Pediatr Nephrol (1995) 9:510-513 �9 IPNA 1995

L i t e r a t u r e r e v i e w - P h y s i o l o g y

The role of H+-ATPases in urinary acidification

Alexandru Constantinescu

Pediatric Nephrology

Albert Einstein College of Medicine, Department of Pediatrics, Division of Nephrology, 1410 Pelham Parkway South K721, Bronx, NY 10461, USA

Received March 6, 1995; accepted March 10, 1995

Key words: Collecting duct - Urine acidification

The kidney plays an important role in the maintenance of acid-base homeostasis. Each day, approximately 4,000 mmol of bicarbonate (HCO3-) are filtered and re- absorbed by the kidney. At the same time, non-volatile acids, corresponding to 50-100 mmol of protons (H+), are produced from protein catabolism. Excretion of H + is de- pendent on the availability of ammonia and other urinary buffers, such as phosphate [1].

Several mechanisms of urinary acidification exist along the nephron. In the proximal convoluted tubule, H + secre- tion is mediated, for the most part, by the secondary active sodium (Na+)/H + exchanger [2]. However, in vitro micro- perfusion studies suggest that there is an additional, pri- mary active H + transport mechanism mediated by a H§ ATPase [3]. Because the H+-ATPase contributes very little to the urinary acidification in this nephron segment, any abnormality of this enzyme would influence very little the ability to reabsorb HCO3-.

On the other hand, active H + extrusion is the main mechanism by which the collecting duct contributes to urinary acidification. The advent of in vitro microperfusion, coupled with immunocytochemical techniques, led to the identification of two distinct populations of cells in the cortical (CCD) and medullary collecting duct (MCD): the intercalated cells (ICs), involved in H+ secretion, and the principal cells (PCs), responsible for Na + absorption and potassium (K +) secretion. The ICs have been found to be of three types: c~-ICs, with an apical H+-ATPase and a baso- lateral chloride (C1-)/HCO3- exchanger; ~-ICs, which are the mirror image of the (z-ICs; and y-ICs, which exhibit C1-/ HCO3- exchanger activity both at the apical and at the basolateral membranes [4]. In addition, in vivo micro- perfusion studies performed in rats on low-K + diet revealed that the distal tubule is capable not only of secreting but also of reabsorbing K § [5]. Soon thereafter, Doucet and Marsy [6] and Garg and Narang [7] were able to detect a K+-dependent H+-ATPase, found to be present in the CCD [8] and outer medullary collecting duct (OMCD) [9]. If the Na+-dependent H + secretion in the proximal tubule is af- fected, the result is proximal (type II) renal tubular acidosis (RTA). A defect in H § secretion by the ~-ICs results in distal (type I) RTA. Distal RTA can also occur as a con- sequence of impaired Na § or C1- reabsorption by PCs, re- sulting in a low intraluminal voltage, which impedes H § secretion, and as a consequence of an increased perme-

ability of the collecting duct resulting in the backdiffusion of H + and increased diffusion of HCO3- into the tubular lumen. Distal RTA can be associated with either hyperka- lemia or hypokalemia. Disturbed function of the H +- ATPase has been implicated in the former and of H+-K +- ATPase in the latter [10].

There are three types of H+-ATPases, differentiated by location, structure, and function. The F-type is found in the mitochondria, where it is responsible for the generation of the mitochondrial membrane potential. In plasma mem- branes of almost all eukaryotic cells, as well as in synaptic vesicles, chromaffin granules, endosomes, lysosomes, and clathrin-coated vesicles, there is a vacuolar (V)-type H +- ATPase. This is responsible for the acidification of the extracellular space (urine in the case of renal epithelial cells) or of the organelles required for optimum activity of certain enzymes, i. e., lysosomal hydrolases [11]. Another type is represented by a member of the P-type ATPases, the electroneutral H+-K+-ATPase. This ATPase is present in the gastric mucosa, but is also found in the kidney and colon, as well as in the plasma membrane of some fungi.

