Respective Roles of H-Atpase and H-K-Atpase in Ion Transport in the Kidney1

Respective Roles of H-ATPase and H-K-ATPase in Ion Transport in the Kidney1

Lal C. Garg2

monia and titrable acid. Both of these functions are L.C. Garg, Department of Pharmacology and Thera- achieved by the acidification of the tubule fluid. Most peutics and Division of Nephrology, Hypertension and of the filtered bicarbonate is reabsorbed in the prox- Transplantation, University of Florida College of Medi- imal tubule. The acidification of the luminal fluid in cine, Gainesville, FL the distal tubule plays an important role in the transport of total ammonia and in the titration of phos- 1991; (J. Am. Soc. Nephrol. 2:949-960) phate and other buffers. During the last several years, it has been established that acidification of the tubule fluid is mediated, at least in part, by an ABSTRACT electrogenic proton pump (H-ATPase) that is located in the luminal membrane of renal tubule cells in both Two types of proton-translocating ATPases, H-ATPase proximal and distal nephron segments, including the and H-K-ATPase, are found in the renal tubular cells. collecting duct. Recent evidence suggests that an H-ATPase is present in both endocytic vesicles and electroneutral H-K-ATPase present in the collecting apical membranes In almost all nephron segments. duct may play a role in the acidification of tubule On the other hand, H-K-ATPase is present only in the fluid. In this article, we will review the biochemical, connecting tubule and collecting duct. There is evi- immunohistological, and physiological evidence for dence to suggest that H-ATPase may be involved the respective roles of H-ATPase and H-K-ATPase in In H secretion in almost all nephron segments. ion transport in various nephron segments. we will H-K-ATPase is involved not only in H secretion but also also consider here the effects of certain physiological in K absorption in the collecting duct segments. Al- and pharmacological factors on the two enzymes. dosterone administration and metabolic acidosis The synthesis and transport of ammonia, which also play an important role in maintaining the acid-base stimulate the activity of H-ATPase in all collecting balance, will not be discussed here. Several excellent duct segments, whereas hypokalemia has only a and recent reviews are available on this topic (1,2). lImited effect on H-ATPase activity. On the other hand, hypokalemia, as well as metabolic acidosis, stimulates H-K-ATPase activity in the collecting duct GENERAL PROPERTIES OF DIFFERENT TYPES OF segments, whereas aldosterone administration H-ATPases alone plays a minor role in the regulation of this There are three types of H-ATPases. The FO-Fi enzyme. The physiological role and regulation of H- type of H-ATPase is present in the mitochondria ATPase in the proximal tubule has not been estab- where it acts as ATP synthase. This type of H-ATPase lished. is also present in bacterial membranes and in chlo- Key Words: H-A TPase, H-K-A TPase, nephron segments, acid roplasts (3). It is composed of 8 to 14 polypeptides. secretion, potassium transport, aldosterone The mammalian enzyme is inhibited by oligomycin, azide, and low concentrations of dicyclohexylcarbo- diimide (DCCD), but not by ouabain (inhibitor of Na- o maintain acid-base balance, the human kid- T K-ATPase) or vanadate (inhibitor of El -E2 type ney not only reabsorbs the filtered bicarbonate ATPases, including Na-K-ATPase). but also generates bicarbonate by excretion of am- The second type of H-ATPase is present in the membranes of intracellular organelles, such as en- Received June 4, 1991. Accepted July 22, 1991. dosomes, lysosomes, and clathrin-coated vesicles (4), 2Correspondence to Dr. L.C. Garg, Department of Pharmacology and Thera- peutics, University of Florida College of Medicine, Health Science Center, P.O. and it acidifies the interior of these organelles. Be- Box 100267, GainesvIlle, FL 32610-0267. cause this H-ATPase is present in the intracellular 1046-6673/0205-0949$03.O0/0 vacuoles, it has been called vacuolar (or V) H-ATPase Journal of the American Society of Nephrology Copyright C 1991 by the American Society of Nephrology (5). The vacuolar H-ATPase is also present in the

Journal of the American Society of Nephrology 949 H-ATPase and H-K-ATPase

plasma membranes of subpopulations of cells in the differences in the enzyme activity of V type mammalian collecting duct and turtle urinary blad- H-ATPases in different organelles may also be be- der where it is responsible for urinary acidification cause of secondary factors present in the organelles. (6,7). In addition, this type of H-ATPase is located in These include the presence or absence of a Cl chan- synaptic vesicles and chromaffin granules (8). The V nd and the lipid composition of the organelle mem- type H-ATPases are composed of nine or more differ- brane (8). The acidification by V type H-ATPase in ent polypeptides. They contain more cystelne resi- various organelles may be controlled by the primary dues than mitochondrial H-ATPase and are Inhibited and secondary factors mentioned above. In contrast, by low concentrations of N-ethylmaleimide (NEM). a the acidification of urine by V type H-ATPase is sulfhydryl alkylating agent (9). In addition, V type probably controlled by the rates of enzyme biogenesis H-ATPases are inhibited by higher concentrations of and by enzyme insertion into and retrieval from the DCCD than is the mitochondrial H-ATPase (ATP syn- plasma membrane (7). There is evidence that the thase) (5). They are not inhibited by azide, ouabain, activity of the H-ATPase and its insertion into and or vanadate. retrieval from the plasma membrane in the collecting The functions of V type H-ATPases in various spe- duct are influenced by adrenal hormones and cies and in various organs in mammals have been changes in acid-base balance (discussed below). reviewed recently (3,5,8). In brief, the major function The third type of H-ATPase is characteristic of the of V type H-ATPases is to acidify the interior of mammalian gastric mucosa (13). It belongs to the El- intracellular organelles. This acidification leads to E2 type of ATPases that form phosphorylated inter- activation of several secondary processes. For cx- mediates and are vanadate sensitive. It is composed ample, it has been demonstrated In several tissues of two subunits and is inhibited by omeprazole and that the acidification of endosomes is important for an imidazopyridine compound (SCH 28080). It resem- the dissociation of ligands from receptors after recep- bles Na-K-ATPase but is not inhibited by ouabain. tor-mediated endocytosis and the subsequent recy- This type of H-ATPase is also present in certain cling of the receptors (5). Under normal conditions, fungi. Recently, ouabain-insensitive K-ATPase activ- endocytosed ligands such as low-density lipopro- ity has been demonstrated in the mammalian col- teins, Insulin, and epidermal growth factor dissociate lecting duct (14,15). The renal K-ATPase is pharma- from their receptors in the acidic pH of the endo- cologically similar to gastric H-K-ATPase (15). somes. However, treatment of cells with weak bases, The gastric H-K-ATPase is responsible for trans- such as NH4C1 or chloroquine, that neutralize acidic port of H into the lumen in exchange for K. This compartments in the cells results in accumulation of transport process is accompanied by a back diffusion ligand-receptor complexes in the endosomes and a of K into the lumen via a K channel and transport of depletion of cell surface receptors. Similarly, the Cl from cell to the lumen via an apical Cl channel. maintenance of an acidic pH within lysosomes is These processes lead to net secretion of HC1 into the necessary for optimum activity of lysosomal hydro- lumen (16). The recognition of the role of H-K-ATPase lases (10). in gastric acid secretion led to the development of The proton-motive force generated by V type omeprazole (PRILOSEC; Merck Sharp & Dohme, H-ATPases in synaptic vesicles and chromaffin West Point, PA), which is used in pathological hy- granules is used for the uptake and storage of neu- persecretory conditions such as Zollinger-Ellison rotransmitters (8). The mechanism of neurotrans- syndrome and in gastroesophageal reflux diseases mitter uptake may vary with the transporter of the (13). The physiological role of renal H-K-ATPase in H neurotransmitter. Thus, like Na-K-ATPase, V type and K transport is discussed in a later section. H-ATPases provide energy for several secondary processes. In fact, it has been suggested that the DISTRIBUTION OF H-ATPase ALONG THE NEPHRON nervous system cannot function without V type H- ATPases because of a lack of accumulation of neu- The distribution of H-ATPase in the various neph- rotransmitters in the absence of H-ATPase (8). ron segments has been studied by both biochemical V type H-ATPases isolated from different kidney and immunohistochemical techniques. Because NEM membrane compartments have been shown to differ has been used as an inhibitor of the V type H-ATPase, in their enzymatic and structural properties. For ex- the enzyme is also called NEM-sensitive ATPase. ample, two H-ATPases isolated from brush borders We will use the term NEM-sensitive ATPase and and microsomes of the bovine kidney differ in their H-ATPase interchangeably. Figure 1 illustrates the 56K subunit, substrate specificity. pH optimum, and locations of the major nephron segments in which sensitivity to inhibition by Cu2’ (11). It has been enzyme activities have been measured in both the rat suggested that the activity of V type H-ATPase In vlvo and rabbit. The distribution of NEM-sensitive may be regulated by two cytosolic proteins, a stimu- ATPase activity along the rabbit nephron is given in latory protein and an inhibitory protein (12). Some Figure 2. Enzyme activity is high in the connecting

