Kidney International, Vol. 9 (1976) p. 149—1 71

Genetic aspects of renal tubular transport: Diversity and topology of carriers

CHARLES R. SCRIVER, RUSSELL W. CHESNEY and RODERICK R. MCINNES

Medical Research Council Group in Genetics, deBelle Laboratory for Biochemical Genetics, McGill University-Montreal Children's Hospital Research Institute, Montreal, Quebec, Canada

Mutations which cause the inborn errors of mem- convoluted and pars recta segments where a major brane transport can provide information about the fraction of electrolyte and organic solute transport normal topology' of renal transepithelial transport. occurs, have a greatly enlarged membranous surface In recent years various reviews of tubular transport formed of closely packed microvilli: the latter in- [1—6] have appeared which discussed the interrelation crease the absorptive surface about forty-fold [7, 8]. between disease and net tubular reabsorption of or- Microvilli are associated with an unstirred layer at ganic solutes.2 Their emphasis was primarily on the the external liquid-solid interface, which is the major functions which served solute transport and less on rate-limiting step in trans brush-border permeation at the diseases associated with the disturbance of trans- low concentrations of many solutes [9]. The unstirred port. In this review we have taken the opportunity to layer also serves as a locale in which enzymes may act describe, and to speculate on, the probable site in the upon substrate, e.g., , before transport tubular cell of the defect in transcellular movement of [10]. Tubular epithelium is also a continuous sheet by the solute in a number of inborn errors of tubular virtue of punctuate contacts, or tight junctions, be- transport. We hope that the speculations will stimu- tween cells at their luminal poles [11]. These junc- late debate, formulation of hypotheses and further tions are probably impermeable to organic solutes experimental evaluation to advance our knowledge. under normal conditions. Therefore, the solute must The table included in this paper provides a catalogue enter epithelial cells to reach the peritubular space of the currently accepted inborn errors of tubular during reclamative transport and to reach the lumen transport. It is these clinical "windows" which, during secretory transport. To achieve this vectorial through the expression of , have revealed process, an asymmetry of net solute flux is required and helped to delineate an impressive array of spe- during reclamative and secretory transports. How cific transport functions in tubular membrane. mutation informs us about the functional organ- ization of two sets of membranes (luminal or brush Cellular uptake and transcellular transport border, and basolateral) and three pools (luminal, cellular and peritubular) ordered in series, and how The epithelial cell of the renal tubule has luminal they achieve these net transtubular fluxes, is of and basilar poles, with definite orientations to the fundamental interest to us in this brief review. fluids in contact with them; its luminal pole faces an Cellular uptake ofsolute.The current view of bio- ultrafiltrate of plasma where solute is topologically logical membranes emphasizes a fluid-mosaic, struc- outside the body while the basilar pole is in contact tural model, in which the proteins form a mosaic in with peritubular interstitial fluid where solute is inside the bilaminar bed of lipid [12]. In the absence of the body. Cells of the proximal tubule, in both the diffusion channels, permeation of the plasma mem- brane by hydrophyllic solutes must be facilitated by Topology—anatomical definition: the structure of a particular carriers [13]. Certain types of membrane protein ap- region or part of the body (Webster). parently serve as carriers [14], thus providing a per- The term "solute" in this paper refers to organic solutes except tinent focus for the effect of mutation on the trans- when clearly indicated otherwise: for example, in the section on port process. Various chemical and kinetic probes phosphate reabsorption. © 1976, by the International Society of Nephrology. [13] reveal the specificity of carriers and their ability Published by Springer-Verlag New York to recognize solutes. Since the transmembrane move-

149 150 Scriverel a! ment of many solutes across the basolateral and equals efflux. In the latter state, [S]1 will be >> [SJ0; it brush border membranes of tubular epithelium, both follows from equations 1 and 2 that Km err must then in vivo and in vitro, can occur against a chemical be >> Km ml The maintenance of this relationship gradient in all parts of the nephron [5,6],there must which is essential to concentrative uptake appears to also be an investment of energy in transport in the be a genetically determined property of the carrier. form of a conjugate-driving force [15]. Efflux across the luminal membrane occurs during Assuming that the solute remains osmotically ac- net absorption of amino acids and by the tive upon entering the intracellular pool, and there is proximal tubule [17—19]; this backflux is quite in no evidence yet to the contrary [16], it follows that keeping with the normal kinetics of efflux depicted in the plasma membrane plays a critical role in the form Fig. 1. However, backflux is necessarily small if con- of a barrier to exodus after concentrative uptake. A centrative uptake is an initial step during net trans- mechanism for attainment of dynamic asymmetry in epithelial reclamation of solute, as is now known to the carrier as it functions on opposite sides of the be the case for the transport of some amino acids and membrane is required. A simple kinetic description of for glucose [17—19]. this event is possible [6] (Fig. I). The Michaelis equa- The ability to generate asymmetry in transcellular tion can be used to describe carrier-mediated and movement of solute, so that a vectorial flux occurs, opposing fluxes2 of solute across the membrane has long been a subject of interest [20]. Transport where entry (or influx) and exodus (or efflux) are across epithelium has many features which are analo- described by separate equations: gous to transport across the plasma membrane; but transepithelial transport also has unique char- Vmax jflf[S]0 lIlt (I) acteristics which distinguish it from simple up- Kmbr +[S]0 take into cells. In tubular epithelium the now well- and recognized substrate specificity of the transport proc- Vmax elf[S]1 ess, presumably invested in the transport proteins or Ueff (2) reactive sites of the plasma membrane [13], and urn- Km elf + [S]1 ned by the appropriate kinetic, chemical and genetic [S]0 and [S]1 are the concentrations of solute on the probes [1—6], is not necessarily identical at opposite outside and inside of the membrane, respectively. poles of the transporting cell. Uptake into the in- When uptake is concentrative, influx exceeds efflux tracellular pool, as defined by equations I and 2, can until the steady state is achieved at which time influx pertain to both luminal and basilar and lateral mem- branes. However, if an identical net flux exists at both sets of membranes, net transcellular flux, from urine to peritubular space, or the reverse would not occur. For example, unless the unidirectional flux of solute outward at the basilar pole of the cell is greater than the unidirectional flux outward at the luminal pole, there is nothing to prevent achievement of a trans- cellular steady state so that backflux becomes the equal of entry at the luminal site serving uptake from glomerular ultrafiltrate, thus making net absorptive transport impossible. During absorption, a mecha- l/Ks 1/Ks. [S1b] [Sal [S0] 1/EPro] nism must be found which maintains the interim Fig. I. Diagramshowing the apparent kinetics of steady-state entry instantaneous uptake ratio below the "true" steady- (influx) and exodus (efflux) under conditions of active transport (accumulation against a chemical gradient) of L- across the state ratio across the luminal membrane; therefore, plasma membrane of a cell. (See text for appropriate Michaelis an explanation for "run out" from the intracellular equations and description of terms.) Where the rate of solute entry space in the direction of the peritubular space is is not higher than the rate of its removal by metabolism, the latter comes to influence transport in the transtubular orientation. Di- required. Two mechanisms seem plausible. 1) Meta- minution of metabolic "runout" may cause the internal solute bolism. If there is a large intracellular pool of the concentration to rise (i.e., change from [S]. to [S]lb); when this solute, membrane transport will not be the rate-limit- change occurs in relation to the luminal membrane, exodus (back- flux) will increase and egressed solute will be removed by the ing step in its transcellular movement. The rate of its flowing column of urine. These considerations may be relevant to intracellular utilization will determine its intracellular the interpretation of solute loss in some hereditary disorders of concentration and, therefore, the number of mole- tubular transport and metabolism and to the concept of maximal rates of tubular absorption (Tm). (From Scriver, Mclnnes and cules of the original solute species available for back- Mohyuddin [97].) flux after uptake. It follows that a disturbance of Renal tubular transport 151

