Single-nephron studies: Implications for acid-base regulation

Kidney International, Vol. 38 (1990), pp. 744—761

NEPHROLOGY FORUM

Single-nephron studies: Implications for acid-base regulation Principal discussant: DAVID Z. LEVINE Ottawa General Hospital and Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada

PO2, 76 mm Hg; bicarbonate, 36 mM; pH, 7.48; pCO2, 41 mm Hg; PO2, 104 mm Hg; and bicarbonate, 31 mM. Measurement of urinary electrolytes revealed: sodium, 91 mM; potassium, 75 mM; and chloride, 3 mM.

Editors JORDAN J. COHEN JOHN T. HARRINGTON JEROME P. KASSIRER

Urine pH was 9.0 (by dipstick). The patient was admitted for further investigation and for therapy.

NIcoLAos E. MADIAS

Body weight and vital signs were carefully monitored, and quantitative urine collections were made every 6 hours. After 12 hours, the urinary potassium excretion fell dramatically while the urinary chloride con-

During the subsequent 48 hours, she received sodium chloride and potassium chloride, initially intravenously and subsequently orally.

Managing Editor

centration increased to 75 mM. By 48 hours, her body weight had

CHERYL J. ZUSMAN

increased by 3 kg, the acid-base disturbance had been corrected, and the serum potassium concentration had returned to normal. The patient eventually admitted to self-induced vomiting.

State University of New York at Stony Brook

and

Discussion

Tufts University School of Medicine

DR. DAvID Z. LEVINE (Professor and Head, Division of Nephrology, University of Ottawa and Ottawa General Hospital, Ottawa, Ontario, Canada): The loss of gastric fluid in this patient produced chronic metabolic alkalosis and its accompanying urinary potassium losses. The acid-base disturbance and the potassium and extracellular (ECF) volume deficits all were corrected by administration of sodium and potassium chloride. Even after more than 30 years of investigational effort, controversy exists as to the underlying mechanisms that account for the .naintenance and repair of this acid-base disturbance. Cogan and colleagues believe that a decrease in glomerular filtration rate exerts a major influence in sustaining the elevated plasma bicarbonate of chronic metabolic alkalosis [1—3]. Luke and Galla, drawing from their extensive experience in animal and

Case presentation A 24-year-old female nursing student was working in the hospital

emergency room when she fainted after a period of dizziness and weakness severe enough to make it difficult for her to reach a chair. She was unresponsive for a short time but had no seizure activity and was not incontinent. She was able to remember the episode. She had experienced three or four similar episodes in the preceding 8 to 10 months. She was told by her family doctor that her electrolytes

were "out-of-whack," but he did not investigate the problem further. She had been amenorrheic for the previous 10 months, but she stated that this was not unusual for her or her sisters. She had had a complete endocrine workup one and one-half years previously, which included a normal pituitary CAT scan. No endocrine abnormalities were detected. The patient denied taking birth control pills, other medications including diuretics, eating an unusual diet, or vomiting. She smoked one pack of cigarettes daily, drank little alcohol, and denied illicit drug use. Her father is healthy; her mother died at the age of 32 of liver carcinoma. On physical examination, the patient was pleasant, cooperative, and not in obvious distress. Her supine blood pressure was 110/80 mm Hg,

human studies, are convinced that chloride plays a unique intrarenal role [4—7]; Schwartz and colleagues proposed such a

role in the 1960s [8]. Others argue that ECF volume and sodium and potassium losses are the pivotal controlling factors [9].

To cast light on renal function in this and other chronic acid-base disorders, many studies of single nephrons have been

carried out, and these are the studies I will focus on in this

dropping to 98/70 mm Hg upon standing. The rest of the physical Forum. First, I will review various difficulties in the interpreexamination was unremarkable except for some mild hirsutism around her breasts and excess hair growth in the pubic region. Laboratory investigations revealed: plasma sodium, 137 mM; potassium, 2.5 mM; chloride, 85 mM; total carbon dioxide, 32 mM; blood

tation of studies on single nephron segments; these problems relate to species differences and experimental design. After briefly describing the segmental contribution to net acid excreurea, 4.6 mM; blood sugar, 7.3 mM; and serum osmolality, 267 tion, I will present the response of different segments in chronic mOsmlkg H20. Other laboratory investigations were normal, including acid-base disturbances. I also will describe our own work, normal plasma calcium and magnesium concentrations. Two sets of involving an in-vivo model of bidirectional bicarbonate flux in arterial blood gas evaluations disclosed: pH, 7.50; pCO2 46 mm Hg; the rat distal tubule, and I will consider new data on bicarbonate secretion and the effects of luminal chloride. Presentation of the Fon'm is made possible by grants from Pfizer, Incorporated; Merck Sharp & Dohme International; and Sandoz, Incorporated.

© 1990 by the International Society of Nephrology

Limitations of studies on nephron segments Species differences. The variable acid-base response among species to selective dietary potassium restriction aptly illus-

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Nephrology Forum: Single-nephron acid-base studies

745

trates the problem of interpreting animal data. Selective dietary potassium deprivation in the rat produces no acid-base distur-

bances after 10 to 12 days despite obvious loss of potassium stores from muscle and the development of lesions of potassium depletion nephropathy [101; more prolonged potassium deple-

Urine

tion does result in metabolic alkalosis. In the dog, selective depletion induces metabolic acidosis, presumably due to inhibition of aldosterone secretion [111. In humans, selective po-

pH

tassium depletion produces a mere 1.8 mM elevation of plasma bicarbonate concentration [121.

Most of our understanding of acid-base disturbances in humans is based on chronic balance studies in unanesthetized

dogs, yet almost no segmental acid-base data from canine nephrons are available. The bulk of single-nephron data derive from experiments in the rat and rabbit. The rat has been well studied with respect to free-flow micropuncture and wholeanimal experiments, whereas the rabbit, providing most of our in-vitro perfusion data, has rarely been studied with free-flow micropuncture or balance techniques. Indeed, rabbits normally

Body

wt g

excrete a net alkali load, whereas humans, dogs, and rats

normally excrete net acid. Thus, even if the effect of various manipulations on a specific nephron segment is similar in the rabbit and the rat, it is unclear whether the findings would be relevant to the physiology of the dog or human. Technical djfficulties and heterogeneity of response. The technical difficulties inherent in micropuncture studies have been reviewed [13]. In-vivo perfusion of distal tubules has provided important data, although it is difficult to collect and analyze small sample volumes from these short, narrow tubules. Even more difficult is the sequential perfusion of two solutions in the same tubule. Repeated puncturing may alter the transepithelial potential difference or introduce other unrecognized effects. Furthermore, if different tubules are compared, histologic variability among tubules can make it difficult to draw

conclusions. The number of distal tubule intercalated cells as one approaches the beginning of the cortical collecting tubule [14] almost certainly varies from one tubule to another, and this variability might underlie the observed scatter in bicarbonate transport rates. With respect to heterogeneity of segmental

response, the in-vivo perfusion data of Lucci et al show transport rates in control distal tubules that vary from a secretory rate of 70 pmol . min' . mm 'to a reabsorptive rate of approximately the same magnitude [15]. We observed a similar variation in direction and magnitude of net distal tubule bicarbonate transport in our laboratory [16]. Data from cortical collecting tubules perfused in vitro from remnant kidneys also can show striking scatter even when two tubules from the same kidney are compared [17]. In the normal rabbit, control values

for bicarbonate reabsorption in cortical collecting tubules (CCTs) can vary from more than +10 pmol. min . mm to —7 pmol min mm' [18], the range of the strong responses seen with deliberate acid and alkali loading. Atkins and Burg showed that rat CCTs secrete bicarbonate in response to alkali loading, but that this secretion is attenuated markedly after one or two hours of in-vitro perfusion [19]. This elapsed time can represent an important experimental variable. Such a change in

Fig. 1. Effects of normal feeding and overnight fasting in SpragueDawley rats. Body weight and urine pH fall as a result of overnight fasting.

of attention. The usual perfusion rate for cortical collecting tubules is approximately 1 nl/min. This rate makes it possible to demonstrate significant effects on bicarbonate reabsorption and secretion, but it is much lower than the normal flow rate passing

through these segments [20]. Accordingly, if the collecting ducts alter their bicarbonate transport rates in response to flow, the data currently available would be quantitatively misleading.

Indeed, Lombard et al showed that when perfusion rates of approximately 5 nI/mm were used, effects of acid or alkali loading could not be discerned in the rabbit CCT [21]. Feeding and diet. One hundred forty years ago, an effect of feeding on urinary acid-base status was reported in some detail by Henry Bence Jones [22]. Bence Jones stated that:

In examining quantitatively the variations at different hours of the day, it was immediately found that there was no fixed degree of acidity of the urine; that the acidity did not vary with the specific gravity, but

that there was a never-ceasing change, an ebb and flow, in the acid reaction of the urine which was quite independent of the specific gravity. The degree of acidity of the urine was found generally to be greatest a short time before food was taken; and after food the acidity was diminished, until about

three hours after breakfast, and four or five or six hours after dinner, when it reached the minimum points; after which it again rose and attained its height previous to food being again taken.