950 Volume 2 ‘Number 5’ 1991 Garg

PCT(SI)OCT 40

(_:l_1:1 >. CORTEX .1 ‘ CTAL PST(S2) ; 30 I- CCD L) ,

- I__Il 1! ,, < II II I, OUTER II MTAL OMCD I 20 STRIPE PST(S3) I I (OS) (OS.) < :__ OUTER I’ MEDULLA . INNER STRIPE I #{149}MTAL OMCO - (IS)(IS)

,‘. I ,, cl IMCD1 1 INNER I MEDULLA IMCD2J 0 PCT PST MTAL CTAL DCT CNT CCD OMCD !MCD IMCD3F Figure 2. NEM-sensitive AlPase activity in individual segments along the rabbit nephron. The abbreviations used for Figure 1 . A diagramatic sketch of the nephron to show the each segment are the same as those described in the location of segments microdissected for assay of AlPases. legend to Figure 1 . The PSI represents 52 segment; MTAL PCI (SI), PCI, initial portion; PSI (52), proximal straight tu- and OMCD represent MTAL (1.5.) and OMCD (l.S.), respec- bule from cortex; PSI ($3), proximal straight portion from tively; IMCD represents IMCD1. Each bar represents mean ± medulla; OMCD (0.5.), OMCD from outer stripe; OMCD(I.S.), SE of three to five animals (data taken from references I 7 OMCD from inner stripe. and 42). tubule (CNT), intermediate in the distal convoluted tubule (DCT), and low in other segments (17). NEM- 40 sensitive ATPase activity in each nephron segment I-. is approximately 40% of total ouabain-insensitive I- ATPase (excluding Na-K-ATPase), which showed a LI similar pattern of distribution along the rabbit neph- 30 ron (18). There are significant species differences in NEM- sensitive ATPase activity along the nephron. The 20 enzyme activities in proximal as well distal tubule segments are higher in the rat than in the rabbit (Figure 3). However, enzyme activities in the collect- 10 ing duct segments are similar in rat and rabbit (17,19-23). Na-K-ATPase activity in the proximal convoluted tubule (PCT) and DCT is also higher in rat than in rabbit (24). The higher activity of NEM-sen- 0 S I S 2 S 3 MTAL CTAI. DCT CCD OMCD 1MCD sitive ATPase and Na-K-ATPase may be related to higher single nephron GFR in the rat. Figure 3. NEM-sensitive ATPase activity in individual seg- Immunohistochemical studies with antibodies ments along the rat nephron. The abbreviations used for each segment are the same as those described in the raised against three subunits of bovine kidney legend to Figure 1. MTAL and OMCD represent MTAL (l.S.) H-ATPase have demonstrated labeling of apical yes- and OMCD (IS.), respectively; IMCD represents IMCD1. Each ides and the apical plasma membrane of the PCT, bar represents mean ± SE of 7 to 15 animals (compiled from thick ascending limbs, DCT, and intercalated cells in references 18, 20, and 37). the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) (25).

in collecting duct segments of both rat and rabbit DISTRIBUTION OF H-K-ATPase ALONG THE (14,15). In both species, H-ATPase activity is present NEPHRON in the CNT, CCD, OMCD, and inner medullary col- Recently, we and others have reported the presence lecting duct (IMCD), but not in other nephron seg- of ouabain-insensitive K-stimulated ATPase activity ments (Figure 4). There are no quantitative differ-

Journal of the American Society of Nephrology 951 H-ATPase and H-K-ATPase

LUMEN BLOOD

>- C., 4 . WE Na + CA- K

(HCO )3

PCI PSI MIAL clAl. DCI t4T ccD MCD

Figure 4. H-K-ATPase activity in individual segments along the rabbit nephron. The abbreviations used for each segment are the same as those described in the legend to

Figure I . The PSI represents 52 segment; MTAL and MCD represent MTAL (IS.) and OMCD (1.5.), respectively (repro- duced from reference 15). ‘ P < 0.05 versus 0.