Urine intracellular metabolism may perturb tubular ab- sorption through modulation of intracellular pooi 1 \ Blood size. Currently available techniques which measure net reabsorption and transepithelial movement are, o"% in essence, "black box" methods which largely fail to account for intracellular events acting on solute. 2) Dissimilar characteristics of luminal and basilar mem- branes. Luminal and basilar membranes are dis- similar both in their morphologic and functional characteristics. Differences at the two poles have been described, for example, in electropotential [21], and in the uptake of amino acids [18, 22—25][19, Fig. 2. A model delineating possible sites for expression of mutant 26, 27] and keto acids [28]. This functional alleles causing hereditary disorders of tubular reabsorption. Appro- asymmetry clearly permits metabolically inert amino priaterecognition of the vectorial flux in secretory transport allows acids, which have no "run out" into other metabolic the mode! to be adapted also to disorders of secretion. Defect IA, mutant site (carrier) blocks entry; defect !B, mutant carrier per- pools, to be reclaimed from urine into blood against a mits excessive exodus (backflux) from internal pool (); defect 2, chemical gradient [29]. Accordingly, proximal tubule blocked catabolic mutant state prevents metabolic "runout" to reclamation may be accomplished by a more per- alternate form (0) leading to accumulation of original solute in the internal pool (A) and exaggerated exodus (backflux) on normal missive efflux at the basilar membrane relative to the carrier; defect 3, defect in exodus at basilar (or lateral) membrane luminal pole. Relatively little work has been done to leads to intracellular accumulation as in defect 2. All lead to examine the role of solute removal by peritubular reduction of net transepithelial flux. Not shown are hormone- modulated defects involving hormone binding at specific sites on blood flow in relation to urine flow, and the relation the basilar membrane, and transduction of signal by internal mes- this may have to transtubular transport kinetics. senger to influence the primary transport process (e.g., phosphate We should recognize also that solutes such as, transport modulation by parathyroid hormone or calcitonin, and amino acids [6, 30, 31] and glucose [19] which are water transport modulation by vasopressin). taken up from peritubular fluid in many portions [16, 26, 27] of the nephron evidently experience only min- transportrequire consideration of two additional imal net efflux across the luminal membrane and do steps: 1) binding of hormone to the appropriate not appear in bladder urine in significant amounts. plasma membrane and 2) intracellular translation of Further evidence for diversification of transport in the hormonal signal. We have selected examples brush border and basolateral membranes in various from Table 1 to describe defective mechanisms at parts of the nephron is seen in the distal tubule, which different stages of the transepithelial transport proc- takes up amino acids, not at all from the lumen and ess. only slowly from peritubular fluid [6]. IA. Disorders of solute uptake of the luminal mem- brane. The casual student of biological transport is Functionalclassification of likely to assume that the hereditary disorders of tubu- inborn errors of tubular transport lar transport express themselves at the luminal mem- brane. We know from studies of electrolyte transport Twomembranes and three solute pools, arranged that other peritubular membranes play vital roles in in series, accommodate transepithelial transport. In solute migration across the tubule; no direct evidence principle, the various inborn errors of reclamative for a luminal membrane defect has been obtained in transport can then be assigned to specific disorders of any disease listed in Table 1; only indirect evidence, membrane function according to Fig. 2. Mutation and only in certain disorders of net reclamation, is at can effect tubular reabsorption at the following hand. The examples we have chosen will highlight a stages: JA) uptake activity (entry) at the luminal number of themes including the diversity of mem- carrier; JB) backflux (luminal exodus) permitted on brane sites used by even a single solute during tubular the carrier; 2)cellularutilization of absorbed solute transport, and the great specificity among sites ex- (pool size) controlled by its metabolic disposal; 3) posed to a wide mixture of solutes. Consequently, unidirectional flux from cell to peritubular fluid at mutation is likely to ablate only a fraction of any basilar and lateral membranes (peritubular exodus). given transport function. Diversity has adaptive ad- Disorders of secretory transport can also be served vantage. by this model with due consideration for the pre- The Hartnup trait. Typical homozygotes have a dominant direction of the fluxes. selective impairment in the intestinal and tubular ab- Hormone-dependent inborn errors of tubular sorption of a particular group of neutral a-amino 152 Scriveret al

Table 1. Heritabledisorders of renaltubular transport inman' Trait Presumed (or Other Apparent (McKusick Substance possible) mutant tissues inheritance cat. No.) affected gene product affected pattern Comment' Hyperdibasic , ornithine Shared "dibasic" Intestine AD/AR Two alleles (different loci?) , amino acid transport liver? Type 1(22270) protein (12600) ("dibasic" group) system (uptake) brain? intolerance; failure to (22270)' thrive, (mitochondrial defect?) bets silent. Type 11(12600) assoc. with mental retarda- tion in recently discovered homoz. Hets have modest dibasic aminoaciduria. Lysine, ornitbine Shared membrane Intestine AR "Negative" reabsorption (22010)' arginine and cyst(e)ine efflux system? brain? of affected amino acid can (skin fibroblasts are occur. Three alleles (same normal locus?) each causing differ- ent phenotypes: in type-I bets, no excess amino acids in urine ("silent"); in type-Ill homoz, intestinal transport intact. Hypercystinuria Cystine Specific system for AR? One pedigree only (23820) cyst(e)ine (uptake) Proline, hydroxy- Shared system for Intestine AR Four alleles (same locus?) (24260)' proline, glycinc iminoacids, I, II, silent hets (& ) (uptake) III, IV hyperglycinuric bets 1, with intestinal defect IV, Km mutant. Hartnup Neutral amino acids Shared system for Intestine AR Two alleles (same locus?) (23450)' (excluding iminoacidslarge neutral amino (skinfibroblasts l intestine affected; II, and glycine) acid group (uptake) arenormal) intestine normal. Hets "silent" in both. Dicarboxylic Glutamic aspartic Shared dicarboxylic Intestine AR aminoaciduria acid transport (glutamate-aspartate system (?) transport defect) (23165) -aminoaciduria - Taurine, (3-alanine No primary transport System defined by com- (hypertaurinuria) 3-AIB defect in man; taurinuria petitive inhibition in hyper- See text inmouse fl-alaninemia in man (McKusick [23740]) (& taurinuria phenotype in mouse). Renal glucosuria Glucose Glucose carrier Intestine normal AR Two forms; Km variant () (uptake) & low-Tm variant (Reubi (03260)' type A). Glucose- Glucose galactose Shared glucose- Intestine AR Minimal renal glucosuria malabsorption galactose carrier and no galactosuria under (23160)' (uptake) usual conditions. Diarrhea principal symptom; mimics disaccaridase deficiency Bartter syndrome Na A Na carrier? Erythrocyte AR Secondary juxtaglomerular (hypokalemic (K secondarily?) (Na content cell hyperplasia, normal alkalosis) (24120)' increased) blood pressure, secondary hyperaldosteronism & hypokalemic alkalosis Albright's hereditary Calcium Regulation of Bone XL (dominant) More then one allele (at osteodystrophy parathyroid hormone (or sex- different gene loci ?) Type I, (pseudohypopara- receptor mechanism influenced AD) no increase in urinary thyroidism, etc.) (or messenger system) cAMP after PTI-I challenge; (30080)' cortical adenyl cyclase present; circulating PTH in- creased. Type II, urinary cAMP response intact but not effective. Renal tubular transport 153

Table 1. (Continued). Trait Presumed (or Other Apparent (McKusick Substance possible) mutant tissues inheritance cat. No.) affected gene product affected pattern Comment2 Familial hypo- Phosphate carrier Intestine? XL (dominant) Low Tm1; "negative" phosphatemic rickets (luminal membrane?)Bone? reabsorption of phosphate (30780) (retaining cellular can also occur, suggesting pool) excessive backflux from cell to urine. Trait responds to phosphate replacement. Renal tubular Bicarbonate Proximal tubular Intestine XL (?) Prevalent in males. Low acidosis (type II) mechanism for (probable) (recessive) capacity (Tm) for reab- (31240) reclamation (or AR?) sorption of HCO3. Daily requirement of -HCO3is 10 to l5mEq/kg. Renal tubular acidosis Bicarbonate Late proximal or AR (?) "Dislocation"(Km) type (type III) early distal tubular assoc. with hypokalemia & (26720) mechanism for re- osteomalacia/rickets. clamation Renal tubular Distal tubular collect- AD Bicarbonate reclamation acidosis (type I) ing duct hydrogen normal. Ability to se- (l7980) ion secretion crete H in distal system mechanism vs. gradient is defective. Daily requirement of HCO3 is Ito 2 mEq/kg. Distal renal tubular H Carbonic anhydrase B Erythrocyte AR Progressive nerve deaf- acidosis with nerve ness is marker finding. deafness (26730)2 RTA responds to HCO3-, Ito 2 mEq/kg. insipidus [-120 Antidiuretic hormone XL Two alleles (different (vasopressin resistant) receptor mechanism (recessive) loci?). Type 1, no urinary (30480) (or messenger system) cAMP response to ADH. ? Type II, female proband urinary cAMP response intact but not effective. Idiopathic Fanconi Generalized effect on Coupling of energy (?) Secondary to AR Adult-onset (22780) and syndrome all solutes & water Tight junction integrity renal phenotype (& AD?) infantile childhood forms (22770) (?) (24270) are differentiated. (22780) Basic defect unknown; probably several alleles.

Symptomatic forms of Fanconi syndromes: a) Same Cystinestorage Same AR Several alleles. Infantile Type I(2l980) (secondary response) (lysosomal defect) (for each type) (type 1) & adolescent (type Type II (2l990) with secondary 11) forms have differing Type Ill (22000) damage to tubule & rates for onset of nephro- glomerulus (later) pathy. "Adult" form (type Ill) has no nephropathy. b) Hereditary fructoseSame -I-phosphate Same AR Nephropathy dependent on intolerance (+ fructose) aldolase (with secondary (hepatic cirrhosis) phosphate depletion in (22960) effects on cellular ATP) kidney. Responds to fruc- tose withdrawal. c) Same Galactose- I-phosphate Same AR "Galactosemia" due to (23040)2 (+ galactose) uridyltransferase (with (cataracts, CNS) secondary effects on does nothaveFanconi cellular ATP) syndrome. Fanconi syn- drome responds to galactose withdrawal. d) Hereditary Same (+ Unknown (with secon- Same (hepatic AR responds metabolites) dary effects on cellular cirrhosis) to tyrosine restriction. (27670) ATP) 154 Scriver et a!