In rat micropuncture experiments, some laboratories rou-

transport rate is not seen in reabsorbing CCTs from rats tinely fast their animals prior to the experiment; others allow gavaged with ammonium chloride or in outer medullary collecting ducts. The flow rate in perfused rabbit tubules in vitro also is worthy

the animals access to food. Starvation and ammonium chloride

loading also have been used interchangeably [23], as have feeding and alkali loading in in-vitro microperfusion experi-

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Nephrology Forum: Single-nephron acid-base studies

Table 1. Examples of responses of nephron segments to chronic acid-base disturbances Chronic acid-base Acid-base status at Segment response, Nephron segment! Sampling stimulus comments Reference species technique experiment Chronic metabolic Proximal/rat In-vivo Metabolic Levine and Nash. JtCO2 decreased, NH4CI loading acidosis even with AJP 255:380-384, acidosis, systemic micropuncture correction of 1973 (81) HC03 loading acidosis Proximal/rat In-vivo Metabolic Cogan et at. id JtCO2 decreased NH4CI loading acidosis 64:1168-1180, micropuncture

Disturbance

1979 (82)

Proximal/rat

In-vivo HCI loading microperfusion

Proximal/rat

In-vivo Renal ablation micropuncture

Metabolic Santella et at. AJP JtCO2 increased 257:F35-42, 1989 acidosis, systemic in early proximal tubule with high (83) HC03 loading filtered load Metabolic Kunau et al. AJP JtCO2 increased for late proximal 249:F62-68, 1985 acidosis, increased (84) segment perfused HC03 Metabolic Buerkert et al. JCI Enhanced NH4 acidosis addition 71: 1661-1675,

Proximal/rat

In-vivo NH4CI loading micropuncture

Metabolic acidosis

Enhanced NH4

Buerkert et at. AJP

In-vivo NH4CI loading microperfusion

Metabolic acidosis

Increased JtCO2

244:F442-454, 1983 (35) Levine. JCI 75:588595, 1985 (16)

In-vivo NH4CI loading microperfusion

Metabolic acidosis

In-vivo NH4CI loading microperfusion

Metabolic acidosis

Early proximal/rat In-vivo NH4CI loading micropuncture

1983 (77)

Distal/rat Distal/rat Distal/rat

secretion

despite Na secretion JtCO2 increased

Lucci et at. AJP 243:F335-F431, 1982 (15)

JtCO2 increased with zero Na perfusate and iv restriction

Vandorpe and Levine. Clin & Invest Med

JtCO2 unchanged

Hamm et at. AJP

12:224-229, 1989 (16)

CCT and MCD/rabbit

In-vitro Renal ablation microperfusion

Normal bath

CCT/rabbit

In-vitro NH4CI loading microperfusion

Normal bath

In-vitro NH4CI loading microperfusion

Normal bath

MCD/rat

In-vitro NH4CI loading microperfusion

Normal bath

JtCO2 increased

IMCD/rat

In-vivo base tip Renal ablation micropuncture

AJP 249:F485489, 1985 (19) Atkins & Burg. AJP 249:F485489, 1985 (19)

Metabolic acidosis

Decreased NH4

Buerkert et a!. JCI

In-vivo microNH4CI loading catheterization

Metabolic acidosis

In-vivo base-tip NH4C1 loading micropuncture

Metabolic acidosis

In-vivo Remote NH4CI microperfusion loading

Metabolic alkalosis

In-vivo papillary Remote NH4CI microloading catheterization In-vivo Chronic micropuncture hypercapnia

Metabolic alkalosis

In-vitro DOCA microperfusion

Lithium in bath

256:F680-687, 1989 (17)

JtCO2 increased

McKinney & Burg. JCI 60:766-768, 1977 (44)

CCT/rat

IMCD/rat IMCD/rat

Rebound alkalosis Distal/rat

IMCD/rat Chronic hypercapnia

Proximal/rat

Distal RTA

CCT/rabbit

JtCO2 increased, stable with time

addition to tubular fluid Net acid secretion increased

Atkins & Burg.

71:1661-1675, 1983 (77)

Bengele et a!. AJP 250:F690-694, 1986 (87)

Normocapnia

Interstitial-toGood et a!. AJP lumen NH3 252:F491-500, gradient increased 1987 (65) JtCO2 increased Vandorpe & Levine. Am Soc. despite alkalemia Nephrol. 1989 (abstr) (90) Net acid secretion Bengele et a!. AJP normal despite 252:F7!2-7!6, alkalemia 1987 (89) JtCO2 increased Cogan. id 74:1942-1947, 1985 (91)

JtCO2 and voltage decreased

Laski & Kurtzman. JCI 72:2050-2059, 1983 (72)

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Nephrology Forum: Single-nephron acid-base studies

Table 1. Continued Disturbance

Nephron segment! species CCT/rabbit

Chronic acid-base stimulus

Sampling technique In-vitro Ureteral ligation microperfusion

Acid-base status at Segment response, Reference comments experiment Normal bath Laski & JtCO2 normal Kurtzman. Mm

Electrolyte Metab MCD/rabbit

In-vitro Ureteral ligation microperfusion

Normal bath

15: 195-200, 1989 (71)

JtCO2 reduced

Laski and Kurtzman. Mm

Electrolyte Metab 15:195-200, 1989 (71)

IMCD/rat

In-vivo base tip micropuncture

Chronic metabolic Late proximal/rat In-vivo alkalosis microperfusion

Proximal/rat

Proximallrat

Distal/rat Distal/rat Distal/rat

In-vivo micropuncture In-vivo micropuncture In-vivo micropuncture In-vivo microperfusion In-vivo micropuncture

Ureteral ligation, pCO2 and Urinary acidification defect disequilibrium pH lithium, amiloride, reduced in amphotericin B lithium, amiloride, and obstruction but not with amphotericin B Metabolic JtCO2 reduced DOCA, Na2SO4, low K, no Cl per unit load alkalosis, HC03 load changed Metabolic JtCO2 increased Furosemide, HCO3, no Cl alkalosis, SNGFR despite systemic alkalemia, changed hypertrophy Metabolic JtCO2 increased Furosemide, HC03, no Cl alkalosis, SNGFR despite systemic alkalemia changed Metabolic K depletion, JtCO2 increased alkalosis despite systemic HC03 loading alkalemia DOCA + NaHCO3 Metabolic tCO2 secretion, alkalosis arrested by zero chloride Metabolic JtCO2 increased Furosemide, HC03, no Cl alkalosis, SNGFR despite systemic alkalemia changed Normal bath tCO2 secretion NaHCO3 loading

CCT/rabbit

In-vitro microperfusion

CCT/rat

In-vitro NaHCO3 loading microperfusion

Normal bath

In-vivo papillary Chronic HC03 microdrinking catheterization

Metabolic alkalosis

DuBose and Caflisch. JCI 75:1116-1123, 1985 (70)

Liu & Cogan. AJP 253:F89-94, 1987 (94)

Maddox & Gennari. JCI

77:709-716, 1986 (96)

Wesson. JCI 84:1460-1469, 1989 (103)

Capasso et al. JCI 80:409-414, 1987 (53)

Levine et al. JCI 85:1793-1798, 1990

Wesson. JCI 84: 1460-1469, 1989 (103)

McKinney & Burg. JCI 60:766-768, 1977 (44)

IMCD/rat

tCO2 secretion, time dependent

Atkins & Burg. AJP

No net acid secretion

Bengele et al. AJP

249:F485-489, 1985 (19) 255:F307-312, 1988 (99)

Experimental models of acid-base disturbances. Many studments in rabbit cortical collecting ducts [24]. In the case of rats, we believe that feeding animals prior to microperfusion exper- ies involve appropriate models of acid-base disturbances such iments produces changes that can importantly alter the direc- as acute and chronic respiratory acidosis and alkalosis, acute tion of distal tubule net bicarbonate movement. Because gavage metabolic acidosis, and chronic metabolic acidosis due to renal with acid elicits predictable changes in plasma bicarbonate tissue loss or acid ingestion. These models, usually in the rat or concentration that have their own time profile, the timing of the the dog, have provided important segmental nephron data experiment after acid feeding can importantly influence the relevant to chronic acid-base disturbances (Table 1). However, serious problems are encountered in studies of acute and experimental result [25]. We found that fasting a rat prior to the acute micropuncture chronic metabolic alkalosis and in studies of potassium depleexperiment results in a predictable fall in urine pH, a difference tion because of a variety of confounding variables. The optimal in plasma bicarbonate concentration of 5 mM when compared way to study potassium depletion is to produce the disturbance with the fed state, and a weight loss of approximately 8% to by dietary restriction while pair feeding and pair watering the 10% of the body weight (Fig. 1) [26]. Madias and Zelman have animals [28]. Similarly, with respect to chronic metabolic commented on the hazards of drawing conclusions from pre- alkalosis, if a steroid model is chosen, there should not be prandial acid-base measurements in acid feeding experiments in confounding influences such as the use of diuretics and potassium restriction. With respect to chloride-depletion metabolic the dog [27].