ences in H-ATPase activity in the CCD and OMCD Figure 5. Bicarbonate absorption in the PCI. CA., carbonic between rabbit and rat, and the enzyme activity is anhydrase. See text for an explanation of various transport inhibited by vanadate, omeprazole, and SCH 28080. processes. The pharmacological characterization of the enzyme suggests that ouabain-insensitlve K-ATPase is simi- Na:H exchanger (29,30), and it has been estimated lar to the well-characterized gastric H-K-ATPase (13). that approximately 35% of bicarbonate absorption Immunohistochemical studies have demonstrated occurs by a sodium-independent process (independ- that a monoclonal antibody against gastric H-K- ent of the Na:H exchanger) (30). Furthermore, lu- ATPase labels intercalated cells in the CCD and minal DCCD, an inhibitor of H-ATPase, was reported OMCD in both rat and rabbit (26). to decrease bicarbonate absorption in the PCT by 2 1 % (3 1). Biochemical studies have demonstrated the ROLE OF H-ATPase IN THE PROXIMAL TUBULE presence of an ATP-driven proton pump in brush- Unlike ouabain, which is a specific inhibitor of Na- border membranes isolated from the rat renal cortex K-ATPase, there is no specific inhibitor of the V type (32,33). In agreement with these findings, H-ATPase H-ATPase that would unequivocally prove its phys- immunolabeling has been demonstrated on the api- iological role in the kidney. Therefore, we rely on a cal membrane of the rat PCT. Taken together, these combination of biochemical, immunohistochemical, results suggest that H-ATPase is responsible for part and electrophysiological experiments to explain the of the proton secretion in the PCT (Figure 5). role of H-ATPase in the various nephron segments. H-ATPase is not only present in the apical mem- Approximately 80% of the filtered bicarbonate is brane but also in the endocytic vesicles in the PCT reabsorbed in the proximal tubule. A major mecha- (25). In addition, immunostaining with H-ATPase an- nism for bicarbonate absorption includes H secretion tibodies has been demonstrated in invaginations at by a Na:H exchanger in the apical membrane (Figure the base of the microvilli (25), which may be areas of 5). This process is energized by the sodium gradient active endocytosis. Endosomal H-ATPase has char- created by the Na-K-ATPase in the basolateral mem- acteristics similar to that of the brush-border membrane. In the luminal fluid, secreted H combines with brane (34). Therefore, endosomes may serve as an filtered bicarbonate to form carbonic acid, which is additional source of H-ATPase for proximal tubular dehydrated by luminal carbonic anhydrase to CO2. acidification by fusing with the luminal membrane Carbon dioxide diffuses into the cell where H is re- and inserting the H pump into this membrane. The generated by a reverse process. Bicarbonate is trans- vacuolar H-ATPase is responsible for the acidic in- ported across the basolateral membrane via a Na terior of the endosomes, which is important for the (HCO3)3 transporter (27,28). receptor-ligand dissociation that occurs after recep- There are several pieces of evidence to suggest that tor-mediated endocytosis. The low pH of the lyso- a part of the bicarbonate reabsorption in the PCT somes is also dependent on a V type H-ATPase. The occurs via a H-ATPase. Bicarbonate reabsorption in acidification of endocytic vesicles and lysosomes the rat PCT is not completely inhibited by luminal plays an important role in the reabsorption and deg- amiloride and t-butyl amiloride, two inhibitors of the radation of filtered proteins in this segment. Thus,

952 Volume 2’ Number 5’ 1991 Garg

H-ATPase in the PCT may be involved not only in H “true” DCT; the middle part is the CNT; and the last secretion into the luminal fluid but also in several part is the initial cortical collecting duct (ICCD) (40). other processes. These three segments are easily distinguishable In It has been reported that metabolic acidosis de- the rabbit but not in the rat. The DCT contains DCT creases NEM-sensitive ATPase in the PCT from su- cells, whereas the CNT has two cell types: CNT cells perficial nephrons, but not in the PCT from juxta- and intercalated cells. The ICCD and the CCD (from medullary nephrons, of the rat (23). The physiological the medullary ray) also have two main cell types, the significance of this observation is not known at the principal cell and the intercalated cell. The interca- present time. lated cells in the CNT are similar to those in the CCD. Bicarbonate absorption has not been studied in the rabbit DCT because this Is a very small segment (<0.5 ROLE OF H-ATPase IN THE THICK ASCENDING mm in length). In a micropuncture study of the rat LIMB OF HENLE’S LOOP DCT, it has been shown that this segment probably Both medullary and cortical thick ascending limbs does not secrete H except in very severe acidosls (41). (MTAL and CTAL) are impermeable to water. Baso- Although NEM-sensitive ATPase activity in the DCT lateral Na-K-ATPase provides the driving force for is greater than in the CTAL or CCD in the rat, the the Na:K:2Cl transporter in the luminal membrane enzyme activity in the DCT is not influenced by (35). Chloride exits the cell passively across the baso- changes in acid-base balance (20,22). lateral membranes via a Cl channel and a KC1 co- The CNT is believed to behave like the CCD with transport system. K is recycled back to the tubular respect to the regulation of H-ATPase (27,42). The lumen through luminal K channels and to the blood CNT is also involved In K secretion (43,44). Morpho- through the basolateral KC1 cotransport system. The logical studies have demonstrated that chronic potas- sum of these processes results in reabsorption of sium loading increases the basolateral membrane NaCl and dilution of luminal fluid. area of CNT cells (45,46). The thick ascending limbs have been thought to play a minor role in the acidification of urine. Re- cently, however, it has been demonstrated that the rat CTAL and MTAL absorb bicarbonate at rates Lumen Principal cell Blood comparable with those In the collecting ducts (36,37). Bicarbonate absorption in the thick ascending limbs Nat, is an active process that occurs by a combination of H secretion across the apical membrane and base Na efflux across the basolateral membrane. The H secretion occurs mainly via a Na:H exchanger, which is inhibited by amiloride (37), as in the PCT (Figure 5). In addition, preliminary observations have provided evidence for the presence of a sodium-independent acid extrusion mechanism, presumably mediated by a H-ATPase (38). K NEM-sensitive ATPase activity in the MTAL and Intercalated cell CTAL is influenced by changes in acid-base balance (Type A) (20,22,33), and recent studies have revealed an inverse linear relationship between NEM-sensitive ATPase activity in MTAL and CTAL and total CO2 in K’ the plasma (39). Taken together, these results sug- K gest that H-ATPase may also be involved in bicarbonate reabsorption in the rat MTAL and CTAL. Cl

ROLE OF H-ATPase IN THE DISTAL CONVOLUTED TUBULE AND THE CONNECTING TUBULE

In the past, the term “distal tubule” has been used to include the nephron segments from the macula densa to the first junction of two nephrons forming Figure 6. Upper part, potassium secretion in principal cell; a CCD in a medullary ray. Later, it was shown that lower part, acid secretion in the type A intercalated cell of this long distal tubule is a heterogenous structure the CCD. C.A., carbonic anhydrase. See text for an expla- that is comprised of three parts. The early part is the nation of various transport processes.

