Calcium oxalate nephrolithiasis: Defective oxalate transport · 1284 Nephrology Forum: Calcium...

Kidney International, Vol. 39 (1991), pp. 1283—1298

NEPHROLOGY FORUM

Case presentation

A 38-year-old man was admitted to the University Hospital of Padova6 years ago because of recurrent nephrolithiasis. The patient had beenin good health except for recurrent renal stones since 1971. He hadpassed more than 15 stones from both sides, all spontaneously, exceptfor one that required a left ureterolithotomy. Chemical analysis of someof the stones revealed calcium, oxalate, and phosphate. The patient hadno history of gastrointestinal disease, excessive sun exposure, or druguse. Physical examination was normal. Laboratory studies revealed aplasma urea of 30 mg/dl; serum creatinine, 1.1 mg/dl; uric acid, 4.6mg/dl; calcium, 10.8 mg/dl; ionized calcium, 1.33 mmol/liter (normal,<1.30); phosphate, 2.2 mg/dl; and C-terminal PTH, 0.4 ng/ml (normalrange, 0.2—0.8 ng/ml). Urine examination was normal. Calciuria was 490mg/day; oxaluria, 47 mg/day; phosphaturia, 1357 mg/day; and uricos-uria, 486 mg/day. Urinary cAMP was 5.13 mol/day (normal, <4.7). Acyanide-nitroprusside test was negative. Radiographs of the chest,abdomen, skull, and hands were normal, as were intravenous pyelog-raphy and renal sonography. No stones were present. Osteodensitom-etry revealed 75% bone mineralization in comparison with controlsmatched for gender and age.

Primary hyperparathyroidism was strongly suspected despite thenormal blood PTH. Accordingly, a 203-technetium scintigram of theparathyroids was obtained, which disclosed an increased tracer uptakecorresponding to the right lower thyroid lobe. Computed tomographyconfirmed the presence of a mass behind the inferior half of the rightlobe of the thyroid gland, and venous sampling of the thyroid veinsdemonstrated an increased concentration of PTH in the right lowerthyroid vein.

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© 1991 by the International Society of Nephrology

At surgery, the lower right and upper left parathyroid glands wereremoved, and a subtotal lobectomy was performed in the upper rightand lower left glands. Histologic examination revealed chief cell hyper-plasm. After parathyroidectomy, the patient's blood and urinary cal-cium and phosphate levels returned to normal. Urinary oxalate excre-tion remained slightly increased (45 mg/day).

The patient remained in good health until 2 years ago, when he wasreadmitted with right renal colic. At that time, his blood calcium was 9.7mg/dl; phosphate, 3.4 mg/dl; and PTH, 0.7 ng/ml. The urinary calciumwas 262 mg/day and phosphate, 867 mg/day. Urinary cAMP was 1.9mol/day. Urinary oxalate was again slightly increased (46 mg/day). Anabdominal plain film and ultrasonogram revealed a calcification in theright kidney 4 mm in diameter. The patient subsequently passed thisstone. He was treated with hydrochlorothiazide and amiloride, and hehas had no further stones over the subsequent 2 years of observation.

The erythrocyte steady-state oxalate self-exchange was high at 1.80x l0 min (normal, <0.55) six years ago and 1.88 x l02 min1 twoyears ago.

Discussion

DR. ARTURO BORSATTI (Professor of Nephrology, Universityof Padova School of Medicine; Chief, Division of Nephrology,Institute of Internal Medicine, University Hospital, Padova,Italy): This patient is unusual in that correction of hypercalci-uria by parathyroidectomy was not sufficient to prevent subse-quent renal stone formation. However, he also had a defect inthe cellular handling of oxalate, as suggested by a high steady-state oxalate self-exchange in red blood cells and mild hyper-oxaluria. The potential role of abnormal cellular handling ofoxalate in the pat hophysiology of calcium oxalate (CaOx) renalstone disease will be the focus of this Forum. I first willconsider the problem of "mild hyperoxaluria" in renal stonedisease. Then I will review the role of oxalate as a stonepromoter, looking for the most reasonable cause of an increasein urinary oxalate. Later I will describe a defect in oxalateself-exchange found in red blood cells of stone formers, andfinally I will try to establish a hypothetical link between whathappens in erythrocytes and what might happen in the cellsinvolved in renal oxalate absorption and secretion.

Mild hyperoxaluria in idiopathic calcium oxalatenephrolithiasis

In 1978 Williams stated that "In only a small percentage ofpatients with recurrent calcium oxalate stones has it beenpossible to demonstrate that urinary oxalate excretion is signif-icantly elevated above the normal range" [11. Ten years later,after the introduction of methods that measure urinary oxalatemore accurately, the same investigator asserted that ". . . mostrecent studies of calcium oxalate stone formers. . . suggest thatthe true incidence of hyperoxaluria is between 10 to 20 percent"

Calcium oxalate nephrolithiasis: Defective oxalate transportPrincipal discussant: ARTURO BORSATTI

Istituto di Medicina Interna, Universitd di Padova, Padova, Italy

EditorsJORDAN J. COHENJOHN T. HARRINOTONJEROME P. KASSIRERNIcoLAos E. MADIAS

Managing EditorCHERYL J. ZUSMAN

State University of New York at Stony Brookand

Tufts University School of Medicine

1283

1284 Nephrology Forum: Calcium oxalate nephrolithiasis

[21. These two statements summarize the enormous method-ologic and conceptual developments in renal stone disease thathave occurred over the last few years. Enhanced accuracy hasbeen achieved by the availability of enzymatic, ion chromato-graphic, or high-performance liquid chromatographic methodsfor the assay of oxalate in biologic fluids, as well as newinformation about the collection and storage of samples [2, 31.In the meantime, various theoretical, experimental, and clinicalobservations have clarified the role of urinary oxalate in cal-cium oxalate lithiasis. We now are quite confident that ". . . inthe formation of the CaOx stones, at the very least, oxalate is asimportant as calcium and deserves equal attention [4].