Table 1. (Continued). Trait Presumed (or Other Apparent (McKusick Substance possible) mutant tissues inheritance cat. No.) affected gene product affected pattern Comment2 e) Wilson's disease Same (with proximal Unknown (seondary 1-lepatolenticular AR Fanconi syndrome responds (27790) and distal RTA) effects on cytochromedegeneration to depletion of copper oxidase system?) storage. f) Lowe's oculocerebro Generalized disfunction Unknown An oculocerebro- XL Basic defect still renal syndrome with defective urinary intestinal-renal (recessive) unknown. Treatment for (30900) ammonia production syndrome (involving tubular reclamation tissues with high defects does not improve y-glutamyl cycle mental retardation or the activity?) cataracts & hydro- phthalmia.

Vitamin D dependencyGeneralized defect. 25-hydroxyvitamin D-(Vitamin D hormone AR Nephropathy dependent on (pseudodeficiency (Secondary response) lo-hydroxylase synthesis occurs in PTH excess & hypocalcemia rickets) (26470) kidney mitochon- (phenocopy occurs in vita- dna; deficiency af- min D deficiency.) fects intestinal absorbtion of cal- cium & initiates PTH response.)

Miscellaneous a) Glucoglycinuria Glucose & glycine Unknown (the two AD Asymptomatic. Normal-Tm (13810) solutes do notsharea (type-B) glucosuria. Possi- common carrier. bility that this is a heterozygous manifestation of a Fanconi-like tubulopa- thy merits consideration.

b) Ludcr-Sheldon Generalized amino Unknown AD Same as for previous entry. syndrome (15250) acids glucose & Symptoms of Fanconi phosphate syndrome have occurred in probands. c) Rowley-Rosenberg Generalized Unknown AR Associated components of syndrome (26850) aminoaciduria syndrome; growth, retarda- tion, muscular hypoplasia, pulmonary involvement & right ventricular hyper- trophy.

A catalogue of 28 inherited disorders of tubular transport is provided herein. Each disease included in the table has a proven (see footnote 3) or suspected pattern of inheritance and is to be found under its own five-digit catalogue number in the appropriate section (autosomal dominant, 10,000 series; autosomal recessive, 20,000 series; and X-linked, 30,000 series) of McKusick's Catalogueof Mendelian Inheritance in Man [161];selected literature citations are given with each entry. Vignettes covering the major clinical features and the genetic aspects of many of these traits will also be found in the Compendiumof Birth Defects, publishedby the National Foundation-March of Dimes [1621. Numerous probable inborn errors of tubular transport are not included in Table I because their tubular manifestations have yet to be clearly understood. These conditions (and their McKusick catalogue number, if available) include the following: Pyroglutamicaciduria (26613)due to a defect in glutathioñe synthesis and secondary overproduction of the pyroglutamic acid (5-oxo-proline) intermediate of the y-glutamyl cycle; idiopathic hypercalcuria(hyperexcretoryform) (23810); some patients with Leigh'snecrotizing encephalopathy (25600); Familialnephrosis (25630)with a generalized tubulopathy; and Immerslund'ssyndrome(26110) with unexplained tubular proteinuria. 2 hets=heterozygote,homoz =homozygote. Proven pattern of inheritance.

acids [6, 32]; nonepithelial cells, such as cultured skin tracellular peptidases; the free amino acids then enter flbroblasts, do not have the defect [33]. Intestinal metabolic pools or leave the cell. The normal plasma uptake of various dipeptides containing amino acids response curve in the Hartnup trait, following dipep- affected by the Hartnup trait is not impaired [34—36], tide feeding, indicates that cleavage and absorption of because dipeptides are transported in the gut and dipeptide-derived amino acids are normal, therefore, kidney, at membrane sites which are independent of efflux of the released free amino acids across the those used by their constituent free amino acids [37, basilar plasma membrane must be intact, and the 38]. Following uptake, dipeptides are cleaved by in- defect in transepithelial absorption must be confined Renal tubular transport 155 to a specific uptake carrier serving the large ently carried-mediated. When the maximum rate of "Hartnup" group of amino acids on the luminal reabsorption (Tm() is reached, the limiting com- membrane (viz. Fig. 26—21 in [6]). In the absence of ponent is, accordingly, uptake at the luminal mem- any comparable studies of dipeptide reabsorption by brane (or intracellular metabolism), not permeability kidney, we reason by analogy that a similar location at the basilar membrane. of the defect in proximal tubule epithelium accounts Tubular reabsorption of D-glucose has long been for the specific Hartnup hyperaminoaciduria. known to observe a Tm in mammalian kidney in vivo Renal glucosuria and glucose-galactose ma!- [46, 47] (Fig. 3). The observed "splay" in the titration absorption. Two autosomal recessive disorders of curve relating filtered load to the threshold for gluco- transport [39, 40] reveal the likelihood that suria (FminG), and the reabsorption rate, has evinced renal tubular epithelium possesses two (or more) ge- much argument. Some consider the observation to be netically distinct mechanisms for glucose transport; compatible with ordinary Michaelis-Menten kinetics this may not be the case in the intestine. The for uptake by a single saturable system; others take it transport defect in each trait almost certainly in- as evidence for anatomical heterogeneity among the volves an uptake system on the luminal membrane. nephrons performing the functions of filtration and The characteristics of D-hexose transport in kidney reabsorption [1]. We believe a resolution of this clas- are complex. Hexose transport mechanisms provide sical argument among renal physiologists is to be substrates for the metabolic systems yielding energy found in the hereditary disorders of glucose trans- for basal renal work, and also for a component of port. renal transport work itself [41]. enter prox- Reubi [48] observed two variations upon the nor- imal tubular cells, in vivo, from luminal and basilar mal titration curve in familial renal glucosuria. The poles [26, 42]. However, luminal and basilar mem- type-A variation is characterized by a low FminG and branes clearly possess differing characteristics for low Tm(;; type-B glucosuria has a low FminG but a hexose transport. By means of the sudden-injection, normal TmG (Fig. 3). A kinetic interpretation [3, 6, multiple-indicator dilution method [26], it has been 49] of glucose reabsorption ascribes the type-A vari- shown that there are D-glucose-preferring (G) sites ant to a reduced number of transport sites, while the (shared with D-galactose) and D-mannose-prefer- type-B variant reflects reduced affinity of the hexose ring (M) sites in the luminal membrane [42]. carrier for glucose. With this in mind, should a hered- Confirmation of these data, and evidence for Na1- itary disorder of glucose transport involve the in- dependent D-glucose transport at the luminal mem- testine as well as the kidney, or should the type-A and brane, has been obtained by a stop-flow micro- type-B phenotypes be found in the same pedigree, perfusion method [43] and by kinetic analysis there would then be little room for the nephron heter- of isolated preparations of brush border membranes ogeneity hypothesis as an explanation for splay in [44]. It is likely also that G sites of luminal and solute absorption curves. basilar membranes are not identical [26, 42]. Further- When in vivo data for hexose transport are com- more, this delineation of hexose transport in the lu- pared with data obtained by the kidney cortex slice minal membrane of kidney leads one to believe that method, additional points of interest are found. its properties are qualitatively different from those The slice method, which exposes only the basilar previously defined for the luminal membrane of in- membranes of proximal tubular epithelium to the testinal epithelium [45]. incubation medium (see following), reveals active By means of a technique using isolated, perfused transport (uptake) of hexoses across these mem- proximal tubule segments, the characterisitcs for true branes [50. 51]; this finding is analogous to the transcellular transport of D-glucose have been re- evidence for active transport in vivo [19]. The slice vealed. D-glucose can be transported against a chem- data also reveal more than one type of D-hexose ical gradient out of the tubule lumen. Active trans- uptake [48]; this finding is also in keeping with the in port is therefore a property of the luminal membrane. vivo observations [26, 42]. Kleinzeller [49] has delin- Glucose reclamation occurs predominantly in the eated a number of homologies between D-hexose convoluted portion of the proximal tubule but it also uptake in vitro by kidney slices and during absorption takes place in the pars recta. The unidirectional fluxes in vivo. However, Silverman, Aganon and Chinard of D-glucose, from cell to lumen, and from cell to [26, 42] have shown with different techniques that peritubular fluid, were dissected from the net trans- basilar membranes and luminal membranes are not epithelial fluxes; outward flux at the basilar pole ex- identical in their hexose carrier properties. Therefore, ceeds exodus at the luminal border by a four-fold the genetic probes of glucose transport in man as- margin. Basilar permeability to D-glucose is appar- sume great importance since they may inform us 156 Scriveret a!