Journal of the American Society of Nephrology 953 H-ATPase and H-K-ATPase

ROLE OF H-ATPase IN THE CCD to erythrocyte band 3 (50-52). It has been shown that respiratory acidosis increases the surface area of the The principal cells are the predominant cell type luminal membrane and decreases the apical tubular (>60%) in the CCD. and they are responsible for Na structures in type A intercalated cells in the CCD, absorption and K secretion. These cells have Na and supporting a role of these cells in acid secretion (53). K conductive pathways in the luminal membrane and There is evidence that type B intercalated cells are Na-K-ATPase in the basolateral membrane (Figure involved in bicarbonate secretion, at least in the 6). The Na channel in the luminal membrane is sen- rabbit. The biochemical mechanism of HCO3 secre- sitive to low concentrations (1 o_7 M) of amiloride. tion in the CCD is not well understood. It has been The CCD is also involved in H secretion, which suggested that the type B intercalated cells is similar occurs via a sodium-independent mechanism (47,48). to the type A cell but has a reversed polarity with a In addition, the CCD of bicarbonate-loaded animals H-ATPase in the basolateral membrane and a Cl- is capable of bicarbonate secretion (49). The inter- HCO3 exchanger in the apical membrane. Immuno- calated cells are thought to be responsible for H histochemical studies have demonstrated that the transport in this segment. It has been shown mor- basolateral membrane of type B intercalated cells in phologically that the CCD contains two subtypes of the rat kidney is stained with H-ATPase antibodies intercalated cells called A cells and B cells (40). There (25). However, the apical membrane of type B inter- are several pieces of evidence to suggest that type A calated cell does not label with antibodies against intercalated cells are responsible for H secretion. A band 3 protein (50-52). In addition, it has been dem- model for bicarbonate reabsorption (or H secretion) onstrated that the Cl-HCO3 exchanger in the B cell is in type A intercalated cells includes an apical H- not regulated by acute changes in peritubular Pco2 or pump and a basolateral Cl-HCO3 exchanger (Figure intracellular pH (54). Furthermore, it has been sug- 6). Immunohistochemical studies have provided sup- gested that the Cl-HCO3 exchanger in the B cell may port for this model (Figure 7). The H pump is a V type act as a Cl self-exchanger (55). All of these observa- H-ATPase (4), and the Cl-HCO3 exchanger is similar tions indicate that the Cl-HCO3 exchanger in these

Figure 7. Light micrograph of rat kidney cortex illustrating W-ATPase immunostaining by a horseradish peroxidase technique using a rabbit polyclonal antibody against W-ATPase from bovine brain (kindly provided by Dr. Dennis Stone). Immunostalning Is observed in the S segment of the proximal tubule where it is located in the apical region beneath the brush border corresponding to coated pits and endocytic vesicles (arrowheads). In the CCD, there is heavy immunostaining of the apical region of type A intercalated cells (closed arrow), whereas type B intercalated cells exhibit diffuse cytoplasmic and basal staining (open arrow). Magnification, xl,000. Courtesy of Dr. Jin Kim.

954 Volume 2’ Number 5. 1991 Garg

cells is different from that present in the basolateral calated cells of the OMCD (50-52). Thus, the cellular membrane of the type A cell. model for H secretion in the OMCD is the same as in McKinney and Burg (49) first showed that bicar- the CCD (Figure 6). Both respiratory and metabolic bonate reabsorption in the CCD is influenced by the acidosis increases the apical surface area of interca- acid-base status of the animal. These observations lated cells in the OMCD (63). have been confirmed by other investigators both in NEM-sensitive ATPase is present in the OMCD of rabbits and in rat (27). NEM-sensitive ATPase activ- both rabbit and rat (17,20). The enzyme activity in ity in the rat CCD is increased during metabolic aci- the OMCD is comparable to that in the CCD (Figure dosis (20,23) and decreased during metabolic alka- 2). There is an increase in NEM-sensitive ATPase losis (22). The enzyme activity in the CCD is inversely activity in the OMCD during acidosis and a decrease proportional to plasma total CO2 (39). These results in the enzyme activity during metabolic alkalosis suggest that the CCD is an important site for renal (20,22). There is an inverse linear relationship be- adaptation to chronic acidosis and alkalosis and that tween NEM-sensitive ATPase activity and plasma changes in the activity of H-ATPase is at least par- total CO2 content in the rat (39). These results sup- tially responsible for this adaptation. Acute respira- port the described model in which a NEM-sensitive H tory acidosis is associated with a migration of mem- pump is responsible for H secretion (Figure 6). brane vesicles to the apical plasma membrane of the Aldosterone administration increases NEM-sensi- CCD, indicating exocytotic insertion of the H-ATPase tive H-ATPase activity in the OMCD of both rabbit into the luminal membrane (53). The opposite may and rat (1 7,64) and stimulates electrogenic H secre- happen during a decrease in bicarbonate absorption tion in the rabbit OMCD (65). These results suggest when peritubular bicarbonate is increased acutely that H secretion in the OMCD is influenced by adre- (56). nal steroids via regulation of H-ATPase activity. NEM-sensitive ATPase activity in the CCD is not Hypokalemia also increases NEM-sensitive activity only stimulated by metabolic acidosis but is also in the rat OMCD (59). The effect is limited to the influenced by adrenal steroids. Aldosterone admin- OMCD and is not seen in the CCD or IMCD. The istration increases and adrenalectomy decreases mechanism of this action of hypokalemia on NEM- NEM-sensitive ATPase activity in the CCD (17,57). sensitive ATPase in the OMCD is not known. The demonstration that changes in acid-base balance can produce a change in NEM-sensitive ATPase activity in adrenalectomized rabbits (42) suggests ROLE OF H-ATPase IN THE IMCD that acidosis (or alkalosis) and aldosterone act inde- The IMCD is a heterogenous structure. It has been pendently of each other. Chronic treatment of the subdivided into three subsegments: the initial one rabbit with deoxycorticosterone, a mineralocorticoid, third (IMCD1 or IMCD1), the middle one third (IMCD2), increases bicarbonate secretion in the CCD, probably and the last one third (IMCD3) (Figure 1). In spite of because of the accompanying metabolic alkalosis the morphological differences between the IMCD2 (58). Hypokalemia per se does not affect H-ATPase and IMCD3, the two subsegments show similar prop- activity in the CCD (59). erties with respect to urea transport and response to Both isoproterenol and 8-bromo-cAMP stimulate hormones (66). Therefore, these subsegments to- net bicarbonate secretion in the CCD (60). The effect gether have been called the terminal IMCD (IMCD). is mediated by stimulation of the Cl:HCO3 exchanger The intercalated cells are absent in the rabbit IMCD in the apical membrane of type B intercalated cell and are present to a very small extent (10%) in the (6 1). Thus, acid-base secretion in the CCD is not only IMCD1 of the rat (67). Traditionally, nonintercalated regulated by H-ATPase activity but also by the activ- cells in the IMCD of all species were considered to be ity of the Cl:HCO3 exchanger in the intercalated cells principal cells. However, morphological reexamina- of the CCD. tion of the IMCD showed that the cells in the IMCD have several characteristics different from the prim- ROLE OF H-ATPase IN THE OMCD cipal cells of the CCD, OMCD, and IMCDI. Therefore, the cells in the IMCD have been called IMCD cells The OMCD has been subdivided into the OMCDO (68). (from the outer stripe) and OMCD, (from the inner There is some evidence to suggest that bicarbonate stripe) (Figure 1). The OMCD1 has been studied more is absorbed independently of sodium in the rat IMCD extensively than the OMCDO. The OMCD is an impor- (69). Metabolic acidosis increases and metabolic al- tant site of sodium-independent H secretion (62). The kalosis decreases NEM-sensitive ATPase activity in intercalated cells of the OMCD are all similar to type the rat IMCD, (20,22). Because intercalated cells (type A cells. Immunocytochemical studies show the pres- A) are present in the rat IMCD,, it is likely that inter- ence of H-ATPase in the apical membrane and band calated cells are sites of NEM-sensitive ATPase mod- 3 protein in the basolateral membrane of the inter- ulation by systemic acid-base changes. Therefore,