The nature of the V-type H+-ATPase was elucidated from studies on isolated membrane vesicles. Membranes isolated from the turtle bladder and the mammalian kidney wer found to exhibit an electrogenic ATP-initiated acid- ification, different from that due to the F- and P-type ATPases [11]. With the help of a multiple-step chromato- graphic procedure, vacuolar bovine kidney H+-ATPase was isolated and was found to have the same structure as the enzyme found in yeast, plant, and coated vesicles [12]. The vacuolar H+-ATPase contains a transmembrane domain (V0) and a catalytic intracytoplasmic domain (V1). Each domain is made up of multiple subunits. The V1 domain contains three subunits A and three subunits B, each with ATP hydrolytic capabilities, and the regulatory accessory subunits C, D, and E [13]. Several investigators have de- tected the existence of isoforms of subunits A [14] and B [15], corresponding to different tissue distribution. The genes encoding all these subunits (A-E) were sequenced and cloned from mammalian, fungal, and plant cells [16]. The gene encoding the subunit E is located very close to the centromere of human chromosome 22, a disease-rich area [17].

The hydrophobicity of the V0 domain made it more difficult to elucidate its structure. It is known, so far, that this domain is comprised of a hexamer of 16 kilodaltons (kDa) proteolipid subunits, along with additional subunits

of 21 and 110 kDa. The latter seems to play an important role in the proper functioning and assembly of the H + ATPase [11 ].

There is a strong homology between the V-type and F-type of H§ with respect to their catalytic sub- unit. A cysteine residue implicated in the binding of ATR located inside the A subunit, is sensitive to oxidation, and its modification by ligands (i. e., N-ethylmaleimide) ren- ders the enzyme inactive. On the other hand, there is no homology between the subunits of the transmembrane do- mains of these two types of H+-ATPases [11].

The H+-K+-ATPase, like all members of the P-family, is a heterodimer, with a- and [~-subunits. The o~-subunit (100 kDa) is responsible for the ATP binding, phosphor- ylation, and interaction with the inhibitors, whereas the [3-subunit (a 60 kDa glycoprotein) appears to play a role in the conformational changes induced by K § and is essential for the functional activity of the enzyme. Isoforms for both o~- and ~-subunits were found in the kidney, stomach, and colon [18, 19]. Fluorescent in situ hybridization was used for the mapping of the gene encoding the ot-subunit of the gastric H+-K+-ATPase to the human chromosome 19q13.1 [20] and that of the [3-subunit to the chromosome 13q34 [21].

In the pages that follow we will summarize key studies pertinent to the structure, function, regulation, and clinical relevance of the V-type H+-ATPase and the P-type H+-K +- ATPase.

Enzyme structure

The differences in structure of the 56-kDa subunit of the vacuolar H+-ATPase led to the speculation that differential expression in various cell types is the result of isoforms of this subunit. The authors used the polymerase chain reac- tion (PCR), DNA sequencing, immunoblotting, and im- munocytochemistry to assess the amplification of isoforms of the 56-kDa subunit in the rat kidney. They obtained evidence of selective amplification of the "kidney" isoform in the ICs, those cells involved in the regulation of H+/ HCO3- transport. The methods used in this study are useful not only for assessing differential expression of enzyme isoforms, but also for investigating cell differentiation in the kidney.

Enzyme localization

The purpose of the study was to determine the location of the H+-ATPase in the rabbit CCD. This was done by elec- tron microscopy and immunogold cytochemistry, using a

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primary polyclonal antibody raised in rabbit against the 70- kDa subunit of the bovine brain isoform and a goat anti- rabbit IgG as a secondary antibody. Transmission electron microscopy of the collecting duct revealed three patterns of distribution of label for H+-ATPase in the ICs. In one type of ICs the label was detected on the apical membrane and in the cytoplasmic vesicles located in the apical domain. In a second type of ICs, more common than the previous one, the label was present on the basolateral membrane and in the cytoplasmic basolateral vesicles. Much more often though, there was a cell type with labeling of the in- tracytoplasmic vesicles, with no real commitment to one membrane domain or another. In the connecting tubule (CNT, shorter in rabbits than in rats) and the initial col- lecting duct (ICT), there was increased labeling of either the apical or basolateral plasma membrane when compared with the CCD. Because the most common cell type had diffuse cytoplasmic distribution of the H+-ATPase, it is possible that, under control conditions, these cells are not involved in H+]HCO3 - transport and exhibit polarity of the transporters only during acidosis or alkalosis. The findings also suggest that the CNT and ICT may play an important role in acid-base regulation.