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Nephrology Forum: Single-nephron acid-base studies

alkalosis, there are difficulties because of nephrocalcinosis [29, 301. In producing experimental metabolic alkalosis, the simultaneous use of steroids, bicarbonate loading, or nonreabsorbable anion administration with or without diuretics takes one further and further from an uncomplicated simple disorder such

[34], Buerkert et al [35], and Graber and colleagues [36] have measured titratable acidity, ammonium, and bicarbonate in different segments of the nephron (Table 1). Because most phosphate is titrated by the end of the proximal tubule, titratable acidity in the final urine represents little of distal nephron

as gastric alkalosis. Furthest removed from such a clinical

H secretion [37]. The most important component of net acid excretion, however, the generation and addition of ammonium to the urine, chronic acid-base disturbances. Indeed, gastric drainage is depends on both proximal and distal nephron sites. This prodisorder is the use of acute sodium bicarbonate loading, a model that seems to have little relation to what occurs in nature or to

cess has been reviewed by Knepper et al [38]. In terms of our main question—what are the intrarenal correlates of chronic Of course, the pattern of secretion of acids or bases in adjustments to acid-base disturbances—nothing is more critical different nephron segments is relevant to understanding adjust- than ammonium handling by the kidney, because it represents ments to acid-base disturbances if such disturbances are gener- the greatest portion of changes in net acid excretion. Briefly, ated or compensated for by the kidney. In addition, data on the virtually all ammonium appearing in the urine is made in physiology of tubular segments provide a complete picture only proximal tubular cells. Proximal tubular cell ammonium is if extrarenal factors or changes in filtration have no influence on secreted into proximal tubular fluid by an active transport final acid-base configuration. process, and luminal ammonium is actively reabsorbed by the Let us assume that a given nephron segment—for example, thick ascending limb in the loop of Henle. After being concenthe proximal tubule—alters its rate of bicarbonate reabsorption trated in the medullary interstitium, ammonia diffuses into the in a given acid-base disturbance. What information is needed to collecting duct lumen. With secreted protons, this ammonia document this change? Clearly it is not sufficient to measure reconstitutes the ammonium that appears in the final urine [35, only the concentration of bicarbonate at some site along the 38, 39]. It is important to note that the role for ammonia proximal tubule, because one needs to know not only how diffusion is not that originally envisioned by Pitts in the proximuch bicarbonate has been delivered, but also how much mal tubule, and that the generation of new bicarbonate is not bicarbonate might have left with water reabsorption. To assess the consequence of trapping of protons by ammonia diffusing these variables, one needs a measurement of the GFR and the from proximal tubular cells into the lumen [38]. Rather, bicarfiltered bicarbonate load of that single nephron, as well as a bonate is generated by the metabolism of oxoglutarate as quantitative sampling of the tubular fluid with measurement of glutamine is metabolized to ammonium. Only by ensuring that inulin concentrations (a marker of water reabsorption) and ammonium reaches the urine is the net gain of bicarbonate bicarbonate concentration [32]. This series of measurements sustained because recirculation of ammonium to the liver would provides an assessment of absolute bicarbonate reabsorption release a proton and thereby result in no net loss of acid [37]. under free-flow conditions and enables one to determine The mechanisms of bicarbonate reabsorption and secretion in changes in bicarbonate reabsorption expressed as pmoL'min. mm different nephron segments also have been reviewed [40, 41], feasible in the rat [311; it produces typical metabolic alkalosis, it is clinically relevant, and it avoids confounding influences.

and I will only summarize them here. With respect to the Accordingly, if we want to quantify the contribution of proximal tubule, a carbonic-anhydrase-dependent Na/H an-

of tubular length [33].

certain tubular segments in chronic acid-base adjustments, the data we need are those that correspond to in-vivo free-flow conditions. Above all, we need to know what the tubule does in the chronic steady state, not what it might do with different kinds of perfusates or what its maximum transport capacity is, however critical such information might be in other contexts.

tiporter plays a prominent role in retrieving more than 90% of filtered bicarbonate. The thick ascending limb actively reab-

sorbs ammonium and bicarbonate in the rat by means of a Na:K:2C1 antiporter on which ammonium can substitute for potassium [41]. Bicarbonate is reabsorbed by a Na/H exchanger [42]. As for distal tubule bicarbonate reabsorption, it is believed

We require measurements of transport rates of bicarbonate that the type-A intercalated cell, possessing an apical proton when the bicarbonate load and tubular flow rate (or perfusion ATPase, is the major determinant of bicarbonate retrieval. rates) are unaltered by the experimental procedure. The studies There is also evidence for bicarbonate secretion that occurs via described in Table 1 should be critically reviewed while taking type-B intercalated cells with an apical chloride/bicarbonate into consideration the "normal" in-vivo segmental bicarbonate exchanger. The cortical collecting tubule both secretes and load. In chronic ammonium chloride acidosis in the rat, for reabsorbs bicarbonate, presumably as a function of the same example, late proximal bicarbonate delivery is only a small two types of intercalated cells. The bicarbonate reabsorptive fraction of normal, so that the rate of bicarbonate reabsorption rate is thought to be modulated by the sodium current, which by the proximal tubule is less than normal unless the load is alters the rate of proton secretion. The outer medullary collectraised experimentally. Accordingly, if the bicarbonate load is ing tubule, which has no sodium current, secretes protons raised, more reabsorption would ensue but the resulting level of electrogenically, causing a lumen-positive transepithelial potenreabsorption would not reflect the situation during chronic tial difference in association with brisk bicarbonate retrieval. Finally, the cells of the inner medullary collecting duct are not steady-state conditions. yet well understood, although cell culture studies look promisAcidification mechanisms in different nephron segments ing. In culture, differentiated inner medullary collecting duct Bicarbonate regeneration and proton secretion. Bicarbonate cells can promote sodium-independent hydrogen ion secretion

reabsorption is only one of the phenomena responsible for and chloride-bicarbonate exchange, and can show carbonic maintaining normal acid-base balance. Karlmark and Danielson

anhydrase activity [43].

Nephrology Forum: Single-nephron acid-base studies

749

Bicarbonate secretion. In 1977 McKinney and Burg made the demonstrated bicarbonate reabsorption during chronic metastriking observation that CCTs harvested from rabbits fed bolic acidosis [15, 16, 52]. Our microperfusion data in normal sodium bicarbonate secrete bicarbonate [44]. Prior to this rats [26] contrast sharply with free-flow data showing bicarbonobservation, abundant evidence from turtle bladder studies ate reabsorption at normal flow [53] and are consistent with the indicated the existence of bicarbonate-secreting mitochondrial- traditional view that the distal tubule participates in the prorich cells. Bicarbonate secretion in the turtle bladder is electro- gressive reclamation of filtered bicarbonate as tubular fluid neutral, active, blocked by acetazolamide and disulfonic stil- traverses succeeding nephron segments. We believe that whether or not rats are fed before the benes, dependent on luminal chloride, and promoted by an

apically situated chloride/bicarbonate anion exchanger [45]. In rats, in-vitro perfused CCTs secrete bicarbonate [19, 46], and in both rabbit and rat CCTs, bicarbonate secretion is thought to be mediated by B-type intercalated cells with the same characteristics as those of the turtle bladder. To assess the significance of bicarbonate secretion for wholeanimal acid-base homeostasis, we must review evidence for its

experiment critically influences the bicarbonate reabsorptive

response of the microperfused distal tubule (Fig. 5). As I

already noted, fasted rats differ from fed rats with respect to urine and blood acid-base status. In fed rats, at high chloride loads, distal tubules secrete bicarbonate in vivo, whereas at normal flow (8 nI/mm), bicarbonate reabsorption cannot be demonstrated. Distal tubules from fasted rats reabsorb bicarexistence in vivo. Of course, CCTs are not accessible to bonate at normal flow and tend to augment reabsorption when micropuncture or microcatheterization techniques, so the cor- the bicarbonate load is increased [26]. Accordingly, shortly tical collecting tubule data that I noted cannot be verified in after night-time feeding, a different "mode" is established vivo. The distal portion of the rat superficial distal tubule has whereby the kidneys render the urine alkaline and the plasma cells in common with the CCT and therefore provides a way of bicarbonate concentration rises. Recently, however, Chan et al assessing whether B intercalated cell bicarbonate secretion reported distal tubule bicarbonate reabsorption in microperfuoccurs in the intact animal. Both in Wistar and in Sprague- sion experiments in fed rats [54]. Differences in response to Dawley rats, intercalated cells are present in the connecting feeding might reflect (1) the quantity and acid content of food segment and in the initial collecting tubule portions of surface eaten, (2) the interval between feeding and sampling, and (3) the distal tubules [47, 48]. The initial collecting tubule is a segment number of B-intercalated cells in perfused distal tubules. How does feeding stimulate bicarbonate secretion? When we that merges with the CCT. On the surface of the kidney are long distal tubules—about 1.5 mm in length—that branch or are gavaged fasted rats with alkali and ensured that the urine was about to branch, and such tubules can be perfused when they alkaline, bicarbonate reabsorption did not occur [261. This finding suggests that alkali loading might result in the release of have two surface loops [49]. We have perfused rat distal tubules in vivo and, because both a substance, systemically or in intercalated cells, that inhibits A- and B-type intercalated cells are present in such surface bicarbonate reabsorption and/or stimulates bicarbonate secre-