Journal of the American Society of Nephrology 955 H-ATPase and H-K-ATPase

the mechanism of H secretion in the rat IMCD1 may change the activity of NEM-sensitive ATPase activity to some extent be similar to that in the OMCD and in the CCD (59), it is likely that the increased bi- CCD (Figure 6). carbonate reabsorption may be mediated by H-K- Recently, we have determined the NEM-sensitive ATPase. In addition, the H-K-ATPase activity is influ- ATPase activity in all three subsegments of the rab- enced by aldosterone. During hyperaldosteronism, bit. The enzyme activity in the IMCD2 and IMCD3 was the enzyme activity is increased in the CNT and CCD, equal to or greater than that measured in the IMCD, but not in the OMCD and IMCD (78,79). Aldosterone (our unpublished observations). Because the subseg- stimulates the activity of Na-K-ATPase in CCD to a ments of the rabbit IMCD do not have intercalated much greater extent than it does H-K-ATPase. There- cells, it is likely that H-ATPase is present in the IMCD fore, under normokalemic conditions, the overall ef- cells. Whether the enzyme is present in the apical fect of aldosterone will be to produce a net secretion membrane or in the intracellular organelles of the of K. However, during K deficiency, there may be net IMCD cell is not known. Recent studies in the isolated reabsorption at least in the OMCD. The stimulatory IMCD of the rat have provided evidence for H secre- effect of aldosterone on H-K-ATPase (79), along with tion, which was increased by chronic acid loading its stimulatory effect on NEM-sensitive H-ATPase (70). Taken together, these results indicate that the (20), will be additive for H secretion in the CCD. IMCD is capable of H secretion despite the absence Metabolic acidosis increases and metabolic alka- of intercalated cells. The mechanism of H secretion losis decreases the activity of H-K-ATPase in the rat in the IMCD is not known at the present time. OMCD (80). The stimulatory effect of metabolic acidosis is additive to the stimulation produced by hy- ROLE OF H-K-ATPase IN THE CNT, CCD, AND pokalemia. and there is an inverse linear relationship OMCD between H-K-ATPase activity in the OMCD and plasma bicarbonate concentration (80). On the basis The rabbit OMCD has been shown to absorb K of rubidium flux measurements, it has been sug- during K restriction (7 1 ,72). The demonstration that gested that there may be an interaction between Na omeprazole (an inhibitor of gastric H-K-ATPase) in- absorption and H-K-pump activity in the CCD (81). hibits K absorption and H secretion in the OMCD of The mechanism of this interaction is not known at rabbits maintained on a low-K diet (73) suggests that this time. at least part of K absorption occurs via H-K-ATPase. The complete physiological and pathophysiological Micropuncture and In vivo microperfusion experi- roles of renal H-K-ATPase have not been established ments suggest that there is H secretion and K absorp- at the present time. However, a recent study has tion in the rat distal tubule during K depletion (74). reported that a decrease in H-K-ATPase activity in The reabsorption of potassium was reduced by SCH the CCD of rats treated with vanadate was associated 28080, an inhibitor of H-K-ATPase. The late distal with the development of distal tubular acidosis (82). tubule in the rat probably represents the CNT and the ICCD. Taken together, these results suggest that renal H-K-ATPase is responsible for K absorption in H-ATPase and H-K-ATPase IN CULTURED RENAL the CNT, ICCD, and OMCD during K depletion. TUBULAR CELLS H-K-ATPase activity is present in CCD and OMCD of both rat and rabbit (Figure 4) (14. 1 5). Immunohis- In addition to microdissected nephron segments, tochemical studies indicate that H-K-ATPase is pres- renal epithelial cells in culture have proven to be ent in the intercalated cells of the CCD and OMCD of useful in understanding the mechanism of electrolyte both rat and rabbit (26). Therefore, it is likely that transport in the renal tubule (83). Both established intercalated cells are responsible for K absorption in cell lines and primary cultures have been used as a exchange for H secretion in the CCD and OMCD tool to study various transporters and to determine (Figure 6). Recently, we examined H-K-ATPase activ- the mechanism of action of hormones. The sources, ity in three subsegments of the IMCD (75) and found nephronal site of origin, hormone responsiveness, that H-K-ATPase activity was higher in the IMCD2 transport and metabolic functions, and electrical than in the IMCD1 and IMCD3. The rabbit IMCD does properties of established cell lines have been re- not have intercalated cells. Therefore, H-K-ATPase viewed recently (83). may also be present in the IMCD cells. Very little effort has been made to examine A low-potassium diet increases and a high-potas- H-ATPase or H-K-ATPase activity in cultured cells. A sium diet suppresses the activity of H-K-ATPase in recent report suggests that Madin-Darby canine kid- the CNT. CCD, and OMCD (14,76). These results ney (MDCK) cells express an apical H-K pump, which suggest that the enzyme is regulated by body potas- is stimulated by aldosterone (84). Proton secretion by sium. Potassium depletion increases bicarbonate MCDK cells is inhibited by omeprazole, an inhibitor reabsorption in the CCD (77). Because it has been of H-K-ATPase, and by barium, a K channel blocker demonstrated that potassium depletion does not that will prevent apical K recycling. Also, there is