Normal & variant Type—A Type—B Glucose—gulactost' gbucosuria glucosuria maiabsorption Fig. 3. Glucose titration curves showing maximum transport (reabsorption)rate (TmG),and venous plasma threshold for glucosuria (Fmin0), in normal subjects and in the hereditary glucosurias. Theoretical type-A and type-B glucosuria titration curves according to Reubi (1954) are Hots shown in left panel; note that the hypo- thetical curves do not conform to the ac- P tual observations in vivo shown in the ) three adjacent panels. Actual type-A Tm6 glucosuria data (2nd panel from left) were 0 redrawn from Elsas, Bossy and Rosenberg Fbomn Fmin6 ta Fmin6 [391; type-B glucosuria data (3rd panel fromleft)were redrawn from Elsas and Rosenberg [49]. The data for renal han- Fm in6 E dling of D-glucose in glucose-galactose I I I I malabsorption (right panel) are taken from 0 250 5000 250 500 0 250 5000 250 500 Elsas et al (0) [40], Beauvais et al () [54] and Abraham et al (A) [55]. Filtered glucose, nag ;ni'C /1. 73rn 2 about the disposition of hexose carriers in the renal "severe" type-A homozygotes retain about one-third tubule in a manner not revealed by any previous the normal Tm0 (Fig. 3); and that type-B probands study. (subject 11-4, Ho!, [58]) have a normal threshold for Glucose-galactose malabsorption is characterized by glucosuria (FminG) which is about one-third the nor- severe impairment of hexose transport in the gut and mal Tm(. minimal deficit in the renal tubule; familial renal The combined evidence both from physiologic ob- glucosuria is a disorder of renal tubular reclamation servations in several mammalian species [19, 26, 42, of D-glucose without an intestinal defect. 47], and from the genetic studies in man suggests the In glucose-galactose malabsorption, an uptake sys- following synthesis and hypothesis. Efficient reclama- tem, which resembles the G-system in kidney brush tion of hexose by the proximal tubule is controlled by border [26, 42],isdeleted in the luminal membrane of the luminal membrane. Three types of luminal hexose intestinal epithelium of homozygotes [40, 52—54]. On carriers operate in parallel in this capacity. One is the the other hand, renal titration studies reveal little [40] M carrier [26, 42], a diffusional system of no further or no [54, 55] deviation of TmG from normal in interest to us here. The second, which we will call the homozygotes (Fig. 3). Endogenous glucose metabo- G1 system, corresponds to the G-system of Silverman lism is normal in the disease [55, 56] and the hexose et al [26, 42]; this carrier interacts with glucose and transport defect is not expressed in the erythrocytes, galactose and is under control of a gene we will call as a representative of nonepithelial tissues [57]. the "glucose-galatose" carrier (G1) locus. We pro- In contrast to these findings in glucose-galactose pose that the integrity of the G1 carrier is unmasked malabsorption, there is no aberration of glucose in one condition—severe type-A glucosuria which transport in the intestine of homozygotes with fami- causes deletion of a second (G2) glucose carrier. lial renal glucosuria [58]. However, the renal titration Evidence for the third carrier, which we call G2, is data reveal a very complex picture compared to the revealed in homozygous glucose-galactose malab- renal findings in glucose-galactose malabsorption. sorption which causes deletion of the G1 carrier and (Fig. 3), Studies in three unrelated pedigrees [39, 58] "unmasks" the activity of the G2 carrier. have revealed a "mild" form of homozygous type-A We estimate that the maximal capacity of the G1 glucosuria inherited from "silent" heterozygotes carrier is about one-third the total capacity for D- (COY pedigree, [58]); "severe" type-A glucosuria in- glucose transport in the normal nephron (Fig. 3). herited from "mild" type-A glucosuric heterozygotes From observations in type-A glucosuria, we deduce (Hold pedigree, [39]) and type-B and "severe" type-A that the affinity of the G1 system for D-glucOSe is less glucosuria in sibs of a pedigree in which there are than that of the principle carrier (G2); this fact is relatives with "mild" type-A glucosuria (Hot ped- revealed by the displacen:mt of the titration curve for igree, [58]. A compelling argument for at least three the residual glucose transport in homozygous type-A mutant alleles, at a gene locus specifying a renal glucosuria (Fig. 3). The G2 carrier appears to be very glucose transport system, is offered by these observa- specific for D-glucose and it has a high affinity and tions. Of particular interest is the evidence that even high capacity for D-glucose transport (Fig. 3). Renal tubular transport 157

Mutation at the G1 locus causes glucose-galactose butions to occur in kidney cortex slices. Specific, net malabsorption; mutation at the G2 locus causes fami- flux orientations across the isolated luminal mem- lial renal glucosuria. The G2 locus may not be ex- brane of the proximal tubule are also necessary to pressed in the intestine according to physiologic and achieve the independent spatial distribution of AIB genetic evidence; or if there is an intestinal G2 carrier, and PAH. The findings indicate that the peritubular, it has not been affected by the which per- cytoplasmic and luminal pools of proximal tubules in mit homozygotes with renal glucosuria to survive and slices exist in series, and that net reabsorptive and to be recognized [58].Morethan one mutant allele secretory fluxes across the luminal membrane remain has already been described for the proposed G2 locus. intact in slices. One type impairs the capacity of the G2 carrier for D- The unique topology of slices was put to use in our glucose and causes "mild" or "severe" Reubi type-A study of hereditary taurinuria in the mouse. Taurine glucosuria; genetic and phenotypic evidence [40, 58] is an inert metabolite in mouse kidney and is there- implies that there exist two different type-A alleles in fore a useful probe of its transport functions. We this respect. The other allele alters the affinity of investigated three inbred strains of mice available the 02 carrier for substrate and causes type-B from the Jackson Laboratory, Bar Harbor, Maine: glucosuria. A/J is a normal taurine excretor (taut±) and Hereditary taurinuria. We divert briefly to discuss C57BL/6J and PRO/Re are two homozygous hyper- the first of three animal models of hereditary loss of taurinuric strains (taut-). Urine taurine is ten-fold organic solute. Such examples are rare [3] and, when greater in taut_ animals, while plasma taurine is com- they occur, the opportunity they provide for in vitro parable in the three strains. Net tubular reabsorption investigation is valuable. of taurine is 96.7 1.3% (mean of the filtered We have used the kidney cortex slice method and in load in A/J, and 83.9 + 0.8% and 78.7 5.0% in vivo clearance studies, in parallel, to delineate the C57BL/6J and PRO/Re, respectively. Intracellular location of an inherited impairment of taurine rec- taurine concentration in outer cortex in vivo is similar lamation [59, 60] which has been observed in the in the three strains. This important finding, when mouse. Taurine is the prevalent 3-amino acid in interpreted according to the kinetics described in Fig. mammalian body fluids and it serves as a marker for 1, indicates that backflux from an expanded in- a well-documented, 3-amino-acid-preferring trans- tracellular pool of taurine is not the cause of hyper- port system in the kidney [38, 60—63]. taurinuria. Wedeen and Weiner have shown that kidney slices Other in vivo findings were of interest. fl-Alanine is do not necessarily expose proximal tubule luminal a competitive inhibitor of taurine transport in kidney membranes to uptake from the incubation medium [38, 60, 63]. -Alanine inhibits taurine reabsorption [64-66]. In response, some investigators have been in vivo, in both taut+ and taut_ strains, indicating the quick to reject the slice method as a useful technique retention of a residual taurine transport activity in to study solute transport in kidney. We have less the nephrons of the latter. reactionary views; we believe the slice method pro- Steady-state uptake of taurine at physiologic con- vides an opportunity to study pools-in-series (Fig. 4) centrations (about 0.5 mM) by thin, outer-cortex which can highlight the topology of transepithelial slices is clearly greater in taut_ than in taut+ tissue. transport. Quick-freeze, soluble-label autoradiog- The higher uptake ratio by taut slices is not the raphy has revealed the distribution of tritium-la- result of altered efflux at the basilar membrane slice; belIed, inert solutes after their uptake by cortex slices efflux from slices is the same in taut+ and taut [64] (Fig. 4, top half). Inulin is confined to an extra- strains. cellular space in contact witji basilar and lateral i-Alanine, which shares the taurine transport sys- membranes of proximal tubular epithelium; only in tem in vitro [38] and in vivo [60—63], is also taken the distal tubule does inulin penetrate the lumen, a- up more avidly by taut slices. /-Alanine which is Aminoisobutyricacid (AIB) is concentrated within vigorously oxidized by taut kidney cortex slices [38] cells of the convoluted and straight portions of prox- is oxidized less by taut_ slices but normally by slice imal tubule, little being found in the lumen; if AIB homogenates with disrupted architecture. fluxes into the luminal pool, it is avidly reclaimed in These data suggest a block in concentrative uptake vitro, p-Aminohippuric acid (PAH) is accumulated of taurine at the luminal membrane of proximal tu- maximally in the lumen of straight segment but also bule (Fig. 4). l-Taurine is not reclaimed efficiently in the convoluted portion of proximal tubules. Integ- from the luminal "lacuna" of slices (the innermost of rity of the punctate contacts between epithelial cells the three poo1s in series) once it has fluxed from cell at their luminal pole is essential for these solute distri- into lumen. Retention of solute in this pool leads to 158 Scriver et a!