segments (Figs. 2 and 3), we have had the opportunity to tion. Vasoactive intestinal peptide or cyclic AMP might be good confirm, in vivo, observations derived from in-vitro perfused candidates for this substance [55, 56]. Vasoactive intestinal peptide stimulates turtle bladder bicarbonate secretion and is present in higher concentrations with the alkaline tide in the turtle in the postprandial state. Cyclic AMP also stimulates bicarbonate secretion in the turtle bladder and in the CCT [56]. Other hormones also might be involved [571. In our studies, we did not control ADH levels, nor did we assess the possibility that altered aldosterone concentrations in response to gavage modulated net bicarbonate reabsorption. Specifically, if gavage with alkali suppresses aldosterone, then a bicarbonate reabsorptive flux might be suppressed. However, because sham gavage with sodium chloride, which might be expected to have the same effect on aldosterone, did not impair bicarbonate reabsorption in fasted rats, we believe that the suppression of bicarbonate reabsorption is due to the bicarbonate rather than to the sodium loading. Chloride modulation of bidirectional bicarbonate flux. Studies on cortical collecting tubules and medullary collecting ducts have implicated chloride as an important modulator of bicarby the distal tubule. The authors also provided additional bonate transport. The brisk bicarbonate secretion in DOCAfree-flow data indicating that glucagon stimulates bicarbonate treated rabbits first demonstrated by Knepper et al [58] was secretion, perhaps via a cyclic AMP-dependent mechanism shown in a subsequent study by Garcia-Austt and colleagues [51]. It is now accepted that the distal tubule has "two parallel [591 to depend on the presence of luminal chloride. Star et al defined the process by which chloride modulates cortical collectand opposing systems for acid-base transport" [41]. In our microperfusion experiments at normal flow (8 nI/mm) ing tubule bicarbonate secretion as involving a strict 1:1 bicarbonwe could not demonstrate bicarbonate reabsorption in distal ate/chloride exchanger that operates independently of transepitubules from normal rats [261. This result was consistent with a thelial voltage or sodium or potassium fluxes [60]. With respect previous report by Lucci et al [15]. Both laboratories easily to bicarbonate reabsorption, Laski and coworkers demoncortical collecting tubules. In 1986, we reported that bicarbonate secretion could be consistently elicited in rat distal tubules in vivo when perfusion was undertaken at high flow rates [50]. Thus, under circumstances of high flow (25 nI/mm) and high loads of bicarbonate, chloride, and sodium, we observed brisk secretion into tubular fluid with normal bicarbonate concentrations. This secretion persisted even when tubular fluid bicarbonate concentration was made equal to that of plasma. Further, acetazolamide blocked this response (Fig. 4). Accordingly, bicarbonate secretion against a concentration gradient into a lumen that has a negative transepithelial voltage (YTE), with blockade by acetazolamide, is consistent with an active transport process. In 1987, Bichara and coworkers reported the first in-vivo free-flow demonstration of bicarbonate secretion during hypotonic volume expansion in the rat [51]. Their data, derived from collections made at early and late distal tubular sites, suggested that normal AVP titers might mask net secretion of bicarbonate

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Nephrology Forum: Single-nephron acid-base studies

•1 \

Fig. 2. Electron micrograph of intercalated cells of rat superficial distal tubules from the University of Ottawa Sprague-Dawley colony. A- and B-type intercalated cells are present. (See Refs. 14 and 47 for classic descriptions of rat intercalated cells. Courtesy of Dr. R.A. Narbaitz and V. KapaL)

strated increased bicarbonate reabsorption by perfusion with unilateral bicarbonate secretory flux. The results of perfusion

with a 100 mM chloride solution can be explained by the increased transepithelial potential difference or a decreased stimulation of the unidirectional secretory flux and/or by reduclow luminal chloride concentrations and proposed that either an

apical chloride/bicarbonate exchange might be the basis for this augmented rate of proton secretion [61]. As for the medullary collecting duct, Stone et al suggested that a low luminal chloride concentration might augment medullary collecting duct acidifi-

cation by generating an inward chloride diffusion potential, which favors proton secretion [621.

We recently measured bicarbonate reabsorptive rates in bicarbonate-secreting and bicarbonate-reabsorbing rat distal tubules during chloride-containing and chloride-free perfusion in vivo [63]. Figure 6 shows that bicarbonate secretion associated with chloride-containing perfusates is abruptly arrested when chloride is replaced. Figure 7 shows that in bicarbonatereabsorbing tubules from rats fasted overnight, perfusion of a solution with zero chloride results in significantly higher bicar-

bonate reabsorptive rates at normal and high flow than does perfusion with a solution containing chloride. Most striking are the results of perfusion studies with a 100 mM chloride solution, which demonstrate that the net bicarbonate reabsorption seen with perfusions containing no chloride can be transformed into net secretion. These results are consistent with the view that in bicarbonate-secreting tubules, the absence of chloride inhibits chloride/bicarbonate exchange [45], thereby curtailing the unidirectional bicarbonate secretory flux. In the case of bicarbon-

ate-reabsorbing tubules, the lack of chloride in the perfusate possibly enhances bicarbonate reabsorption, either by augmenting a transepithelial potential difference, thereby favoring increased proton secretion, or by suppressing an opposing

tion of the normally negative transepithelial potential difference by high flow [64]. Whatever the mechanism of modulation of

both reabsorptive and secretory fluxes by luminal chloride, I believe these in-vivo data will provide an important link between the in-vitro studies already noted and the renal effects of chloride administration in chronic metabolic alkalosis. Relationship of hydrogen secretion and bicarbonate retrieval to ammonium handling. I have stressed that major adjustments in net acid secretion must involve altered ammonium handling, whereas the emphasis in this presentation thus far has been on bicarbonate retrieval. Reabsorption of filtered bicarbonate depends on proton secretion, a process seemingly unrelated to the ammoniagenesis that results in the generation of "new bicarbonate" [371, provided that ammonium finds its way into the urine. Thus, only excreted ammonium is associated with new alkali generation. So why is proton secretion important with respect to chronic adjustment to acid-base perturbations? After all, continuing acid excretion during chronic acid loading and transient generation of new bicarbonate in chronic hypercapnia depend not only on bicarbonate retrieval, but on increased ammonium excretion. The answer is that for collecting duct ammonium accumulation to proceed, a sufficiently acidified tubular fluid must be present so that ammonia can continue to diffuse into the lumen without generating a very high urine pH [65]. Thus, one link between almost complete absorption of filtered bicarbonate by proton secretion and efficient ammonium excretion is that both depend on effective proton secretion

751

Nephrology Forum: Single-nephron acid-base studies A

B

Perfusate:

mM + Diamox

[tCO2] = 31 mM Perfused Collected 80

42

[tCO2l 38

24 ni/mm'

mM

60

Plasma

40

E

E 20 0

0

E —20 0.

—40 —60

—80 7 nI/mm

24 nI/mm

7 nI/mm

24 nI/mm

Fig. 4. Effects on distal tubule bicarbonate flux (JtCO2) of perfusion with 31 mM tC02 with and without acetazolamide blockade. Insert shows collected [tCO2] rises above plasma LtCO2I. (See text and Ref.

50.)

Perfusate tCO2 = 28 mM

80 Fasted rats

60 40 lami

E

20

Fig. 3. Electron micrograph of intercalated cells of rat superficial distal tubules from the University of Ottawa Sprague-Dawley colony. A-type intercalated cells contain microplicae and subapical vesicles,

some coated with studs. B-type intercalated cells are more electron dense and have well developed pallisading structures in the basolateral region. (See Refs. 14 and 47 for classic descriptions of rat intercalated cells. Courtesy of Dr. R.A. Narbaitz and V. Kapal.)

K

0 E

8 nI/mm

—20 —40 -

in the distal nephron. Another potentially important bicarbonate-ammonium interaction is the inhibition of bicarbonate secretion in cortical collecting tubules by luminal ammonium [381.

It also is possible that stimulated antiporter activity in the

—60 -

rammn

Fig. 5. Effects of feeding and overnight fasting on distal tubule bicar-

proximal tubule is responsible for increased rates of both NaIH bonate fluxes (JtCO2) in vivo. Normally fed rats reabsorb little or no bicarbonate at normal flow and secrete bicarbonate at high flow. exchange and ammonium secretion [66]. Overnight fasted rats reabsorb bicarbonate at both normal and high flows. (See text and Ref. 26.)

Pathogenesis of RTA syndromes based on segmental defects Overview. In 1974, Halperin and colleagues proposed a new approach to the pathogenesis of distal renal tubular acidosis [67]. This hypothesis stimulated not only a renewed interest in medullary collecting ducts, or papillary collecting ducts—were intrarenal mechanisms associated with RTA, but a fresh way of involved in different types of distal RTA. It is of interest that looking at patients. Halperin et al drew on classic views these proposals have used whole-animal postulates to generate regarding the origin of the high urinary pCO2 in alkaline urine to investigations on single segments of the distal nephron, and that propose that the urinary pCO2 could be used as a measure of in-vitro data from these segments have been incorporated into hydrogen secretory capacity in the distal nephron. This ap- the design of clinical tests attempting to identify different RTA proach was extended by others who postulated that different subgroups. As I already noted, studies of acidification of luminal fluid in segments of the distal nephron—cortical collecting tubules,

Nephro!ogy Forum: Sin gle-nephron acid-base studies

752

B

A

80

25-

E E

0 E

-25

0 0 0 -,

55

60

E

40

-50

0S

-75

0

-100

-75

[CI] = 0 mM

0.

20

[CI] = 45 mM

-,

o Perfusate (CII, mM

Fig. 6. Effects of luminal chloride replacement on distal tubule bicarbonate secretion (-JtCO2) in fed (A) and DOCA-treated (B) rats. Replacement of perfusate chloride arrests the secretory flux. (See text and Ref. 63.