956 Volume 2 - Number 5’ 1991 Garg

preliminary evidence for H secretion mediated by H-K-ATPase activity in the CCD, administration of H-ATPase and by H-K-ATPase in another cell line, aldosterone is associated with potassium wastage RCCT-28A (85). However, neither H-ATPase nor and excess acid excretion, whereas aldosterone defi- H-K-ATPase has been characterized biochemically in ciency is associated with retention of both potassium these cells. and acid (90). Because aldosterone also increases the activity of Na-K-ATPase, which is responsible for K secretion in the CCD, and because the activity of Na- REGULATION OF URINARY ACIDIFICATION AND K K-ATPase in the CCD is greater than that of H-K- EXCRETION ATPase, the effect of aldosterone on K secretion will be greater than on K absorption, resulting in K wast- There is a complex relationship between the con- ing. centration of aldosterone, potassium, and total CO2 in the plasma and transport of H and K in the collecting duct segments. The available data suggest SUMMARY AND CONCLUSIONS that H secretion in the CCD and OMCD depends on In summary, there are at least two H-ATPases H-ATPase and H-K-ATPase, both of which are stim- (an electrogenic H-ATPase and electroneutral H-K- ulated by metabolic acidosis (Figure 6). In addition, ATPase) that mediate Na-independent H secretion in both morphological and physiological studies mdi- the distal nephron. Both of these ATPases are present cate that a decrease in plasma pH is a major stimulus in the intercalated cells of the collecting duct. The for urinary acidification. The intracellular signal(s) activity of both types of H-ATPases is regulated by for the activation of the H pump and H-K pump plasma aldosterone, potassium intake, and changes during acidosis has (have) not been established. in acid-base balance. H-ATPase is also present in These intracellular signals may stimulate the inser- other segments of the nephron, but less is known tion of the two pumps into the apical membrane or about its regulation in those segments. induce the synthesis of additional pumps, or both. Chronic administration of DOCA to rabbits increases H secretion in the OMCD (65). In addition, ACKNOWLEDGMENTS H-ATPase activity in the CCD and OMCD of the rabbit My work Included in this review was funded by the National Science is stimulated by aldosterone treatment (1 7). These Foundation and the American Heart Association, Florida Affiliate. I results suggest that aldosterone receptors shown to thank Dr. Kirsten Madsen for critical review of this manuscript. I be present in the CCD and OMCD (86) may not only also thank Dr. Neelam Narang for preparation of Figures 2 and 3. be present in the principal cells but are also present Ms. Judy Adams and Ms. Jeanette Janney provided excellent 5cc- in intercalated cells. Mineralocorticoids produce kal- retarlal assistance. iuresis and antinatriuresis by increasing in Na-K- ATPase activity in the CCD (18,87). The increase in REFERENCES Na absorption will increase lumen negative voltage, which will stimulate H secretion indirectly in the 1 . Knepper MA, Packer R, Good DW: Ammonium transport in the kidney. Physiol Rev 1 989;69: CCD. Thus, aldosterone increases H secretion di- 179-249. rectly via stimulation of H-ATPase and indirectly by 2. DuBose TD Jr. Good DW, Hamm LL, Wall SM: stimulation of Na-K-ATPase in the CCD. Ammonium transport in the kidney: New phys- Urinary K excretion is the net result of K secretion iological concepts and their clinical implications. J Am Soc Nephrol 1991;1:1 193-1203. in the CCD and K absorption in the OMCD. K secre- 3. Nelson N: Structure, molecular genetics and ev- tion in the CCD depends on Na-K-ATPase in the olution of vacuolar H-ATPases. J Bmoenerg Bio- basolateral membrane of principal cells, and K ab- membr 1989;21 :553-571. sorption in the OMCD is mediated by the H-K-ATPase 4. Stone DK, Cider BP, Xie X: Structural properties present in intercalated cells. Potassium loading in- of vacuolar proton pumps. Kidney mt i 990;38: 649-653. creases plasma aldosterone, which in turn will stim- 5. Forgac M: Structure and function of vacuolar ulate K secretion. In addition, potassium loading it- class of ATP-driven proton pumps. Physiol Rev self stimulates Na-K-ATPase (88) and K secretion (89) 1989;69:765-796. in the CCD of adrenalectomized animals. In contrast, 6. Gluck 5, Kelly S, A1-Awqati Q: The proton trans- potassium loading decreases H-K-ATPase activity in locating ATPase responsible for urinary acidification. J Biol Chem 1982;257:9230-9233. the CCD and OMCD (76), suggesting a decrease in K 7. Schwartz GJ, A1-Awqati Q: Regulation of trans- absorption. The opposite is true for hypokalemia, epithelial H transport by exocytosis and endo- which is associated with an increase in H-K-ATPase cytosis. Annu Rev Physiol 1 986;48: 153-156. activity and in K reabsorption (43). Thus, it seems 8. Nelson N: Structure and pharmacology of the proton ATPases. Trends Pharmacol Sd 1991; that plasma K is the principal regulator of urinary K 12:71-75. excretion. 9. Gluck 5, Caidwell J: Immunoaffmnity purifica- In spite of the stimulatory effect of aldosterone on tion and characterization of vacuolar H-ATPase

Journal of the American Society of Nephrology 957 H-ATPase and H-K-ATPase

from bovine kidney. J Biol Chem 1987;262: Kidney Int 1990;38:584-589. 15780-15789. 29. Chan YL, Giebisch G: Relationship between so- 1 0. Olbricht CJ, Cannon JR. Garg LC, Tisher CC: dium and bicarbonate transport in the rat prox- Activities of cathespins B and L in isolated neph- imal convoluted tubule. Am J Physiol 1981; ron segments from proteinuric and nonprotein- 240:F222-F230. uric rats. Am J Physiol 1 986;250:F1055-F1062. 30. Preisig PA, Ives HE, Cragoe EJ, Alpern RJ,

1 1 . Wang Z, Gluck 5: Isolation and properties of Rector FC: Role of Na/H antiporter in the rat bovine kidney brush-border vacuolar H-ATPase. proximal tubule bicarbonate absorption. J Clin J Biol Chem l990;265:21957-21965l. Invest 1 987;80:970-978.

12. Gluck 5, Zhang K: Isolation and partial charac- 3 1 . Bank N, Aynedijian HS, Mutz BF: Evidence for terization of bovine kidney vacuolar H ATPase a DCCD-sensitive component of proximal bicar- cytosolic regulatory proteins. [Abstractj. J Am bonate reabsorption. Am J Physiol 1985;249: Soc Nephrof 1 990; 1:740. F636-F644. 1 3. Sachs G, Carisson E, Lindberg P. Wailmark B: 32. Kinne-Saffran E, Beauwens R, Kinne R: An Gastric H, K-ATPase as therapeutic target. Annu ATP-driven proton pump in brush-border mem- Rev Pharmacol Toxicol 1988;28:269-284. branes from rat renal cortex. J Membr Biol 14. Doucet A, Marsy S: Characterization of K-ATP- 1 982;64:67-76. ase activity in distal nephron: Stimulation by 33. Tui-rini F, Sabolic I, Zimolo Z, et al. : Relation- potassium depletion. Am J Physiol 1 987;253: ship of ATPases in rat renal brush-border mem- F4 1 8-F423. branes to ATP-driven H secretion. J Membr Biol 1 5. Garg LC, Narang N: Ouabain-insensitive K-ATP- 1989; 107: 1-12. ase in distal nephron segments of the rabbit. J 34. Sabolic I, Burckhardt G: Proton ATPase in rat Clin Invest l988;81:1204-1208. renal cortical endocytic vesicles. Biochim Bio- 1 6. Reenstra WW, Bettencourt JD, Forte JG: Mech- phys Acta 1988;937:398-410. anism of active Cl secretion by frog gastric mu- 35. Greger R: Ion transport mechanisms in thick cosa. Am J Physiol 1986;250:G455-G460. ascending limb of Henle’s loop of mammalian 1 7. Garg LC, Narang N: Effects of aldosterone on nephron. Physiol Rev 1985;65:760-797. NEM-sensitive ATPase in the rabbit nephron 36. Good DW, Knepper MA, Burg MB: Ammonia segments. Kidney Int 1 988;34: 13-17. and bicarbonate transport by thick ascending 18. Garg LC, Knepper MA, Burg MB: Mineralocor- limb of rat kidney. Am J Physiol 1984;247:F34- ticoid effects on Na-K-ATPase in individual F44. nephron segments. Am J Physiol 1981 ;240: 37. Good DW: Sodium-dependent bicarbonate ab- F536-F544. sorption by the cortical thick ascending limb of 19. Garg LC, Narang N. Proton translocating (H)- rat kidney. Am J Physiol 1 985;248:F82 1 -F829. ATPase in proximal tubules of rat nephron. In: 38. Good DW, Kurtz I: Effects of chronic metabolic Bach P, Lock EA, eds. Renal Heterogeneity and acidosis and minerlocorticoids on H/HCO3 trans- Target Cell Toxicity. New York: John Wiley & port in rat medullary thick ascending limb lAb- Sons; 1985:103-106. stracti. Kidney Int 1 989;35:454. 20. Garg LC, Narang N: Stimulation of an N-ethyl- 39. Garg LC, Narang N: H-ATPase activity in neph- maleimide-sensitive ATPase in the collecting ron segments of the rat during metabolic acidosis