Spaces

HF Lumen

Inulin AIB PAH

Tau+ Tau

DR

Time Time

A/i C57BL PRO/Re Renal tubular transport 159

the higher uptake ratio observed in vitro. The in vivo data indicate "sequestration" of taurine in the urine pool, a finding also compatible with a luminal mem- brane transport defect. In vitro and in vivo data are thus concordant. We believe that in vivo and in vitro data have been used in parallel, in this case, for topological assignment of a hereditary transport de- fect to a specific membrane surface in the mamma- lian nephron. Appropriate studies—both with quick- freeze, soluble-label autoradiography to discern whether labelled taurine accumulates in excess in the lumen of taut- slices, and with isolated brush border membranes to study taurine binding—will either af- firm or dispute these conclusions. lB. Defects in the integrity of the (luminal) plasma Fig. 4. An interpretation of enhanced taurine uptake as observed in membrane to efflux. No hereditary disorder of tubular kidney cortex slices obtained from hyperlaurinuric mice (C57BL/6J and PRO/Re strains designated taut in text), when compared with transport has been proven in this class. However, normal mice (A/f, designated taut). Upper half. Figure shows there is precedent for genetic control of exodus in distribution of 3H-inulin, 3H- (or '4C-) a-amino-isobutyrate (AIB) prokaryocytes [67, 68]; and two disorders in man are and 3H-p-aminohippuric acid (PAl-I) after incubation of rat cor- tex slices. Inulin does not penetrate the lumen of the proximal reasonable candidates for this type of defect. tubule, and is confined to extracellular peritubular and glomerular Wong, Kashket and Wilson [671 describe a genetic spaces; AIB is concentrated by proximal tubule cells. PAH is maximally concentrated in the lumen of proximal tubule. A con- defect of thiogalactoside transport in Escherichia coli, cept of "pools-in-series" is implied by these findings (see text). and Hectman and Scriver [68] found a mutant strain (Rephotfigraphed from Wedeen and Weiner 163].)Lowerhalf: of Pseudomonasfluorescens defective in -alanine ac- Figure shows the enhanced distribution ratio (DR) during time cumulation. Both mutants are unable to concentrate course for taurine uptake by slices. The. interpretation for this anomaly is shown in the two sketches at the bottom; it utilizes the the relevant free solute against a gradient in the in- "pools-in-series" hypothesis. The shaded area indicates the inulin tracellular pool, yet both retain the pertinent carrier space; the adjacent clear area is the cytoplasmic pool; the luminal in their plasma membranes. It was surmized in both pool is drawn at the apex bounded by luminal membrane and tight junctions (shown by solid bars). The luminal pool can be entered that the mutant carrier was unable to prevent solute from the extracellular space only through the cytoplasm. Taurine efflux following uptake into the intracellular pool. becomes trapped in the luminal pool (stippled area) of taut_ slices. Four relevant permeation fluxes are shown: influx across basilar With these precedents in mind, it has been proposed membrane (J1); efflux into luminal pool (J2); reclamation flux from [69, 70] that classical cystinuria and X-linked hypo- luminal pool (J3); and efflux across basilar membrane (J4). J3 phosphaturia may be disorders in which the relevant, exceeds J2 under normal conditions so that little amino acid is specific carriers in the luminal membrane are mutant retained in the luminal pool. Assuming J3 =[Tau1JX permeability1,, where Ic is the lumen-to-cell movement, accumula- so as to allow abnormal backflux, while still retaining tion of taurine in luminal pool will occur if luminal membrane their carrier functions for facilitated entry. permeability1, is decreased, all other events being unchanged. The change in J3 in taut_ kidney is presumed to be the result of an Cystinuria is an autosomal recessive disorder char- hereditary impairment of transport at the urinary surface of the acterized by defective transport of the diamino dicar- taurine carrier in the luminal membrane (from Chesney RW, Scri- boxylic amino acid cystine, and the diamino mon- ver CR, Mohyuddin F, J. Cnn Invest, vol. 57, 1976, in press). ocarboxylic amino acids lysine, ornithine and arginine [71]. Two or more mutant alleles exist at the gene locus controlling the transport function in- volved in cystinuria [72]. Net tubular reabsorption of the four amino acids is greatly impaired in mutant homozygotes and in "genetic compounds". It is par- tially impaired in heterozygotes for two of the three proposed alleles [72]. While FminLYS and TmLYS may both be zero in homozygous cystinuria, Lester and Cusworth [73] have shown that lysine infusion will still provoke enhanced excretion of ornithine, arginine and cystine. They, and others [74, 75], have also shown that the endogenous renal clearance of cystine and the other affected amino acids can exceed the clearance of in- 160 Scriveret a! ulin, so that the "negative reabsorption"3 of amino enters the cell [77, 88]. Each of these latter acids occurs in cystinuria. On the other hand, observations suggests that intracellular cysteine is the cyst(e)ine uptake is only slightly depressed, if at all, in source of excess urinary disulfide in cystinuria. slices prepared from human cystinuric kidney [72, 76, These disparate observations at first defy coherent 77, 78]; the representative dibasic amino acid lysine interpretation. Moreover, the genetic evidence (Table observes a reduced rate of uptake without change in I) tells us that mutation, in the form of isolated its apparent Km for uptake [72, 76, 78]. The carrier hypercystinuria, and as hyperdibasicaminoaciduria, retains its ability to interact with the dibasic amino can impair tubular reabsorption of cystine and the acids. By contrast with these findings in kidney, the dibasic amino acid quite independently, a finding uptake of both cystine and the dibasic amino acids is which is explicable only if we assume that the luminal clearly impaired in the intestine in homozygous cys- membrane contains reactive sites which are tinuria [721. cyst(e)ine-specific and dibasic amino acid-specific, The original hypothesis of Dent and Rose [79], and each under the control of separate genes. However, to of Robson and Rose [80], stated that cystinuria is a reiterate, the physiological evidence [83] tells us that defect in a selective transport (uptake) system of the a third species of uptake site in the luminal tubule shared by cystine and the dibasic amino acids. membrane of the mammalian nephron is shared by However, subsequent work in vitro has shown that the five naturally occurring amino acids, but that this cystine and the dibasic amino acids do not share a site is apparently not present in the basilar membrane common system for uptake across the basilar plasma [61, 81, 82]. Loss of only the luminal membrane membrane of the tubule as it is exposed in slices of system [83] (shared by cysteine, cystine, lysine, orni- mammalian kidney [60, 81, 82]. On the other hand, thine and arginine) would indeed account for the cystine, cysteine and the three dibasic amino acids all cystinuria trait as we know it. But this simple inter- interact with each other at the luminal membrane, in pretation would not explain the stimultaneous obser- the normal proximal tubule, presumably on a shared vation of zero FminLYS, zero TmLYS, negative reab- site [83]. The discordance between the properties of sorption of amino acids and competitive interaction uptake sites on luminal and anti-luminal membranes between the four amino acids during reabsorption. is further ramified when efflux is considered. An alternative and seemingly unifying interpretation Schwartzman, Blair and Segal [84, 851 showed that is to propose that cystinuria is a defect in a shared cysteine and the dibasic amino acids shared a mem- effiux system of the plasma membrane so that the brane efflux site in kidney cortex slices. The relevant intracellular amino acids (cysteine, lysine, properties of the efflux system appear not to be dupli- ornithine and arginine) experience exaggerated cated completely at the corresponding influx site; nor exodus. This interpretation explains most of the in does the efflux carrier experience the customary prop- vitro data and would account for backflux into urine erties of counterfiow. These properties might be ac- from the intracellular pool to yield negative reabsorp- comodated by the behavior of carriers on the outer tion under certain conditions. The relevant amino surfaces of luminal and basilar membranes when ex- acids still interact on a shared luminal membrane posed in the slice model (see above, hereditary tauri- uptake system which permits competitive inhibition nuria discussion) except for the following facts. to occur under certain conditions. We know also that a mixed disulfide (cysteine- The second candidate for defective luminal efflux is homocysteine) is prominent in cystinuric urine [86]; X-linked hypophosphatemia (familial hypophospha- that the renal arterial:venous extraction ratio is nor- temic rickets). In this "phosphopenic" form of rick- mal for cysteine, and for cystine, in cystinuria [87]; ets [70] tubular reabsorption of phosphate is selec- and that the intracellular cysteine:cystine ratio in nor- tively affected [891; a corresponding defect in mal human and cystinuric renal cortex is normally intestinal absorption may also exist [901. The vener- about 10: 1, regardless of the form in which cyst(e)ine able hypothesis of Albright ascribed the defect in tubular reabsorption of phosphate in familial hypo- phosphatemia as a consequence of secondary hy- The term "negative reabsorption" is sometimes used when it is perparathyroidism initiated by a primary disorder of found that the excretion rate (UV) of a solute exceeds its load in vitamin D-dependent calcium absorption in the in- glomerular filtrate (F), whereas the customary relationship is F > uv. The term "net secretion' could also be used in this instance testine [911. However, this is implausible as there are provided it was not implied that UV > F was the result of a no elevated serum concentrations of C-terminal im- specific energy-dependent secretion process. When used in this munoreactive PTI-I in mutant hemizygotes unless cal- paper, negative reabsorption implies an abnormal finding; secretion is reserved for normal functions in which UV > F is the cium homeostasis has been altered [70]. Moreover, customary relationship. the anticipated generalized defect in tubular reab- Renal tubulartransport 161 sorption of solute which accompanies "calciopenic" luminal membrane effiux hypothesis, in cystinuria secondary hyperparathyroidism [70] is not observed and X-linked hypophosphatemia for example, should in the X-linked trait. Furthermore, normal urinary consider this possibility. cyclic-AMP excretion under basal conditions [89, 92] 2. Disorders of intracellular pool