-20

[CI] = 100 mM 8

25 Perfusion rate, ni/mm

Fig. 7. Effects of increasing flow rate with different perfusate chloride

concentrations on distal tubule bicarbonate reabsorption (JtCO2) in

rabbit CCTs show that H secretion in vitro is related to fasted rats. (See text and Ref. 63.) sodium transport in that the lumen-negative potential generated by active sodium reabsorption favors the secretion of hydrogen and potassium ions [40]. In contrast, in outer medullary collect-

one of inadequate H + secretion rather than an inability to

ing ducts, H secretion is accompanied by Cl secretion and is sodium independent [40]. In this segment, the transepithelial potential difference is positive, likely as a result of electrogenic proton pumping. In both segments, mineralocorticoid stimulates H secretion, but only in the CCT is there a component

maintain a W concentration gradient. DuBose and Caflish have validated the use of the U-B pCO, and have studied papillary collecting duct function under conditions of lithium and amiloride administration, and with unilateral ureteral ligation [701. Of course, there can be no question that the urinary pCO2 eleva-

related to the sodium current. The CCT both secretes and tion I referred to is real; nor can one deny that the papillary reabsorbs bicarbonate, but the maximal rate of reabsorption is collecting tubules contribute to that elevation, even though much less than that in the medullary collecting duct. At low their function sometimes is impaired [701. However, the papildoses (l0— M), amiloride alters the lumen-negative potential lary collecting tubule does not have a transepithelial voltage and sodium conductance of the CCT. Using this information, that is significantly different from zero; in fact, the mechanism Batile and coworkers have proposed that sodium-dependent of proton secretion, or indeed the cells responsible for proton CCT acidification can be stimulated by furosemide and sodium secretion, are not yet well understood. Whatever effect amilosulfate and can be used to test whether this portion of the ride has in this segment, it is not likely related to abolition of a collecting duct is responsible for an acidification defect [681. sodium-generated negative VTE. The effect might be related to Halperin et al also have recognized the importance of the the presence of a Na/H exchanger; such an exchanger has been transport characteristics of distal nephron segments and have found in papillary collecting duct epithelia in cell culture [431. Is there any direct evidence that in distal RTA the segment stressed that ammonium excretion, in the end, must be defecinvolved with voltage-dependent proton secretion is functioning tive for significant systemic acidosis to persist [691. Halperin et al proposed that the urinary pCO2 could be a subnormally, as would be predicted from the approach by measure of distal nephron proton secretion when the bicarbon- BatlIe [68]? Laski and Kurtzman have directly assessed the ate concentration in the lumen of the distal nephron is adequate. possibility that in unilateral obstruction, it is the inhibition of The rationale for this hypothesis is that secreted protons react sodium reabsorption in the cortical collecting tubule that causes with bicarbonate in the lumen and generate carbonic acid. the acidification defect via the voltage-dependent pathway [71]. Because of the lack of carbonic anhydrase in the lumen of the Previously, they had shown that lithium impairs CCT voltage cortical and medullary collecting duct, the dehydration of [721. They studied rat CCTs at various time intervals after carbonic acid is delayed and occurs deep in the medulla, unilateral obstruction and compared bicarbonate reabsorption thereby raising the pCO2 of the urine. Most would now agree with tubules of the contralateral unobstructed kidney. They that the urine minus blood pCO2 differences (U-B pCO2) showed a significant decrease in medullary collecting duct provides a very useful measure of overall distal nephron hydro- bicarbonate reabsorption, a finding consistent with other data gen ion secretion without any distinction between the CCT, [73]. Surprisingly, however, the bicarbonate reabsorption of medullary collecting duct, and inner medullary collecting duct. cortical collecting tubules was not reduced; indeed, it was Accordingly, even when no hydrogen ion gradient is present, higher in some instances when compared with that in contrasome patients with RTA are unable to significantly raise their lateral tubules. The possibility that this result could be related urinary pCO2 above normal (U-B pCO2 = 0); the defect thus is to a species difference must be seriously considered, as is the

Nephrology Forum: Single-nephron acid-base studies

case "whenever isolated tubule data are applied to answer

753

ing ducts might stimulate acid secretion in the setting of

questions of physiology arising from studies of whole animals papillectomy. Usually, in acidosis, the papilla participates in other than rabbits" [59]. Laski and Kurtzman also noted that acid secretion to a greater degree than normal in acidifying the failure of sodium sulfate infusions in whole animals to tubular fluid as well as playing a vital role in ammonia trapping. increase acidification might reflect an already maximal rate of In summary, renal ablation studies indicate that hypertroacidification in the sodium-sensitive segment. phied proximal tubules have increased transporter activity, In summary, for experimental models of distal RTA syn- which retrieves the higher-than-normal bicarbonate load predromes, acidification defects in the papillary collecting duct are sented to surviving nephrons. The distal tubule also participates established. Medullary collecting duct proton secretion is sup- in this process, whereas collecting ducts might not. The inpressed in ureteral obstruction, whereas lithium can suppress crease in proximal ammoniagenesis has a limited salutary effect cortical collecting tubule acidification. It is anticipated that on overall generation of bicarbonate because of impaired reenfuture studies of RTA models will provide additional evidence trapment of ammonia along the collecting ducts. of impaired acid secretion by segments of the distal nephron Chronic metabolic acidosis and will enable us to identify the intrarenal defects responsible for the excretion of inappropriately akaline urine. Chronic acid loading is the usual means of studying the renal response to chronic metabolic acidosis. The increase in renal Remnant kidney studies ammoniagenesis that occurs in this situation is critical to the When renal mass is reduced, major changes at the segmental adaptive response and generally is well recognized [80]. Good nephron level enable surviving nephrons to increase their et al have shown that the transepithelial ammonia concentration transport functions. This compensatory increase in function gradients in the inner medulla of the rat play a critical role in allows for almost-normal extracellular fluid volume composi- ensuring that adequate ammonium appears in the urine [65]. In tion despite the reduction of more than 85% of renal mass in the in-vivo exposed papilla, the ammonia concentration differsome preparations. The changes that occur in remnant kidneys ence indicates that this gradient is an important force, resulting involve alterations in cellular transporters, ammonium metab- in adequate ammonium excretion. Thus, interstitial accumulaolism, and changes in cell structure. tion of ammonia is a primary determinant of ammonia secretion For the proximal tubule, surviving nephrons hypertrophy in into the inner medullary collecting ducts in vivo. A major remnant kidney models, and this alteration presumably creates feature of the adjustment to chronic acid loading is an increase a larger surface area for proton pumping. Studies of proximal in the interstitium-to-lumen gradient of ammonia and the resulttubule brush-border vesicles from rats and dogs have shown an ing greater diffusion of ammonia into the lumen to form ammoincrease in the Na/H antiporter activity [74]. Although the nium, which is excreted. For the proximal tubule, the filtered bicarbonate load is proximal tubules of surviving nephrons hypertrophy and increase their reabsorption of the increased filtered bicarbonate greatly decreased because of the reduction of the plasma load, the net effect for the whole kidney is still a decrease in bicarbonate concentration, so that even with vigorous retrieval whole-kidney proton secretion because of the decrease in of bicarbonate by the proximal tubule, the rate of hydrogen ion secretion is much lower than normal [81, 82]. When the filtered nephron mass. In the distal tubule, studies of remnant kidneys by Zalups et load in acidotic rats is increased by either microperfusion a! have shown that cells of the initial collecting tubule increase techniques or systemic alkali infusions, the rate of bicarbonate their surface area in a manner consistent with an increased reabsorption increases [83, 84]. ability for A-type intercalated cells to secrete more protons With respect to the distal tubule, three microperfusion stud[75]. The most comprehensive functional data on distal tubule ies have elucidated in-vivo acidification characteristics [15, 16, acidification comes from a study by Kunau and Walker [76]. 62]. These studies, however, did not use perfusate bicarbonate After three-quarters nephrectomy, the superficial distal tubule concentrations that were comparable to the very low bicarbonof the rat showed increased acidification, even when the larger ate concentration that reaches the distal tubule in acidotic rats bicarbonate load and more negative transepithelial voltage was given ammonium chloride. These studies show that (1) in accounted for in separate control experiments. Hamm and chronic ammonium chloride acidosis, bicarbonate reabsorption coworkers studied the in-vitro response of cortical collecting in the distal tubule is greatly increased, (2) reversal of the tubules and medullary collecting ducts from rabbits after uni- direction of normal potassium and sodium fluxes does not lateral nephrectomy and infarction of the remaining kidney [17]. influence this rate of reabsorption, and (3) restriction of normal Surprisingly, net bicarbonate reabsorption did not change in water abstraction or use of perfusates containing no sodium

either segment. With respect to abnormalities of ammonia handling, Buerkert and colleagues have shown that reentrapment of ammonia along collecting ducts is impaired in a rat remnant kidney model, despite increased ammoniagenesis by

also does not diminish the stimulated rate of acidification. In-vitro microperfusion of rat and rabbit CCT obtained from animals with metabolic acidosis have shown increased bicarbonate reabsorption [41, 85]. Papillary collecting duct acidifi-

cation has been extensively studied by Bengele et al, who With respect to the function of the papilla in maintaining measured all the components of net acid excretion [86, 87]. In acid-base balance, the data of Sabatini are puzzling [78, 79]. chronic metabolic acidosis, acid secretion in the inner medulOne month after chemically induced papillary necrosis, no lary collecting duct was two to three times normal, as evidenced abnormalities were detected in distal nephron acid excretion by by the microcatheterization technique. This finding is consiswhole-kidney acidification tests. I suspect some plasticity in the tent with the base-tip papillary micropuncture data already proximal tubular cells [77].

kidney's response, whereby the cortical and medullary collect-

reviewed [65].

