of transport functions has never lost its fascination for me, and I am profoundly grateful to the many colleagues at Cornell Medical College and at Yale University School of Medicine who shared my passion for the kidney.

Potassium transport--an update.

Potassium homeostasis depends on the coordinated interaction between tightly regulated potassium transfer in and out of the extracellular fluid compartment, and renal excretion or retention of potassium. Potassium transport along the nephron involves extensive proximal tubule reabsorption of potassium. Potassium is also reabsorbed along the thick ascending limb of Henle's loop. Regulated potassium secretion, or potassium reabsorption in exchange for hydrogen ions along the connecting tubule and collecting tubule, is responsible for potassium excretion. Renal potassium transport is modulated by potassium intake, several hormones, acid-base factors and distal nephron sodium delivery. WNK family kinases have also emerged as factors regulating sodium and potassium transport in the distal nephron.

Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA-regulated apical chloride channels in cortical collecting duct.

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK.

CFTR is required for PKA-regulated ATP sensitivity of Kir1.1 potassium channels in mouse kidney.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel plays vital roles in fluid transport in many epithelia. While CFTR is expressed along the entire nephron, its function in renal tubule epithelial cells remains unclear, as no specific renal phenotype has been identified in cystic fibrosis. CFTR has been proposed as a regulator of the 30 pS, ATP-sensitive renal K channel (Kir1.1, also known as renal outer medullar K [ROMK]) that is critical for K secretion by cells of the thick ascending limb (TAL) and distal nephron segments responsive to aldosterone. We report here that both ATP and glibenclamide sensitivities of the 30 pS K channel in TAL cells were absent in mice lacking CFTR and in mice homozygous for the deltaF508 mutation. Curcumin treatment in deltaF508-CFTR mice partially reversed the defect in ATP sensitivity. We demonstrate that the effect of CFTR on ATP sensitivity was abrogated by increasing PKA activity. We propose that CFTR regulates the renal K secretory channel by providing a PKA-regulated functional switch that determines the distribution of open and ATP-inhibited K channels in apical membranes. We discuss the potential physiological role of this functional switch in renal K handling during water diuresis and the relevance to renal K homeostasis in cystic fibrosis.

A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse.

The increasing number of available genetically manipulated mice makes it necessary to develop tools and techniques for examining the phenotypes of these animals. We have developed a straightforward and rapid method for the isolation of large quantities of single tubule fragments from the mouse kidney. Immunohistochemistry, electron microscopy, and fluorescence microscopy were used to evaluate the viability, functional characteristics, and morphology of proximal tubules (PT), and collecting ducts from cortex (CCD) and inner stripe of the outer medulla (ISOMCD). Tubules were isolated using a modified collagenase digestion technique, and selected under light microscopy for experimentation. Electron microscopy and trypan blue exclusion showed that a large portion of unselected proximal tubules were damaged by the digestion procedure. The selected tubules, however, all excluded trypan blue, indicating that the plasma membrane had remained intact. Immunocytochemistry on isolated CCD showed normal distribution of H(+)-ATPase, pendrin, and anion exchanger-1 (AE-1) staining. The pH-sensitive dye 2',7'-bis(2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure Na(+)-dependent and -independent intracellular pH (pH(i)) recovery rates in PT, and in single intercalated cells of CCD and ISOMCD fragments. Na(+)-dependent pH(i)-recovery was 0.144+/-0.008 (PT), 0.182+/-0.013 (CCD), and 0.112+/-0.010 pH units/min. (ISOMCD). Na(+)-independent pH(i) recovery was found in all three segments (PT: 0.021+/-0.002, CCD: 0.037+/-0.002, ISOMCD: 0.033+/-0.002 pH units/min) and was sensitive to concanamycin. In summary, we have developed a new technique for rapid and straightforward preparation of large quantities of defined tubule fragments from mouse kidney. Using this technique, the first measurements of plasma membrane vacuolar H(+)-ATPase activities in mouse PT and collecting duct were made. This technique will facilitate further characterization of kidney function in normal and genetically manipulated animals.

Regulation of the expression of the Cl-/anion exchanger pendrin in mouse kidney by acid-base status.

BACKGROUND: Pendrin belongs to a superfamily of Cl-/anion exchangers and is expressed in the inner ear, the thyroid gland, and the kidney. In humans, mutations in pendrin cause Pendred syndrome characterized by sensorineural deafness and goiter. Recently pendrin has been localized to the apical side of non-type A intercalated cells of the cortical collecting duct, and reduced bicarbonate secretion was demonstrated in a pendrin knockout mouse model. To investigate a possible role of pendrin in modulating acid-base transport in the cortical collecting duct, we examined the regulation of expression of pendrin by acid-base status in mouse kidney. METHODS: Mice were treated orally either with an acid or bicarbonate load (0.28 mol/L NH4Cl or NaHCO3) or received a K+-deficient diet for one week. Immunohistochemistry and Western blotting was performed. RESULTS: Acid-loading caused a reduction in pendrin protein expression levels within one day and decreased expression to 23% of control levels after one week. Concomitantly, pendrin protein was shifted from the apical membrane to the cytosol, and the relative abundance of pendrin positive cells declined. Similarly, in chronic K+-depletion, known to elicit a metabolic alkalosis, pendrin protein levels decreased and pendrin expression was shifted to an intracellular pool with the relative number of pendrin positive cells reduced. In contrast, following oral bicarbonate loading pendrin was found exclusively in the apical membrane and the relative number of pendrin positive cells increased. CONCLUSIONS: These results are in agreement with a potential role of pendrin in bicarbonate secretion and regulation of acid-base transport in the cortical collecting duct.

A trail of research on potassium.

A complex pump-leak system involving both active and passive transport mechanisms is responsible for the appropriate distribution of potassium (K) between the intra- and extracellular fluid compartments. In addition, the kidneys, and to a lesser extent the colon, safeguard maintenance of the narrow range of low K concentrations in the extracellular fluid. Early renal clearance studies showed that K is normally both reabsorbed and secreted by renal tubules, and that regulated secretion is the major source of K excretion. Studies at the tubule and cell level have localized secretion and reabsorption of K to principal and intercalated cells in the collecting ducts. Measurements of the electrochemical driving forces across individual cell membranes have permitted the characterization of specific ATPases, K channels and K cotransporters and also provided insights into the molecular structure of individual transporters that regulate K excretion.