size. Interference is partial evidence against the defect being a selective with metabolic disposal ("run out") of solute could hyperresponsiveness of the tubule to normal levels of lead to expansion of the intracellular pool in the circulating PTH [93]. Mutant hemizygotes also mani- presence of continuing uptake (Fig. 2). Conse- fest a low Tm1 and negative reabsorption of ortho- quently, the effective concentration of solute which phosphate [89, 92, 94]; these two findings focus interacts with the luminal carrier at the intracel- interest on a primary transport defect involving lular interface could increase; under this condi- orthophosphate. tion, backflux into the lumen and the moving column The fortunate discovery by Eicher and Southard, of urine must increase, all other events being equal, as at the Jackson Laboratory, of an X-linked mutation the efflux component at the luminal membrane re- which causes hypophosphatemic rickets in the mouse sponds to the elevated, internal solute concentration has allowed us to investigate, in preliminary fashion, (Fig. 1). In this respect, intracellular metabolism of the mechanism of hyperphosphaturia in this pre- solute comes to influence its transtubular migration. sumed model of the human disease. Renal tubular One cannot avoid thinking of the possibility, that reclamation of orthophosphate is equally diminished under this circumstance the Tm of a solute may be in the mutant mouse and in man. Concentrative up- influenced by its renal metabolism [97, 98]. take of phosphate by cortical and medullary slices Renal tubular reabsorption of amino acids has appears to be normal in the mutant hemizygous been carefully measured in and sar- mouse; and the intracellular concentration of total cosinemia, two blocked-catabolic mutant states in and inorganic phosphate also appears to be normal man, to determine whether impaired catabolism of [95]. the solute impedes its reabsorption [99—102]. In A hypothesis is proposed to accomodate the find- phenylketonuria the hepatic conversion of phenylala- ings in X-linked hypophosphatemia. We suggest that nine to tyrosine is almost completely blocked [103]. the mutation permits excessive luminal efflux (back- hydroxylase activity, with about one- flux) of cytoplasmic phosphate ion to account for fifth the specific activity of the hepatic enzyme, is negative reabsorption in the trait. We also suggest found in human renal cortex [104]. However, it is not that the equilibrium of phosphate between four pools known whether the renal enzyme is an isozyme or in series may determine its net transepithelial flux; the whether it is deficient in phenylketonuria. It is known luminal, cytoplasmic, mitochondrial and peritubular from measurement of phenylalanine reclamation that spaces comprise the four phosphate pools. Partition there is no abnormality of tubular reabsorption of of phosphate in the mitochondrial pool may be found this amino acid in phenylketonuria [99, 100, 101]. to be important in the transepithelial movement of A similar observation has been made for sarcosine phosphorus, modulating it in a manner analogous to reabsorption in sarcosinemia [102]. It is likely that that proposed for transepithelial movement of cal- less than 10% of total body sarcosine oxidation takes cium [96]. The hypothesis requires us to know place in mammalian kidney [102, 105]; a severe loss whether concentration of phosphate in the cytoplasm of sarcosine oxidation in one of our patients was not available to backflux is dependent to any extent on accompanied by impaired renal reabsorption of this the amount in the mitochondrial pool. It may be of amino acid [102], indicating that renal oxidation of significance that mitochondria, particularly of prox- this amino acid is unimportant in its reabsorption. imal tubule cells, are palisaded at the basilar mem- Accumulation of sarcosine in the mutant state ac- brane [7], forming an interface between peritubular tually enhances glycine reabsorption [102] even and cytoplasmic pools. Events which diminish or in- though sarcosine and glycine interact competitively crease phosphate activity in mitochondria may come on their shared uptake sites [6, 102]. Counterfiow to influence phosphate activity in cytoplasm in a between the raised intracellular sarcosine and urinary series model of transport and may help to explain the glycine on the luminal carrier appears to be a satisfac- various effects of calcium and PTH infusions re- tory explanation for the behavior of glycine reabsorp- ported in X-linked hypophosphatemia [89, 92, 93]. tion in sarcosinemia. The latter finding also suggests It is implied that a disorder of luminal membrane a normal backflux exchange activity at the luminal backflux could be expressed in any portion of neph- membrane in sarcosinuric kidney and no loss of car- ron and could come to influence net reabsorption in rier integrity, It follows from the examples of phenyl- both the proximal and distal tubules. Any test of the ketonuria and sarcosinemia that an initial increase in 162 Scriver et a! the amount of an amino acid in its peritubular and PRO/Re mutant. The in vivo topology of trans- luminal pools does not come to influence the capacity epithelial transport permits this phenomenon to be for its net tubular reabsorption, beyond the normal observed in PRO/Re kidney whereas the in vitro kinetics of concentration-dependent uptake [13] at topology does not. the plasma membrane. With the hindsight afforded by the PRO/Re Another animal model (the third and last to be model, it is of interest that the venous plasma thresh- mentioned in this review) has provided a valuable old (FminPRO) for prolinunia in some human ho- opportunity to examine directly the effect on net mozygotes with autosomal recessive tubular absorption of an initial increase in appears to be slightly lower than the threshold ob- intracellular pool size [97]. The mutant homozygous served in normal persons [108]. That finding suggests PRO/Re mouse has hyperprolinemia and less than 1% that renal proline oxidase activity may subtly in- of the normal proline oxidase activity in kidney [106, fluence proline reabsorption even in man. It is also 107]. Under normal conditions, proline is reclaimed apparent from these observations that the classical avidly from urine by the mammalian nephron [61, Tm concept will require reevaluation since renal 108, 109]; the kidney also takes up proline from pen- metabolism of a solute does indeed influence its rate tubular plasma in vivo [110]. In the PRO/Re mouse, of tubular absorption. the endogenous proline concentration is eight times 3. Disorders of exodus at the antilurninal pole. This normal in plasma and four times normal in kidney type of disorder would impede net transtubular mi- cortex; by contrast it is fifty times normal in the urine gration of solute (Fig. 3). The result would be en- [97]. The in vivo, steady-state uptake of proline across hanced intracellular accumulation of the substrate the basilar plasma membrane is not impaired and leading to exaggerated backflux at the luminal sur- efflux at this surface is normal; the integrity of the face. Thus far, only experimental models of this well-documented proline transport systems in these mechanism have been reported. membranes [Ill, 112] is also retained in PRO/Re The rapid-injection, multiple-indicator dilution kidney. Appropriate studies also reveal the luminal technique has shown [10, 42, 113] that most solutes membrane uptake of proline to be intact in the which interact competitively to augment their frac- PRO/Re mouse [97]. tional excretion do so at the luminal membrane. Proline oxidation in normal mouse kidney is of However, an equivalent competition between solutes such large capacity that the intracellular proline pool during unidirectional exodus at the basilar plasma is kept at a low level [97]; consequently, the normal membrane [84, 85] could also impede net metabolic outflow comes to influence proline uptake reabsorption. The effect of artificially blocked exodus rather dramatically. Normal slices do not observe on fractional excretion has been studied in the ligated any expansion of the soluble proline pool until the ureter preparation in vivo [31, 114]. L-Arginine en- external substrate concentration is greatly elevated; hances renal clearance of L-lysine in the ligated dog and saturation of tubular reabsorption in vivo (Tmpro) [31] in part by causing the intracellular lysine concen- is not observed in the normal mouse until the filtered tration to increase through competitive interaction proline load is augmented far beyond the limit which between lysine and arginine at the efflux site in the is required to delineate the Tmpr in man. basilar membranes. Corresponding experiments in The extraordinary hyperprolinuria which charac- the rat [114] showed that L-lysine provokes cellular terizes the PRO/Re mouse can be understood only accumulation of S-labelled products derived from by taking into account the elevated intracellular con- extracellular L-35 S cystine, and increases the renal centration of free proline [97]. When we apply Mi- clearance of L-cystine. These findings illustrate the chaelis kinetics (Fig. 1), we see excessive prolinuria in general theme of this review, that renal uptake and the PRO/Re phenotype as a simple consequence of transtubular migration are independent phenomena the primary elevation in the concentration of in- [115]; they also support the likelihood that blocked tracellular proline in vivo (depicted as SibinFig. 1). efflux of solute from the basilar pole of the epithelial Efflux of proline is thus enhanced on the same normal cell can impede net reabsorption. It remains for in- carrier that moves it normally into the cell either from vestigators to find a disease of tubular function in urine, or from peritubular fluid. However, when man which fits this interpretation. exodus occurs across the luminal membrane, proline will emerge into a moving column of fluid; since its Generalized disorders of tubular transport distal tubular reclamation is insignificant [6], the "lost" proline will appear in bladder urine. Fractional The renal Fanconi syndrome [116] of various eti- excretion of proline is accordingly elevated in the ologies, and the X-linked, oculo-cerebro-renal syn- Renal tubular transport 163 drome of Lowe, Terry and MacLachlan [117] are ex- amples of generalized disruptions of tubular + 200 transport activities. The Fanconi syndrome can be defined as the in- tegrated clinical manifestations, of whatever cause, resulting from excessive urinary loss of three or more + IOU classes of solutes including the amino acids, mono- saccharides, electrolytes (phosphate, calcium, bi- carbonate, potassium, sodium), uric acid and protein (particularly /3-globulins); water loss also occurs. 