duct segments of the rat nephron by metabolic and alkalosis. Contrib Nephrol 1 99 1 , in press. acidosis. Can J Physiol Pharmacol 1985;63: 40. ‘FIsher CC, Madsen KM. Anatomy of the kidney. 1291-1296. In: Brenner BM, Rector FC, eds. The Kidney. Vol.

2 1 . Alt-Mohammaed AK, Marsy 5, Barlet C, Kha- 1 . Philadelphia: WB Saunders; 1991:3-75.

douri C, Doucet A: Characterization of N-ethyl- 4 1 . Lucci MC, Pucacco LR, Carter NW, Dubose TD maleimide-sensitive proton pump in the rat kid- Jr: Evaluation of bicarbonate transport in rat ney. J Biol Chem 1986;261:12526-12533. distal tubule: Effects of acid-base status. Am J 22. Garg LC, Narang N: Decrease in NEM-sensitive Physiol l982;243:F335-F341. ATPase activity in collecting duct by metabolic 42. Garg 1.1C, Narang N. Aldosterone-independent alkalosis. Can J Physiol Pharmacol 1990;68: effects of dietary potassium on N-ethylmaleim- 1119-1123. ide-sensitive ATPase in distal nephron segments 23. Sabatlni 5, Laski ME, Kurtzman NA: NEM-sen- of adrenalectomized rabbits. In: Kovecevic Z, sitive ATPase activity in rat nephron: Effect of Guder WG, eds. Molecular Nephrology: Biochem- metabolic acidosis and alkalosis. Am J Physiol ical Aspects of Kidney Function. New York: Wal- 1 990;258:F297-F304. ter de Gruyter & Co.; 1987:51-56. 24. Garg LC, Mackie 5, Tisher CC: Effect of low 43. Gieblsch G, Malnic G, Berliner RW. Renal trans- potassium diet on Na-K-ATPase in rat nephron port and control of potassium excretion. In: segments. Pflugers Arch 1 982;394: 113-117. Brenner BM, Rector FC, eds. The Kidney. Vol. 1. 25. Brown D, Hirsch S, Gluck 5: Localization of a Philadelphia: WB Saunders; 1991:283-317. proton-pumping ATPase in rat kidney. J Clin 44. Kaufman JS, Hamburger RJ: Potassium (K) se- Invest 1988;82:21 14-2 126. cretion in the connecting tubule (CNT) lAb- 26. Wlngo CS, Madsen KM, Smolka A, Tisher CC: stractj.J Am Soc Nephrol 1990; 1:686. H-K-ATPase immunoreactivity in cortical and 45. Stanton BA, Biemesderfer D, Wade JB, Gie- outer medullary collecting duct. Kidney Int blsch G: Structural and functional study of the 1 990;38:985-990. rat distal nephron: Effects of potassium adap- 27. Alpern RJ, Stone DK, Rector FC. Renal acidi- tation and depletion. Kidney Int 1981; 19:36-48. fication mechanisms. In: Brenner BM, Rector 46. Kaissling B, Le Hir M: Distal tubular segments FC, eds. The Kidney. Vol. 1. Philadelphia: WB of the rabbit kidney after adaptation to altered Saunders Co.; 1991:318-379. Na- and K-intake. Cell Tissue Res 1982;224: 28. DuBose TD: Reclamation of filtered bicarbonate. 469-492.

958 Volume 2 ‘ Number 5- 1991 Garg

47. McKinney TD, Burg MB: Bicarbonate absorption sine trisphosphatase. J Lab Clin Med 1987; by rabbit cortical collecting tubules in vitro. Am 109:34-39. J Physiol 1978;234:F141-F145. 65. Stone DK, Seldin DW, Kokko JP, Jacobson HR: 48. Koeppen BM, Helman SI: Acidification of lu- Mineralocorticoid modulation of rabbit medul- minal fluid by the rabbit cortical collecting tu- lary collecting duct acidification. A sodium-in- bule perfused in vitro. Am J Physiol 1982;242: dependent effect. J Clin Invest 1983;72:77-83. F521-F531. 66. Knepper MA, Star R: The vasopressin-regulated 49. McKinney TD, Burg MB: Bicarbonate transport urea transporter in renal inner medullary by rabbit cortical collecting tubules: Effect of collecting duct. Am J Physiol 1 990;259: acid and alkali in vivo on transport In vitro. J F393-F40 1. Clin Invest 1977;60:766-768. 67. Clapp WL, Madsen KM. Verlander JW, Tisher 50. Schuster VL: Organization of the collecting duct CC: Intercalated cells of the rat inner medullary intercalated cells. Kidney Int 1990;38:668-672. collecting duct. Kidney Int 1 987;3 1:1080-1087.

5 1 . Alper SL, Natale J, Gluck 5, Lodish HF, Brown 68. Madsen XM, Clapp WL, Verlander JW: Struc- D: Subtypes of intercalated cells in rat kidney ture and function of the inner medullary collect- collecting duct defined by antibodies against ing duct. Kidney Int 1988;34:441-454. erythroid band 3 and renal vacuolar H-ATPase. 69. Ullrich KJ, Papavassiliou F: Bicarbonate reab- Proc Nat! Acad Sd USA 1989;86:5429-5433. sorption in the papillary collecting duct of rats. 52. Verlander JW, Madsen KM. Low PS, Allen DP, Pflugers Arch 1981;389:271-275. Tisher CC: Immunocytochemical localization of 70. WalE SM, Sands JM, Flessner MF, Nonoguchi band 3 protein in the rat collecting duct. Am J H, Spring KR, Knepper MA: Net acid transport Physio! 1988;255:F1 15-F 125. by isolated perfused inner medullary collecting 53. Verlander JW, Madsen KM, Tisher CC: Effect ducts. Am J Physiol 1990;258:F75-F84.