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Nephrology Forum: Single-nephron acid-base studies

Data from distal nephron segments indicate that acidification

is only indirectly related to the handling of sodium. The possibility of sodium-independent, whole-kidney net acid excretion has been addressed by Harrington et a! [88], Using

intact animals, these investigators attempted to assess the validity of predictions regarding sodium-independent mm-

Chronic respiratory acidosis

In 1971, we reported the first measurement of absolute net bicarbonate fluxes in a nephron segment under free-flow conditions [33]. In these free-flow micropuncture studies in hypercapnic rats, the filtered load of bicarbonate was not changed, and the absolute net reabsorptive rates corresponded to the in-vivo situation. In the same study under acute conditions, we also infused the animals with bicarbonate and found a greatly increased rate of bicarbonate reabsorption. We concluded that the rate of bicarbonate reabsorption by the proximal tubule in acute hypercapnia, in the absence of bicarbonate loading, is not increased. This finding corresponded to the observation in the intact animal that the increase in plasma bicarbonate could be accounted for by extrarena! buffering. With respect to the effects of chronic respiratory acidosis, Cogan has shown a marked stimulation of proximal tubule bicarbonate reabsorption, which persists for hours after the pCO2 is reduced to normal [91]. These experiments exemplify

eralocorticoid stimulation of proton secretion in cortical and medullary collecting tubules and noted that the medullary collecting duct reabsorbs bicarbonate more vigorously than does the cortical collecting tubule. The results of a carefully conducted balance study on acidotic dogs stimulated to secrete acid maximally, with and without access to dietary sodium, showed that no anticipated mineralocorticoid effect ensued— including marked potassium losses—when dietary sodium was restricted [881. Thus, the sodium independence of collecting duct hydrogen secretion is not consistent with the findings in these studies in intact animals. The data may well raise the problem of species differences (that is, dog versus rabbit), but the information also suggests that it might be appropriate to re-examine conclusions from amiloride "blockade" of the the overall approach I wish to stress in this review. These sodium-dependent negative transepithelial potential difference investigators demonstrated in a chronic acid-base disorder that in other dog studies involving mineralocorticoid administration. the proximal tubule reabsorbs more bicarbonate than normal at I believe it is possible that even if sodium-independent acidifi- a filtered load corresponding to that occurring in vivo. Further, cation occurs in the distal tubule and the medullary collecting this response can persist even when the hypercapnic stimulus is duct, there may indeed be brisk participation by the cortical no longer present. The cellular process presumably involves thick ascending limb, where proton secretion proceeds via increased antiporter activity, as has been shown in chronic metabolic acidosis [74]. NaIH exchange [42].

Finally, a comment is in order as to whether systemic acidosis per se is the signal for stimulated acidification. In

Chronic metabolic alkalosis

metabolic acidosis, it is tempting for us to presume that the low

bicarbonate concentration in the peritubular environment faChronic metabolic alkalosis has been a source of controversy vors bicarbonate efflux. Indeed, in-vitro studies have shown over the last quarter of a century. Careful consideration of hard that such an effect can occur acutely [18]. After chronic data from single nephron segments is helpful in the evaluation ammonium chloride loading, however, Bengele et al found that of opposing interpretations of nephron function in this disturduring the phase of rebound alkalosis, the papillary collecting bance. The major controversies have involved (1) whether duct does not secrete less net acid than in control conditions glomerular filtration rate decreases sufficiently to maintain an [89]. This study shows, for a single nephron segment, normal elevated plasma bicarbonate concentration, or whether inammonia secretion in the presence of metabolic alkalosis. creased reabsorption of bicarbonate by renal tubules is the Recently, we also showed that distal tubules from fed rats important sustaining factor, (2) the role, if any, chloride plays in reabsorb bicarbonate during the rebound alkalosis that occurs 2 maintaining or correcting the disturbance, (3) the role, if any, of days after ammonium chloride feeding [90], These in-vivo changes in sodium, potassium, extracellular fluid volume, and observations are supplemented by in-vitro data: CCTs from (4) the underlying signal for stimulated renal tubular bicarbonrabbits fed ammonium chloride can reabsorb bicarbonate ate reabsorption, bearing in mind that at no time is systemic briskly under in-vitro conditions, in which the acidifying influ- blood pH acid. With respect to a role for extracellular fluid ence is no longer present [851. Thus, it is likely that cellular and volume and potassium losses, I am not aware of any segmental metabolic changes, that is, A-type intercalated cell adaptation data that can bear on the current controversies. Concerning the and stimulated ammoniagenesis, can persist for hours or days, need for sodium to sustain increased bicarbonate reabsorption, even in the absence of an acidic milieu. the question of sodium-independent proton secretion in distal In summary, in chronic metabolic acidosis caused by acid nephron segments already has been referred to in my discussion loading, all nephron segments have an increased capacity to of metabolic acidosis. reabsorb bicarbonate but have little impact on correcting the What signals augment bicarbonate reabsorption? The comdisturbance unless the bicarbonate load is also increased. ments I have made about rebound alkalosis are particularly Rather, new bicarbonate is generated by stimulation of ammo- pertinent to the situation in chronic metabolic alkalosis, as is niagenesis by proximal tubular cells and the subsequent in- the observation that in-vitro cortical collecting tubules continue creased diffusion of ammonia into the lumen of the papillary to secrete bicarbonate or acid at increased rates in the absence collecting ducts. It is likely that both sodium-dependent and of initiating stimuli. In contrast to these findings are the sodium-independent acidification events occur in chronic met- observations by Gifford et al that the in-vitro perfused CCT abolic acidosis, whereas rat studies on the rebound alkalosis taken from a rat made acutely alkalotic by peritoneal bicarbonthat follows ammonium chloride loading indicate that bicarbon- ate/chloride exchange secretes bicarbonate at a greater rate ate retrieval is not inextricably regulated by an acidic blood pH.

than normal [92].

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Nephrology Forum: Single-nephron acid-base studies

Is bicarbonate reabsorption increased in the whole-kidney in chronic metabolic alkalosis? Reductions in glomerular filtration

the disturbance. Our in-vivo data from rat distal tubules indicate that luminal chloride is likely an important modulator.

rate have been reported in chronic metabolic alkalosis in humans [1, 2]. Perhaps the fall in GFR, and hence the fall in bicarbonate filtered load, accounts for the elevation in plasma bicarbonate concentration. However, in a study by Rosen et al [4], correction of metabolic alkalosis was clearly associated with bicarbonaturia, even when reductions in filtered load and GFR were allowed to persist. In the dog, chloride-depletion metabolic alkalosis induced by hemofiltration was associated with increased bicarbonate reabsorption by the kidney, although GFR was reduced [93]. With respect to the proximal tubule, several laboratories have considered whether the bicarbonate load is normal or decreased in chronic metabolic alkalosis. Cogan and Liu reported that in rat models of chronic metabolic alkalosis GFR is decreased; further, no evidence for increased proximal bicar-

Conclusion

In this Forum, I have tried to evaluate experiments on nephron segments from animals subjected to chronic acid-base

disturbances. By being aware of species differences, noting details of the experimental design, and above all, ensuring that the experimental situation closely simulates in-vivo conditions, we can gain relevant insights into clinical disturbances. At the end of his review on the molecular biology of renal

hydrogen ion transport, Gluck commented that "our understanding of the cause and treatment of acidification disorders and adaptive responses of the kidney will depend on knowing the function and regulation of these transport proteins at the

cellular and molecular levels. Progress to this goal will require the isolation of these ion transporters, the elucidation of their bonate reabsorption could be demonstrated [3, 94]. In chloride- structural and biochemical properties, and the study of their ion depletion metabolic alkalosis in the rat, Galla and Luke also transport kinetics, synthesis, degradation, and traffic both to reported decreased GFR associated with the metabolic alkalo- and from the plasma membrane" [74]. This is surely a well-stated job description. But it is essential sis [5]. Cogan has argued that it is the decrease in filtered load that we always have a view of the integrated renal response to rather than stimulated tubular bicarbonate reabsorption that what single segments do and how transport proteins contribute maintains chronic elevations of plasma bicarbonate. In support to their function. If we do not strive to explain the acid-base of this hypothesis is the striking observation that administration

of atrial natriuretic peptide can restore GFR to normal and correct the metabolic alkalosis, presumably without having any

direct effect on bicarbonate handling at single-segment sites [95]. In contrast, Maddox and Gennari found increased proximal bicarbonate reabsorption in rat models of chronic metabolic

adjustments so well described in dogs and humans, we risk collecting a wealth of exciting molecular insights of uncertain in-vivo significance.