0 Substances not normally observed in any quantity in urine may also appear in excess, including lithium, magnesium, insulin and vitamin D and even ly- sozyme [118]. The increase in fractional excretion of these solutes is largely due to reduced net tubular reclamation. However, impairment of tubular secre- 200 300 tion is also observed in the syndrome; for example, I 50 TmPAHisdepressed [119]. Morphologic abnormalities may accompany the Fanconi syndrome. The swan-neck lesion, which in- 1.00 volves the initial portion of the proximal tubule, is a hallmark [120]. In all likelihood, this lesion is second- ary to the underlying cause of the syndrome. The 0.50 atrophy of the absorptive surface in the anatomial Normal increase lesion further reduces the membrane activity avail- U able for transport. Many species of transport sites are 100 200 300 nonetheless still active, in the atrophied epithelium, Filtered L —proline. glinoles113FF! 1/j. 2 since the normal competitive interactions between Fig. 5. Upper part: Renal titration curves relating proline reabsorp- molecules, which share reactive sites, are retained in tion to filtered load in normal subjects and three patients with tije the Fanconi syndrome [79, 121] (Fig. 5). idiopathic Fanconi syndrome. Thelowernormal range for Tmpao 1109]is shown by the shaded area. Reabsorption is depressed at all A reduced Tm value characterizes the tubular concentrations of proline in ultrafiltrate in the Fanconi syndrome. transport of various solutes in the Fanconi syndrome In two subjects, excreted proline exceeded the filtered load when (Fig. 5). This finding alone could be attributed to a the plasma proline concentration was raised indicating "negative" reabsorption under these conditions. Lower part: Proline inhibits simple reduction in the activity or the number of glycine competitively on a shared, high-capacity system for uptake in carriers in the membrane (equationl). However, the normal subject /109, 111/. The upper normal range for frac- negative reabsorption has also been observed at high tional excretion of glycine in the presence of proline is shown by the shaded area. Glycine excretion is abnormally increased, even at plasma solute concentrations in some patients with normal endogenous levels of proline in the Fanconi syndrome, the syndrome (Fig. 5) and this finding implies more indicating excessive inhibiton of reclamation, or excessive back- than the loss of carrier activity. The work of flux, of glycine on its tubular transport systems [109, 111]. The response to proline inhibition is unusual in two subjects and nega- Bergeron, Vadeboncoeur and Laporte with the ma- tive reabsorption occurred. The latter finding indicates retention of leic acid model of the Fanconi syndrome [25, 122, membrane sites at which counter-flow inhibiton of uptake may be 123] is of particular interest in the latter context. occurring simultaneously. Negative reabsorption of other amino acids was not observed indicating a selective interaction between Maleic acid is a noncompetitive inhibitor of solute proline and glycine at a site whose ability to retain solute has been uptake by kidney in vitro [1251, and it causes the compromised in the Fanconi syndrome. Fanconi syndrome in vivo [1241. Within a few hours of exposure to sodium maleate (400 mg/kg i.p.), concentration of the amino acid while its fractional there is a profound deterioration in the network of excretion is elevated [122]. Exaggeration of these perimitochondrial membranes [123]; this anatomical findings to the point of "negative reabsorption" can abnormality and the Fanconi syndrome appear and be imagined. then abate in parallel after the maleate injection. "Negative reabsorption" in the human Fanconi Following peritubular capillary injection of in syndrome (Fig. 5) and transtubular backflux of solute the maleate-treated rat, the amino acid appears in with reduced cellular accumulation in the maleic acid tubular urine at a rate indicating direct transtubular model in the rat suggest that two fundamental mech- flux [25]. There is also a reduction in the cellular anisms are potentially adrift in the syndrome. The 164 Scriveret al defect in tubular transport may encompass enhanced which appears to exist in HFI, highlights the energy exodus at the luminal surface after uptake of solute at requirements of mechanisms serving transtubular either pole (see' defect lA, Fig. 2). The mechanism transport. From this perspective it is worth recall- may be a loss of dynamic asymmetry in the carrier ing that solutes of different species which do not secondary to a disorder of energy metabolism and its share common carriers in kidney membranes can still coupling to the carriers. The other defect concerns impede each others' reabsorption [132] or uptake integrity of punctate contacts between cells; integrity [133] by mechanisms which do not involve is maintained by a variety of factors including cellular competitive interaction [133]. Competition for avail- metabolism. able energy may occur under such circumstances, a Hereditary fructose intolerance (HFI) [126] is a mechanism which may explain the unusually intense disease which illustrates how metabolism can be com- inhibition of amino acid reabsorption upon exposure promised in the Fanconi syndrome. The metabolic to solute loads in the Fanconi syndrome (Fig. 5). abnormality induced by fructose in HF! is initiated by cellular storage of fructose-i-phosphate (F1P) in Disorders of net tubularsecretion liver, kidney and bowel [126]. The Fanconi syndrome accompanies this metabolic event [127] waxing and There is no known primary hereditary disorder waning with exposure to and withdrawal of D-fruc- affecting the active tubular secretion of organic so- tose in the diet. Tissues normally assimilate fructose lutes, with the possible exception of some forms of and vigorously convert it to glucose and lactate. hyperuricemia. However, one can speculate that or- Fructokinase catalyzes the phosphorylation of fruc- ganic anions which escape excretion by nonionic dif- tose to F1P; disposal of F1P requires fructose-l- fusion [134], and which require conjugated transport phosphate aldolase which is present in splanchnic systems for their tubular excretion, are eligible sub- tissues and muscle, The aldolase (B-isoenzyme) of strates for inborn errors of tubular transport. The splanchnic tissues has strong cleavage activity toward recent finding that the proximal tubule contains li- F1P and strong condensing activity toward gandin [135], an organic anion-binding protein which dihydroxyacetone phosphate and D-glyceraldehyde is antigenically similar to the hepatic Y protein, yields phosphate [129]. HF! is characterized by deficient a candidate for an organic anion carrier in kidney. aldolase-B activity. An inherited deficiency of ligandin would provide a The critical enzymes required for fructose metabo- significant test of its importance in tubular secretion lism in human kidney are found in the cortex but not of organic anions. In the meantime, we can question in medulla [128]. This finding constitutes strong in- whether ontogeny of renal ligandin activity, similar direct evidence that renal cortex metabolizes fructose to that of hepatic Y protein [136], is an explanation by the FIP route, and that storage of FTP would for the abnormally low secretion of substances such occur in this region of kidney in HF!. The association as PAH, chloramphenicol and penicillin in the hu- of a precise anatomical location of fructose man newborn. An alteration in ligandin-dependent metabolism in kidney, and provocation of the Fan- organic acid excretion might even explain why a por- coni syndrome by exposure to fructose in HFI, is tion of gouty individuals with normal uric acid pro- intriguing. The normal activity of fructokinase per- duction have reduced net renal urate clearance with mits large amount of FiPto accumulate in HF! upon resultant hyperuricemia [137]. Finally, if it is an im- exposure to fructose. This response is accompanied portant organic anion carrier, we could understand by a fall in serum inorganic phosphorus while cellular better why hyperuricemia occurs in type-I gly- (ATP) is consumed to form cogenosis, diabetes mellitus, lactic acidosis, maple the F1P [129]. This relationship in HF! constitutes a syrup urine disease and fructose-i ,6-diphosphatase "futile" hydrolysis of ATP, with depletion of high- deficiency when there is endogenous accumulation of energy phosphate and inorganic phosphate pools arid the organic acids peculiar to each of these conditions it is analogous to the situation in galactosemia in [133]. which the Fanconi syndrome also occurs [130]. Inherited defects of distal tubular acidification are Parathyroid hormone plays an important modu- likely to represent true disorders of net tubular secre- lating role in the pathogenesis of the Fanconi syn- tion. Classical renal tubular acidosis (type I or cRTA) drome in HFI [131]. Administration of D-fructose occurs when the normal hydrogen ion gradient be- may not provoke the Fanconi syndrome in the ab- tween tubular urine and plasma is not maintained. sence of the hormone [131]. Parathyroid hormone Accordingly, there is an inability to lower the urine stimulates renal adenyl cyclase and ATP consump- pH no matter how marked the systemic acidosis tion. The "threshold" defect in ATP metabolism, [139]. Three abnormalities of hydrogen ion secre- Renal tubular transport 165 tion could occur: 1) H secretion may be normal but (ARVDD) [70] is a disorder in which the renal syn- with excessive back diffusion, so that the normal H thesis of la, 25-dihydroxycholecalciferol, a hormonal gradient (800: 1-1000:1) between urine and plasma is form of vitamin D, is apparently defective. A "calcio- dissipated; 2) H secretion may be normal but only penic" form [70] of postnatal rickets develops, ac- up to a limited capacity, above which no further companied by secondary hyperparathyroidism; a acidification can occur; 3) H secretion may be de- generalized defect in proximal tubular transport creased at any urine pH. Recently findings suggest follows. All abnormalities disappear when phar- that the third mechanism is an attractive explanation macologic doses of vitamin D, or quasi-physio- of cRTA [140]. Long ago, Pitts and Lotspeich [141] logic doses of la-hydroxyvitamin D analogues, are postulated that the elevated urinary Pco2 in alkaline given. The disorder of tubular transport in ARVDD urine, which is generally 35 mm Hg higher than in is related, in some manner, to the constellation of plasma, is the result of distal H ion secretion fol- vitamin D hormone and calcium depletion and of lowed by delayed, noncatalyzed, dehydration of PTH excess. Depression of extracellular calcium ion HZCO3 to form CO2 and H2. The gradient between [147] or elevation of cytoplasmic calcium [148] in- urine and plasma was assumed to be dependent on creases plasma membrane permeability. Calciotropic impermeability of the collecting duct to C020. Pak hormones which can modify cytoplasmic calcium al- Poy and Wrong [142] were the first to observe that ter tubular transport of amino acids and