of acute respiratory acidosis on two populations 7 1 . Stokes JB: Sodium and potassium transport of intercalated cells in rat cortical collecting across the cortical and outer medullary collect- duct. Am J Physiol 1987;253:Fl 142-Fl 156. ing tubule of the rabbit. Am J Physiol 54. Werner ID, Hamm LL: Regulation of C1 and 1982;242:F5 1 4-F520. HCO exchange in the rabbit cortical collecting 72. Wingo CS: Potassium transport by the medullary tubule. J Clinlnvest 1991;87:1553-1558. collecting tubule of the rabbit: Effects of varia- 55. Tago K, Shuster VL, Stokes JB: Stimulation of tion in K intake. Am J Physiol 1987;253:F1 136- chloride transport by HCO3-CO2 in rabbit cortical Fl 141. collecting tubule. Am J Physiol 1 986;25 1 :F49- 73. Wingo CS: Active proton secretion and potas- F56. sium absorption in the rabbit outer medullary 56. Breyer MD, Kokko JP, Jacobson HR: Regula- collecting duct. J Clin Invest 1989;84:36l-365. tion of net bicarbonate transport in rabbit cor- 74. Agulian 5, Unwin R, Capasso G, Giebisch G: tica! collecting tubule by peritubular pH, carbon Potassium (K) reabsorption and proton (H) se- dioxide tension, and bicarbonate concentra- cretion in the rat distal tubule during potassium tions. J Clin Invest 1986;77:1650-l660. depletion (LK) [Abstract]. J Am Soc Nephrol 57. Khadouri C, Marsy 5, Batlet-Bas C, Doucet A: 1990; 1:645. Effect of adrenalectomy on NEM-sensitive ATP- 75. Garg LC, Narang N: Heterogeneity of ouabain- ase along rat nephron and on urinary acidifica- insensitive K-ATPase along the inner medullary tion. Am J Physiol 1987;253:F495-F499. collecting duct (IMCD) [Abstract]. Physiologist 58. Garcia-Austt J, Good DW, Burg MB, Knepper 1 990;33:A 104. MA: Deoxycorticosterone-stimulated bicarbon- 76. Garg LC, Narang N: Suppression of ouabain- ate secretion in rabbit cortical collecting ducts: insensitive K-ATPase activity in rabbit nephron effects of luminal chloride removal and in vivo segments during chronic hyperkalemia. Renal acid loading. Am J Physiol 1985;249:F205- Physiol Biochem l989;l2:295-301. F212. 77. McKinney TD, Davidson KK: Effect of potas- 59. Garg LC, Narang N: Effects of low-potassium sium depletion and protein intake on renal tu- diet on NEM-sensitive ATPase in the distal neph- bular bicarbonate transport in vitro. Am J Phys- ron segments. Renal Physiol Biochem 1990; iol l987;252:F509-F516. 13: 129-136. 78. Garg LC, Narang N: Effects of aldosterone on 60. Schuster VL: Cyclic adenosine monophosphate- ouabamn-insensitive K-ATPase activity in rabbit stimulated bicarbonate secretion in rabbit cor- nephron segments [Abstract]. J Am Soc Nephrol tical collecting tubules. J Clin Invest 1985; 1990;l:684. 75:2056-2064. 79. Garg LC. Effects of aldosterone on H-K-ATPase

6 1 . Hayashi M, Yamaji Y, lyon M, Kitajima W, Sa- activity in rabbit nephron segments [Abstract]. ruta T: Effect of isoproterenol on intracellular In: Bonvalet JP, Farman N, Lombes M, Rafestin- pH of the intercalated cell in the rabbit cortical Oblin ME, eds. Aldosterone: Fundamental As- collecting ducts. J Clin Invest 1 99 1 ;87: 1 153- pects. Paris: John Libby; 1991:328. 1157. 80. Komatsu Y, Garg LC: Stimulation of ouabain- 62. Stone DK, Seldin DW, Kokko JP, Jacobson HR: insensitive K-ATPase in the rat medullary col- Anion dependence of rabbit medullary collecting lecting duct by potassium depletion and meta- duct acidification. J Clin Invest 1 983;7 1:1505- bolic acidosis [Abstract]. FASEB J 1991 ;5:A752.

1508. 8 1 . Zhou X, Wingo CS: Rubidium efflux by the cor- 63. Madsen KM, Tisher CC: Structural-functional tical collecting duct: Effect of active Na transport relationship along the distal nephron. Am J [Abstract]. J Am Soc Nephrol 1 990; 1:696. Physiol 1986;250:Fl-F15. 82. Sabatini S, Daphnis E, Kurtzman NA: Vanadate 64. Mujais SK: Effects of aldosterone on rat collect- causes distal renal tubular acidosis by inhibiting ing tubule N-ethylmaleimide-sensitive adeno- cortical collecting tubule H-K ATPase [Abstract].

Journal of the American Society of Nephrology 959 H-ATPase and H-K-ATPase

J Am Soc Nephrol 1990; 1:658. 87. Katz A: Renal Na-K-ATPase: Its role in tubular 83. Gstraunthaler GJA: Epithelial cells in culture. sodium and potassium transport. Am J Physiol Renal Physiol Biochem 1988; 11:1-42. 1 982;242:F207-F2 19. 84. Oberleithner H, Steigner W, Silbernagal 5, Vo- 88. Garg LC, Narang N: Renal adaptation to potas- gel U, Gstraunthaler G, Pfaller W: Madin-Darby sium in the adrenalectomized rabbit: Role of canine kidney cells. III. Aldosterone stimulates distal tubular sodium-potassium adenosine tris- an apical H/K pump. Pflugers Arch l990;416: phosphatase. J Clin Invest 1985;76: 1065-1070. 540-547. 89. Muto 5, Sansom 5, Giebisch G: Effects of high 85. Bello-Reuss E, Tyler P: Acidification mecha- potassium diet on electrical properties of cortical nism by the peanut-lectin (+) cell line RCCT-28A collecting ducts from adrenalectomized rabbits. (P+) (Abstract]. J Am Soc Nephrol 1990; 1:646. J Clin Invest l988;8l :376-380. 86. Doucet A, Katz Al: Mineralocorticoid receptors 90. Kurtzman NA: Disorders of distal acidification. along the nephron. Am J Physiol 1981 ;24l: Kidney Int 1990;38:720-727. F605-F61 1.

“Study with me, then, a few things In the spirit of truth alone, so that we may establish the manner of

Nature’s operations .... For this essay which I plan will shed light upon the structure of the kidney. Do not stop to question whether these Ideas are new or old, but ask, more properly, whether they harmonize with Nature. And be assured of this one thing, that I never reached my idea of the structure of the kidney by the aid of books, but by the long, patient and varied use of the microscope. I have gotten the rest by the deductions of reason, slowly, and with an open mind, as is my custom.”

Marcello Malpighi. De viscerum structura exercirtatio anatomica. Bologna cx typ. J. Montij, 1666; as translated by J.M. Hayman, Jr.

960 Volume 2 - Number 5’ 1991