Questions and answers DR. NIcoLAos E. MADIAS (Chief, Division of Nephrology, New England Medical Center, Boston, Massachusetts): David,

alkalosis [96]. A recent abstract also suggests increased antiporter activity of apical membrane vesicles in this situation as you know, considerable work has been carried out in the [97]. laboratory of our institution that has served to formulate a Does chloride have a specific role in maintaining and correct- "non-homeostatic" theory on the regulation of renal acid ing chronic metabolic alkalosis? Galla and Luke's review of excretion. According to this theory, changes in renal acid their extensive experimental data suggests that even when excretion in response to acid-base stresses do not appear to be

chloride-depletion metabolic alkalosis has been maintained for driven by alterations in systemic pH. How does this theory fare 7 days, chloride is needed to maintain and correct the distur- in the light of the insights on segmental acidification that you so bance independent of hypokalemia or changes in glomerular elegantly summarized for us? filtration rate or extracellular fluid volume [5]. DR. LEVINE: If we recall that the "non-homeostatic" view of I already have reviewed aspects of bicarbonate handling and renal acid excretion is applied to the chronic steady-state

its modulation by chloride in the distal tubule. Clearly, the dependence of bicarbonate secretion in DOCA-induced alkalosis on luminal chloride is relevant to whether and how chloride

modulates the repair of gastric alkalosis. Galla et al recently attempted to describe distal nephron segmental bicarbonate handling during chloride correction of metabolic alkalosis [6]. They showed that the bicarbonaturia occurring after chloride administration did not involve the accessible distal tubule, and they suggested that chloride might exert its effect on collecting duct segments. In a preliminary report, however, Wesson and Babino proposed that chloride handling is altered at the distal tubule site during correction of chloride-depletion metabolic

situation, indeed much segmental data support it. The main features of the theory are that systemic blood pH does not regulate acid secretion, but that both chloride and sodium play a major role. First, I already have referred to situations in which

either distal tubules or papillary collecting ducts reabsorb bicarbonate at normal or increased rates at a time when the systemic blood pH is alkaline [53, 89, 90]. Second, in addition to the in-vitro finding that bicarbonate-secreting segments depend

on luminal chloride for bicarbonate exchange, our recent in-

vivo data show that perfusion with zero luminal chloride

decreases bicarbonate secretion in alkalotic rats and stimulates bicarbonate reabsorption in normal fasted rats. The extensive studies by Galla and Luke also establish a role for chloride in alkalosis [98]. Let me summarize. In chronic metabolic alkalosis, some, but the maintenance and repair of metabolic alkalosis consistent not all, studies suggest that decreased filtered load contributes with the predictions of the "non-homeostatic" theory [5]. to the elevated plasma bicarbonate concentration, that chloride Third, the role of sodium reabsorption and of a sodium current deficits stimulate nephron segments to reabsorb bicarbonate, in modulating proton secretion by the distal tubule and the and that at one or more distal nephron segments, provision of cortical collecting duct is accepted. In the thick ascending limb chloride is likely associated with bicarbonaturia and repair of there is NaJH exchange [41], although for other distal segments,

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Nephrology Forum: Single-nephron acid-base studies

coupling between proton secretion and sodium reabsorption is indirect. Of interest is the in-vivo work of Kunau and Walker [100], who showed that perfusion of distal tubules with amiloride dramatically reduces the lumen-negative potential difference, and that associated with this reduction is a 50% decrease

hydrochloric acid depletion should account for the possibility that secretion as well as reabsorption of bicarbonate might be altered. Two, chloride plays a role in modulating bicarbonate transport. DR. KASSIRER: If we make the assumption that there is little

in the rate of bicarbonate reabsorption. Although there are or no change in glomerular filtration rate in the setting of studies—usually acute experiments—in which segmental acid- selective chloride depletion, can we conclude that excessive ification seems to be influenced by ambient pH, the chronic reabsorption in the proximal nephron and cessation of bicarin-vivo studies that I have cited are more relevant to the events

the "non-homeostatic" theory was designed to explain. In short, the theory fares quite well. DR. JOHN T. HARRINGTON (Chief of Medicine, NewtonWellesley Hospital, Newton, Massachusetts): Recently, a great deal of attention has been paid to the notion that ammonia excretion from the kidneys is simply passive and that the liver is the prime acid-base organ. Would you offer a general comment on that concept? Second, do any of the studies that you reviewed shed any light on that argument? DR. LEVINE: The arguments of Atkinson and Bourke [101] have generated controversy by denying a significant role for the kidney in regulating acid-base balance. The data I reviewed that most closely relate to this view pertain to the production of

bonate secretion in the distal nephron account for the sustained alkalosis? DR. LEVINE: Although the data conflict, it is likely that the

proximal tubule reabsorbs more bicarbonate than normal in sustained metabolic alkalosis. With respect to the question of

bicarbonate secretion, it remains to be seen whether this process is an important feature of chronic metabolic alkalosis. During DOCA alkalosis associated with bicarbonate ingestion, there is net bicarbonate secretion in vivo by rat distal tubules [63]. In contrast, in another chronic model of metabolic alkalosis [103], distal tubules reabsorb more bicarbonate in vivo than in control conditions. Accordingly, we need specifically designed experiments to determine whether selective chloride depletion is associated with cessation of bicarbonate secretion in distal nephron segments. In addition, we must be aware that

ammonium by the kidney as a result of the metabolism of glutamine. With the generation of this ammonium, there is a the "distal nephron" consists of at least five histologically

stoichiometrically linked production of new bicarbonate, which distinct nephron segments (thick ascending limb, distal tubule, contributes to acid-base regulation. I cannot add to the other cortical collecting tubule, outer medullary collecting duct, and published arguments emphasizing the role of the kidney in inner medullary collecting duct). Each of these segments might contribute in its own way to whole-kidney bicarbonate handling acid-base homeostasis [102]. in the different types of metabolic alkalosis. DR. JEROME P. KASSIRER (Associate Physician-in-Chief, DeDR. KASSIRER: Then what is the mechanism for the increased partment of Medicine, New England Medical Center): What

special insights into the pathophysiology of selective hydrochloric acid depletion have evolved from microperfusion and micropuncture studies?

potassium loss and the high potassium clearance under those circumstances?

DR. LEVINE: I know of no data from single nephron segments

a natriuresis occurred, as did bicarbonaturia, while she was

DR. LEVINE: In the patient under discussion, a kaliuresis and

that directly apply to the chronic metabolic alkalosis of several hypokalemic and alkalotic. Certainly, the acute loss of sodium days' duration that follows selective hydrochloric acid drain- bicarbonate into the urine must be accompanied by high flow age. The chronic studies providing in-vivo micropuncture data rates along nephron sites that normally secrete potassium. This have involved one or more of the following experimental high flow rate would lead to a greater secretion of potassium maneuvers: injection of steroids, ingestion of bicarbonate or down a concentration gradient into the lumen. Another factor non-reabsorbable anions, diuretic administration, dietary deple- that has been discussed recently is the possibility that a tion of chloride, or dietary depletion of potassium. Further, hydrogen-potassium ATPase might play a role in clinical acidthere is disagreement, even in comparable studies, regarding base potassium interactions [104]. Wingo proposed that such a the role of GFR in altering renal bicarbonate load. Most of these pump exists in intercalated cells in the medullary collecting tubule of the mammalian nephron [104]. The difficulty with studies are presented in Table 1. The most recent measurements of proximal and distal tubule invoking the action of this pump to explain potassium loss is bicarbonate handling in chronic metabolic alkalosis indicate that we expect that more protons will be secreted when more that bicarbonate reabsorption is increased at both these neph- bicarbonate is presented, as in the acute phase of metabolic ron sites [103]. In this study, rats were fed a diet low in both alkalosis, and this should lead to increased, rather than depotassium and chloride; they received furosemide and drank creased, potassium reabsorption. On the other hand, to the supplemental potassium. This situation is different from gastric extent that such cells might be rendered alkaline during chronic drainage experiments, in which fluid volume, sodium, and metabolic alkalosis, we might assume that the rate of proton potassium are replaced. The ideal experiment would be to study pumping would decrease, and hence potassium would be lost in rats after gastric drainage [311 and undertake free-flow or the urine. My supposition is that chronic metabolic alkalosis is microperfusion collections in a fashion that preserves the flow attended by an increased rather than a decreased rate of proton rate and luminal fluid concentrations of sodium, potassium, secretion in the chronic steady state. DR. MADIAS: It is clear that the work of Luke and Galla has chloride, and bicarbonate at various nephron sites. In short, the identified the distal nephronal segments as the locus of the experimental protocol must simulate the in-vivo conditions. Notwithstanding these issues, two general insights emerge maintenance of chloride-depletion alkalosis. I have two quesfrom the studies I reviewed. One, any consideration of the tions. (1) Is information available indicating that the intracellupathogenesis of the metabolic alkalosis following selective lar concentration of chloride might be playing a role in modu-

Nephrology Forum: Single-nephron acid-base studies

757

lation of this phenomenon? (2) In this disorder, decreased findings were consistent with an electroneutral chloride-coudelivery of chloride to the medullary collecting duct might pled potassium secretion mechanism located in the apical stimulate a bicarbonate reabsorptive flux mediated by the membrane. Finally, they also determined that because the secretion of protons in the company of a chloride conductance. addition of barium to the perfusion solutions did not impair the