Functional studies have identified a K + absorptive flux in the CCD and OMCD obtained from K§ animals. Immunocytochemical techniques revealed labeling of the ICs of the CCD and OMCD for H+-K+-ATPase, using an- tibodies raised against the gastric form of this enzyme. Molecular techniques served to determine the sequence of the gastric, colonic, and urinary bladder enzymes, but did not yield conclusive results regarding the expression of isoforms. The authors of the present study used PCR-based cloning, DNA sequence analysis, reverse transcription, PCR of RNA from nephron segments obtained by micro- dissection, and in situ hybridization to elucidate the cellular expression and localization of the H+-K+-ATPase. The re- sults demonstrate full homology between the c~-subunit of the gastric and kidney H§ Sequence differences were found between colon, toad bladder, and kidney iso- forms, consistent with pharmacological differences (sensi- tivity to inhibitors) described by other investigators. The cr mRNA was localized to ICs of the CNT, CCD, MCD, and inner medullary collecting duct (papillary duct), as well as to the PCs of CNT and CCD. The role of H+-K +- ATPase in the renal pelvic epithelium remains unclear, but the enzyme may affect the solubility of urinary salts, and therefore be implicated in urolithiasis.

Enzyme function

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Studies by Doucet and Marsy [6] revealed K+-dependent H§ activity in the late distal convoluted tubule (DCT), CCD, and MCD of rats fed a K+-deficient diet. Similar results were reported in rabbits [7]. The authors of this study performed micropuncture and in vivo micro- perfusion experiments in rats fed a normal or a low-K + diet. The early and late distal tubule segments were perfused with control solution, followed by solutions containing specific inhibitors of H § transport. The transepithelial vol- tage and luminal pH were determined before and after exposure to inhibitors. It was found that the early portion of the DCT possesses a Na+/I-I + exchanger, sensitive to 5-(N,N-hexamethylene)amiloride (HMA), an amiloride analogue. Bafilomycin, a specific inhibitor of the vacuolar H+-ATPase, reduced the HCO3- reabsorption (H + secretion) only at high concentrations (2x10 -6 M). This suggests that the Na+/H + exchanger plays the main role and that the H +- ATPase plays a minor role in early distal tubule acidifica- tion in the rat. On the other hand, the late portion of the distal tubule, which contains PCs and ICs, exhibited bail- lomycin-sensitive (i. e., H+-ATPase), Sch 28080-sensitive (i. e., H+-K+-ATPase), and HMA-sensitive (i. e., Na+/H + exchanger) pathways of acidification. The inhibition of H § ATPase by bafilomycin did not increase the lumen-negative transepithelial voltage (Vt), as would have been expected, becanse the contribution of H + secretion to the generation of this Vt is minor. This could also be explained by the presence of an electroneutral exchange of H § for another cation. By perfusing the late segment with Sch 28080- containing solution, the authors found that the Vt was sig- nificantly decreased in K+-depleted animals, suggesting the presence of an H+-K§ K § and H + trans- port. This study does not allow us, however, to ascertain the relative role of each cell type in acid-base homeostasis. This was investigated in the study that follows.

Enzyme regulation

The effects of respiratory acidosis and respiratory alkalosis (both acute and chronic) on the kidney H +- and H+-K +- ATPase activities were studied in rats. Because of previous suggestion that aldosterone may affect H + secretion [22], the animals were divided into one group with adrenals in- tact and a second group which underwent adrenalectomy, followed by dexamethasone and aldosterone replacement for 1 week. After induction of respiratory acidosis or al- kalosis, the nephron segments [PCT, medullary thick as- cending limb (mTAL), CCD, and MCD] were micro- dissected from collagenase-treated kidneys, and the enzy- matic activity was determined radiochemically using 32p-ATR Six hours (acute) of respiratory alkalosis led to downregulation of both enzymes, whereas acute respiratory acidosis did not affect their activities. Chronic ( > 6 h) hypercapnia (PCO2 > 40 mmHg) led to activation of H +- ATPase in all the segments studied, but with the greatest increments in the collecting duct segments. H+-K+-ATPase activity was doubled in CCD and MCD, with no change in the PCT and MTAL enzyme activity compared with very low control values. The effects were independent of the presence or absence of aldosterone. It is therefore possible that humoral or cellular factors other than aldosterone are responsible for these responses.