other solutes the normal Pco2 gradient was absent in patients with [149]. Depression of cytoplasmic calcium also in- distal RTA. This finding was extended by Halperin et creases tight-junction permeability [150]. Depression al [140] who examined the urinary Pco2 gradient of cytoplasmic calcium is likely to occur in ARVDD, after loading their patients with bicarbonate to max- the result being a change in membrane and imize H secretion. In their opinion [140] the most transtubular permeability, so that reclamation of so- tenable explanation for the negligible Pco2 gradient lute may be depressed or backflux enhanced. It is also which they found is deficient H secretion, without apparent from recent work [96] that the excess of production of carbonic acid for delayed dehydration PTH and the depletion of vitamin D hormone, which to CO2. Others [143] have provided counter-evidence characterized ARVDD, will combine to deplete mito- to this hypothesis, and suggest that the low urinary chondrial and cellular calcium and further alter cal- Pco2 in cRTA may represent excessive backflux of cium-dependent cellular permeability. H2C03 in alkalosis, and of H during acidosis. Pseudohypoparathyroidism of the classical type Absence of erythrocyte carbonic anhydrase B has (PHP, type I) is an example of tubular unrespon- been described in a family with distal RTA and deaf- siveness to PTH. In this disease, there is a fail- ness [144]. This observation is intriguing, in view of ure of PTH infusion to augment urinary cyclic 3', the suggestion of Maren [145] that distal bicarbonate 5'-adenosine monophosphate (AMP) [151] and frac- reclamation is entirely dependent on intracellular tional excretion of phosphate is diminished. Endo- carbonic anhydrase activity, which provides H ions genous serum PTH levels are typically elevated while to combine with HC02. Distal tubular carbonic anhy- serum calcium remains low and phosphorus is ele- drase has not yet been shown to be abnormal in these vated. Recent studies [152] have shown that the PTH patients. receptor and adenyl cyclase in renal cortex are appar- ently intact in PHP-type 1; it is the mechanism of cellular response to PTH and the effect on solute Disorders accompanied by altered tubule-hormone interaction transport which are defective. On the other hand, cyclic AMP-dependent mechanisms in the tubules Hormones regulate several tubular transport func- responsive to hormones other than PTH seem to be tions. Such transport activities will be modified if the intact although this facet of the problem has not been tubule is hyperresponsive to normal (or elevated) rigorously examined to our knowledge. It is of inter- serum concentrations of the relevant hormone; or if est that the renal adenyl cyclases responsive to PTH, the tubule receptor and translation system are unre- calcitonin and vasopressin are preferentially located sponsive to the normal regulatory action of the hor- in outer cortex, inner cortex and medulla, respec- mone. Both phenomena can be illustrated by inher- tively [146]. The basis for the insensitivity of adenyl ited disorders which involve parathyroid hormone cyclase to PTH in the intact tubule in PTH-type I (PTH). PTH-responsive, membrane-dependent activ- remains obscure. ities appear to be localized to the basilar surface of A variant of PUP known as type II PHP, has been outer cortical tubule segments [146]. reported [153, 154]. In this trait, fractional excretion Autosomal recessive vitamin D dependency of phosphate remains insensitive to PTH infusion, 166 Scriveret a! but a brisk increase in urinary cyclic AMP testifies to port what others have proposed recently [31, 97, 114, a responsive renal adenyl cyclase. PHP type II is 115]; that accumulation of solute by renal tubule cells further distinguished by a normal serum concen- and its transtubular transport are each essentially tration of inorganic phosphate, a feature which different processes, the former being most evident in remains unexplained at present. Many facets of the the straight protion, the latter in the convoluted por- tubular metabolism and handling of vitamin D, phos- tion of proximal tubule [115]. The normal topology phate and calcium, as well as of cyclic AMP of absorbing epithelium provides the framework for metabolism regulated by PTH, are pertinent to the these concepts which, being of fairly recent origin, are interpretation of PHP, but are beyond the scope of still likely to be a source of controversy as well as a this review. stimulus for further investigation. On the other hand, if they are valid, it should not be surprising that Commentary tissues lacking the topological orientations of epithe- In this review we have used some of the inborn hum (e.g., erythrocytes, blood leukocytes and cul- errors of tubular transport listed in Table 1 to illus- tured skin fibroblasts) fail to show any abnormality trate, in a specific manner, the various components of of solute transport wherever the investigation has transtubular movement of solute. These experiments been performed, as in cystinuria [155],Hartnupdis- of nature, as well as the steadily increasing body of ease [33], iminoglycinuria [156], glucose-galactose laboratory experimentation in man and animals, sup- malabsorption [57] and X-linked hypophosphatemia - -' [157], for example. 7 Various elegant experimental methods have pro- 0.10 vided the important evidence for normal backflux across the luminal membrane, transtubular fluxes in 0.08 - 6 both directions and differential handling of solute uptake and migration in different regions of the kid- a. 0.06 ney (e.g., [19,, 25,31,83, 114, 115].) Even the // 5 traditional "black box" clearance method appears to 0.04 - honor this theme (Fig. 6). As a result of this new 4 awareness, our interpretation of altered Tm values, 0.02 // fractional reabsorbtion rates relative to solute con- 0-i 3 centration and other clinical indices of altered net I I I I I tubular transport in the inborn errors of tubular 15 45 75 105 135 165 195 function is likely to undergo refinement in the future. Time, mm All of this will benefit diagnosis, counselling and Fig. 6. Renalclearance studiesin the rat after bolus injection of 1'C- treatment of patients with hereditary disorders of labelled x-amino-isobutyrate (AIB), an inert synthetic amino acid. tubular transport. Fractional excretion (left ordinate) rises above the 15-mm value The expression of mutant alleles which affect the (set at zero to accommodate interindividual variation between 0.03 and 0.05 at 15 mm), to approach a steady state after 90 mm while gene products controlling the various stages of trans- the plasma concentration of A1B is falling (.—., (right ordinate). tubular transport is also complex [3, 61. The gene loci 3H-inulin clearance remained stable during the experiment. The which determine the structure and quantity of the reciprocal fall in fractional reabsorption of AIB with time could reflect saturation of an intrarenal binding process; this explanation relevant proteins are likely to exhibit one or more is unlikely since reinjection of AIB, or elevation of MB plasma mutant alleles per inborn error of transport. Genetic concentration, did not alter the phenomenon. AIB equilibration between peritubular fluid and epithelium followed by backflux into heterogeneity underlies a great many monogenic dis- the lumen is a more likely explanation. AIB concentration fell in eases [158] and the hereditary disorders of tubular the cortex between 30 and 195 mm (from 1284 + 25 cpm/mg of wet transport are no exception. Consequently, it s not wt of prepared slice, mean SD [N = 161 to 953 + 26cpm/mg[9N surprizing that, for example, three different mutant = 16], whereas the concentration remained unchanged in medulla (from 830 + 218 cpm/mg [N = 8] to 923 274 cpm/mg [N = 8]). alleles, each apparently at the same "cystinuria" gene When considered with the plasma AIB levels, these figures indicate locus, occur among subjects with cystinuria [72]; and that although the distribution ratio of AIB is the same in Cortex and that four mutant alleles, all apparently at the locus medulla at 3'/2 hr, it is significantly less in the medulla than the cortex at 30 mm. These tissue changes were not an artefact of specifying the shared transport system for imino changing urine or plasma AIB concentration. The findings suggest acids and glycine, are now required to explain the that a slowly accumulating tissue poo1 of AIB in the medullary different forms of familial renal iminoglycinuria [6, portion of the nephron is a potential source for enhanced luminal exodus (backflux) and, thus, of the observed increase in fractional 109]; and that different alleles at two independent excretion (Mclnnes RR, Scriber CR, unpublished data, [149]). gene loci controlling glucose transport are needed to Renal tubular transport 167 explain glucose-galactose malabsorption and renal Acknowledgments glucosuria [39,40J. The discovery of two different phenotypes in the pseudohypoparathyroidism trait, This investigation was supported by the Medical one with blunted renal cyclic nucleotide formation Research Council of Canada. Dr. Chesney and Dr. and one without, again argues for mutant alleles Mclnnes held Fellowships from the MRC and the which probably occur at different gene loci and which Cystic Fibrosis Foundation of Canada, respectively, control different components of the relevant trans- while at McGill University. Dr. Chesney is presently port processes. It follows that recognition of genetic with the Department of Pediatrics, University of heterogeneity will also help to direct the physician Wisconsin School of Medicine, Madison, Wisconsin. toward more precise diagnosis, counselling and treat- Dr. Mclnnes is now with the Genetics Unit, Massa- ment. chusetts General Hospital, Boston, Massachusetts. Appropriate dissection of tubular function in man We acknowledge the role our colleagues Michel Bergeron, Andre Borle, FazI Mohyuddin, Leon through genetic "probes" has revealed an important Rosenberg, Stanton Segal and Susan Tenenhouse general underlying theme. Loss of a specific transport function rarely leaves the homozygous proband de- have played in the development of the ideas ex- void of all function for the particular transport in pressed in this paper; any errors are our sole respon- sibility, not theirs. question [160]. For example, homozygotes with renal iminoglycinuria retain a significant fraction of pro- Reprint requests to Dr. Charles R. Scriver, Medical Research line reclamation under endogenous conditions [109]; Council Group in Genetics, de Belle Laboratory for Biochemical and homozygotes with severe type-A glucosuria re- Genetics, McGill Unioersity-Montreal Children's Hospital Research tain one-third of their maximum glucose reabsorptive Institute, 2300 Tupper Street, Montreal, Quebec, Canada H3HI P3. capacity in kidney and have lost none in the intestine [39, 58]. 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