Given that the urine is virtually chloride free, is there a increase in potassium secretory flux, the K-Cl cotransport mechanism for recapturing the secreted chloride at the distalmost part of the nephron? DR. LEVINE: Two recent studies measured intracellular chloride concentrations in intercalated cells. Gifford et al measured intracellular chloride in intercalated cells from cortical collect-

pathway likely operates in parallel with the conductive pathway, which is inhibited by barium. DR. HARRINGTON: You referred in passing to the intercalated

cells and their role in hydrogen secretion. I've never quite understood how the message gets to the intercalated cell. What

ing ducts of rats subjected to acute metabolic alkalosis by drives it, either in the lumen or within the cell, to turn on chloride/bicarbonate peritoneal exchange [1051. The intracellular chloride concentration did not differ from that measured in control animals, but it was much higher than that measured in principal cells either during control or metabolic alkalosis. Beck et al also measured intracellular chloride in intercalated cells of

hydrogen secretion or bicarbonate reabsorption? DR. LEVINE: The regulation of bicarbonate secretion and proton secretion by intercalated cells is a matter of continuing investigation. As I already noted, zero lumen chloride should impede the function of an apically situated chloride/bicarbonate

rat distal tubules, either during hydropenia or during acute exchanger that is present in B-type intercalated cells. For hypertonic sodium bicarbonate infusions [1061. Intracellular A-type intercalated cells, if peritubular bicarbonate concentrachloride concentrations of intercalated cells from these alkalot- tion is abruptly increased, the exit of bicarbonate from the cell ic rats were significantly lower than in the control animals. It would be impeded, the cell would become alkaline, and apical

will be of interest, of course, to obtain measurements of proton pumping would be reduced. Experimental evidence intracellular chloride of the two types of intercalated cells supports the possibility of such acute effects on A- or B-type during chloride-depletion metabolic alkalosis of several days' duration. Whether changes in intracellular chloride concentra-

intercalated cells [18]. Less obvious, however, is the signaling mechanism underlying the secretion of bicarbonate by distal

tion can alter the HC03/C1 exchanger or vacuolar H tubules in fed rats or by turtle bladder epithelium in the ATPase is a matter of current study [56, 107].

postprandial state, a more chronic condition. Vasoactive intes-

Concerning the second part of your question, Diezi and tinal peptide and other hormones might play a role [55]. The colleagues have provided data that bear critically on our dis- work by Schuster on cyclic cAMP stimulation of bicarbonate cussion [108]. In-vivo papillary micropuncture was performed secretion in B-type intercalated cells further supports the view on rats subjected to dietary chloride restriction for 7 to 10 days that these cells are hormone targets [56]. My earlier comments without any other confounding factors such as the use of to Dr. Madias concerning intracellular chloride concentrations diuretics, steroids, or alkaline loading. The papillary collecting are also relevant to your question. duct data show that chloride is reabsorbed against a significant DR. MADIAS: What might be the teleology behind the stimuconcentration gradient, most likely by an active transport lation of the sodium/hydrogen antiporter activity in the proxiprocess. These rats were hypochioremic, presumably alkalotic, mal tubule during metabolic acidosis, given that the filtered load and excreted little chloride in the urine. of bicarbonate in that disorder is suppressed? Might it be that a DR. RONALD D. PERRONE (Division of Nephrology, New substantial fraction of the antiporter's activity is actually exEngland Medical Center): Wright and colleagues reported a pended in effecting sodium/ammonium exchange? chloride-linked mode of potassium secretion in the distal tubule DR. LEVINE: The idea is very appealing. Nagami has meaand collecting duct [109]. I believe that they ascribed these sured proximal tubule ammonia production and secretion rates findings to a luminal potassium chloride symporter. This theory [66, 112]. Compelling data indicate that ammonia production relates to one of the concerns brought up a few moments ago. increases during NH4C! acidosis and that NH4 can substitute Are you aware of any more data in this area? for H on the Na/H antiporter. However, no direct evidence DR. LEVINE: Thank you for referring to this again; I should yet suggests that increased rates of Na/NH4 exchange rather have touched on this in my earlier answer. In our previous than increased ammoniagenesis accounts for the greater luminal review [40], we discussed the early findings of Velazquez et a! accumulation of NH4 during metabolic acidosis. I suspect that [110], who reported that when sulfate replaces chloride in the this is likely, however. DR. MADIAS: Do we know anything about the behavior of perfused rat distal tubule, net potassium entry increases. They proposed that this increase might result from a reduction in the juxtaglomerular nephrons during adaptation to acid-base reabsorption of potassium, possibly coupled to chloride. The stresses? study you refer to now shows that the increase in net potassium DR. LEVINE: I am not aware of any studies in which the deep secretion occurs in both early and late portions of the distal nephrons were examined during chronic acid-base disturtubule [109]. However, of more interest is other work on the bances. Two papillary micropuncture studies have been permechanism underlying this net entry of potassium when chlo- formed under acute conditions, however. Frommer et a! studied ride is removed from the lumen. Ellison et al measured unidi- in-vivo bicarbonate handling by the papillary collecting ducts rectional potassium fluxes and thereby determined whether the during acute metabolic alkalosis induced by sodium bicarbonate net potassium entry associated with zero lumen chloride con- infusion [1131. They concluded that juxtamedullary nephrons centrations was a result of increased secretion or decreased contributed more bicarbonate to the final urine than did the reabsorption [111]. They found that the absorptive flux does not superficial nephrons. Also, Galla and coworkers used their change but that the unilateral secretory flux increases. Their acute model of peritoneal chloride/bicarbonate exchange to

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study the function of deep nephrons during chloride correction are very low, medullary collecting duct acidification increases; when chloride concentration is raised, acidification decreases [40]. Accordingly, the delivery of high chloride loads to the medullary collecting ducts should minimize the reabsorption of bicarbonate arriving from proximal nephron segments or after secretion by the cortical collecting tubules or distal tubules, As nephrons [6]. DR. HARRINGTON: You commented on the role of the prox- I already noted, in chloride depletion, chloride also can be imal tubule in producing ammonia but stressed the importance captured by an active transport mechanism in the papillary of diffusion of ammonia into the medullary portions of the collecting ducts. Finally, we should keep in mind that several kidney. What interferes with that diffusion? Is it simply gross hormones can alter the function of intercalated cells that secrete

of the acute metabolic alkalosis [6]. The bicarbonaturia that resulted from chloride provision was likely the result of increased excretion from the collecting ducts rather than increased delivery from either the superficial or juxtamedullary

scarring, or is there a cellular defect that interferes with bicarbonate or hydrogen ions [56, 74], and the provision of ammonia moving into the medulla in the remnant kidney chloride might alter the concentration of such hormones or the model? DR. LEVINE: In the remnant kidney study by Buerkert et al,

response of cells to them. DR. JORDAN J. COHEN (Dean, School of Medicine, State the ablation of renal tissue was caused by infarction of the University of New York at Stony Brook, Stony Brook, New upper pole, the lower pole, and the posterior surface of one York): What directions would you suggest that future research kidney by ligating branches of the renal artery [77]. One week take? later, the other kidney was removed to reduce total mass by DR. LEVINE: I hope that granting agencies will recognize that two-thirds. Although scar tissue was present on the margins of in-vivo work, provided it is excellent, is still critically important the infarcted area, microscopically the renal papillae available for understanding how the whole kidney works. Those who feel for study did not appear different than papillae from the control compelled to leave in-vivo work to join their expert colleagues group. Because of this finding, it is not likely that scarring in the cell culture and molecular biology arena might hesitate a explains the failure of the kidney to reentrap ammonia along the moment and ask whether they could not continue to make collecting ducts. Further, the fall in luminal pH between the end important contributions by the study of appropriate in-vivo of the distal tubule and the base of the collecting duct also was models of chronic acid-base adjustments. Such an endeavor similar in the normal animals and those with remnant kidneys. would provide the missing segmental nephron data that I have Because the concentration of ammonium in the samples ob- tried to identify in this review. tained near the tip of the loop of Henle was lower in the remnant kidneys than it was in the control kidneys, these researchers Acknowledgments proposed that ammonium was swept away by altered hemodyHelpful suggestions were made by Drs. Mark Knepper, Mitchell namics in the papilla, thereby resulting in the papilla' s failure to Halperin, Steve Nadler, John Gennari, Marty Cogan, Ed Alexander, establish high interstitial concentrations of ammonium. It is Linda Peterson, and John Seely. Mrs. Phyllis Morrissey cheerfully likely that this process, rather than impaired diffusion of NH3, typed endless drafts of the manuscript. underlies the inadequate delivery of ammonium into the urine. Of course, measurements of the diffusibility of NH3 across Reprint requests to Dr. D.Z. Levine, Professor and Head, Division of renal structures in various disease states would be of interest. Nephrology, University of Ottawa and Ottawa General Hospital, DR. MADIAS: Do you have any suggestions about what might

be the critical signal that the kidney registers during chloride administration to modulate its function toward repairing meta-

Health Sciences Building, 451 Smyth Road, Ottawa, Ontario, Canada

KIH8M5

bolic alkalosis? How is chloride administration perceived by the

kidney? What sort of events might take place to change the kidney's functions and lead to correction? DR. LEVINE: Let's speculate on what might happen at different nephron sites during the repair of chronic metabolic alkalosis of several days' duration, a situation that one encounters clinically. The administration of chloride accompanied by fluid will expand extracellular fluid volume. Because GFR often

decreases in such patients, saline administration raises the filtered load of bicarbonate in this setting, decreases proximal reabsorption, and thereby makes more bicarbonate available for renal excretion. We also can expect that the luminal fluid traversing distal nephron segments will have a higher concentration of chloride. The B-type intercalated cells located in the

distal tubule and cortical collecting ducts could respond to increased luminal chloride concentrations by secreting bicarbonate and reabsorping chloride, as these cells have an apical bicarbonate/chloride exchanger. The medullary collecting duct contains A-type intercalated cells, which actively pump protons

into the lumen; this entry of protons is accompanied by net chloride secretion. That is, when lumen chloride concentrations

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