The authors used excitation ratio fluorometry of the in- tracellular pH (pHi) indicator, BCECF, to measure the pHi recovery of ICs of CCD from an acute acid load. Individual ICs were differentiated from the PCs, in split open tubules, by using the Nomarski differential interference contrast optics. ICs, mitochondria-rich cells, appear in various shapes and with darker cytoplasm, whereas PCs appear polygonal in shape and lighter. Recovery of ICs from an acute acid load involved ouabain-insensitive, Sch-in- hibitable (H+-K§ and Na+-dependent (presumably Na§ § exchanger) mechanisms. This study provides evi- dence for the presence of the H+-K+-ATPase in ICs of the adult rabbit CCD, which may function in control condi- tions. The polarity of this transporter remains to be deter- mined.

A multitude of factors are involved in the regulation of the H § transporting enzymes in different cell types. A cytosolic activator [23] and a cytosolic inhibitor [24] are apparently regulating the H+-ATPase activity. K + depletion and acid- base abnormalities induce changes in the activity of the H +- K+-ATPase. Obstructive uropathy, diuretics or lithium have a different effect on each of these enzymes.

Nitric oxide (NO) is produced from L-arginine by a family of enzymes, NO synthases (NOS). Studies on mouse peritoneal macrophages suggest that the vacuolar H +- ATPase, involved in the regulation of pHi, is disturbed by NO produced by an inducible form of NOS. In the kidney, NO participates in the regulation of renal vascular resis- tance and in tubuloglomerular feedback. Cells of juxta- glomerular apparatus possess an inducible form of NOS. LLC-PKt cells produce NO by a constitutive NOS, whereas proximal tubule cells and inner MCD cells in primary culture express a cytokine-induced form of NOS. Because CCD is a nephron segment involved in H+/HCO3 - trans- port, the authors assessed the role of NO in this process. Rat CCDs, obtained from collagenase-treated kidneys, were exposed to exogenous NO donors (nitroprusside and 3-morpholino-sydnonimine hydrochloride, SIN-l). The

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activity o f the H+-ATPase was measured by the fluoro- metric method (ATP hydrolysis coupled to oxidation of NADH). In addition, the authors used immunocytochemical techniques to evaluate the differential labeling of the tub- ular cells with antibodies against NOS. NO was found to inhibit the bafilomycin-sensitive H+-ATPase in the ICs of CCD. The mechanism(s) responsible for this finding is not known, but it could be that the NO is involved in the trafficking of the enzyme, since vacuolar H+-ATPase is targeted to specific membrane domains and inserted in the membrane to serve for H § extrusion in acidosis or en- docytosed from the plasma membrane in conditions of al- kalosis.

Clinical implications

Urinary tract obstruction (for 24 h) and chronic adminis- tration of amiloride or lithium cause distal RTA due to a diminution of the lumen negative potential; this, in turn, diminishes H + and K + secretion. The degree of acidosis differs in these conditions: while lithium administration is associated with a marked metabolic acidosis and normo- kalemia, chronic administration of amiloride or urinary tract obstruction are associated with mild degrees of aci- dosis and hyperkalemia. The authors studied the H +- and H+-K+-ATPase activities in tubules microdissected from adult rats, which were subjected to the aforementioned conditions. They found that H+-ATPase and H+-K+-ATPase are inhibited by lithium, leading to metabolic acidosis. But lithium also inhibited Na+-K+-ATPase, thus counteracting the hypokalemia that would have resulted from a lower K + absorption via H+-K+-ATPase. Amiloride administration, on the other hand, preferentially inhibited the H+-ATPase, without affecting the H+-K+-ATPase activity. Because of the diminished Na+ absorption, the Na+-K+-ATPase activity was low. This, together with a normal H+-K+-ATPase ac- tivity, resulted in hyperkalemia. Urinary tract obstruction had dissimilar effects on these enzymes. While Na+-K +- ATPase and H+-ATPase activities were inhibited in both CCD and MCD, the H+-K+-ATPase was stimulated in the CCD and inhibited in the MCD. Thus, the mechanism re- sponsible for metabolic acidosis in this case is the impaired H + secretion in the MCD. Hyperkalemia was due to the inhibition of the Na+-K+-ATPase and stimulation of H+-K +- ATPase activities in the CCD. While humans differ from rats in response to these conditions, this study offers a model for further investigation of the role played by these enzymes in acid-base and K + homeostasis.

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