Robertson and Peacock first reported that small increases ofoxalate excretion (40—100 mg/day) predisposed to the formationof renal stones [5]. These researchers described this conditionas "mild hyperoxaluria." Later reports give a frequency of"mild hyperoxaluria" in patients with stones of 12% to 63%[5—8], 37% [9], and 50% [101. In some series, the frequency is aslow as 17% [11, 121. The scatter in these values probably resultsfrom the use of different selection criteria (the inclusion ofpatients with recurrent stones or those with single stones), fromgeographic and racial differences, from different analyticalapproaches, and last but not least, from differences in choosingthe cut-off point that defines "mild hyperoxaluria,"

The issue of the upper limit of normal for urinary oxalate is animportant one. Some investigators have defined mild hyperox-aluria as a urinary oxalate excretion more than two standarddeviations above the mean, yet two considerations raise doubtabout this definition. First, the physico-chemical properties ofoxalate (which I will discuss later) are such that even smallincrements in urinary oxalate might result in quite importanteffects on the saturation of calcium oxalate because the effect ofraising oxalate concentration on saturation of oxalate in theurine is linear [3]. Second, if one takes into account thecumulative frequency plots for oxaluria given by Larsson andTiselius [3] and calculates similar plots from the data of Rob-ertson et al [81, the interval between 1 and 2 SD includes farmore stone formers than normal controls. The ideal approachshould have been the receiver operating characteristic (ROC)curve, that is, the analysis of the curve obtained plotting thepositive versus false-positive rate for selected values of ox-aluria, which, to my knowledge, has never been attempted.

These considerations lead me to speculate that hyperoxaluriamight be etiologically as important in renal stone disease as ishypercalciuria. What I will try to establish next is the role ofoxalate as a stone promoter, and the mechanisms of hyperox-aluria.

Oxalate as a stone promoterOxalic acid, a strong organic acid, forms an insoluble salt

with calcium at physiologic pH. The clinical importance ofoxalate in renal stone disease is related to the insolubility of thecalcium oxalate salt. Because not more than 50 micromoles ofoxalate can be dissolved in one liter of water, the concentrationof oxalate in the urine must be considered a strong promoter ofcalcium oxalate precipitation.

Several studies have evaluated the importance of urinaryoxalate as a determinant of calcium oxalate precipitation. In1978, Robertson and coworkers analyzed the frequency distri-bution of some urine constituents in a group of recurrent stone

formers and found that oxalate was a more important risk factorin making stones than was calcium [8]. In a later study,Robertson and Peacock demonstrated a relationship betweenoxaluria and the number of stone episodes per year as well asquantity and size of crystalluria, a relationship that was lostwhen calcium was considered by itself [5]. The importance ofoxalate in the urinary saturation of calcium oxalate also wasstudied by Finlayson [13]. Utilizing a mathematical approachthat takes into account the possibility that oxalate makescomplexes with other urinary ions, Finlayson found thatchanges in urinary oxalate concentrations are 15 times as potentas equimolar changes in calcium concentration in effectingcalcium oxalate saturation.

An increase in urinary oxalate can derive from: (1) increaseddietary intake; (2) increased metabolic production; (3) aug-mented intestinal absorption; and (4) a renal leak. I will nowconsider these possibilities in detail.

Oxalate is a common component of foods, but the oxalatecontent of most foods is low (less than 0.01 mmol/liter).Nonetheless, some foods (spinach, rhubarb, tea, beer, choco-late, peanuts) are rich in oxalate; as a consequence, theiringestion might induce hyperoxaluria. Finch et a! examined theurine at 37° and 4°C for crystals in groups of normal subjectseating a low'oxalate diet, an unrestricted diet, and a low-oxalatediet with the addition of oxalate-rich foods [14]. They found nooxalate crystals in the urine in the first two groups, whereas10% of subjects supplemented with oxalate-rich foods showedoxalate crystals at 37°, and 40% had crystals at 4°C. Robertsonfound that calcium oxalate stone formers ingest slightly butsignificantly more oxalate than do controls [151. He concludedthat this dietary factor cannot play a major role in the causationof the "mild hyperoxaluria" because urinary oxalate does notincrease until ingested oxalate exceeds an intake of approxi-mately 2 mmol/day. Griffith et al, in a case-control study, wereunable to find any significant difference in dietary oxalate intakebetween hyperoxaluric stone formers and controls [16]. Barkerand colleagues examined the relationship between diet andrenal stones in 72 areas of England and Wales and did not findany relationship between diet and stone formation [171. Thus,available data fail to support a role for increased dietary intakeof oxalate as the main cause of hyperoxaluria.

Could increased oxalate excretion in patients with calciumoxalate nephrolithiasis derive from an exaggerated dietary intakeof oxálate precursors? The two most important precursors ofoxalate are ascorbic and glyoxylic acids. Ascorbic acid accountsfor 35% to 50% of the daily urinary excretion of oxalate [18], butits metabolic turnover is limited, so the small quantity commonlyingested with foods cannot result in increased oxalate synthesis[19]. Even the impact of megadoses of vitamin C on oxalateproduction is far from established. Major points of confusion arethe methods used to assay oxalate, the fact that ascorbate isunstable at room temperature and at physiologic pH in the urine,and that it undergoes a nonenzymatic oxidation to oxalate [201.The consequence of high ascorbate intake on urinary oxalaterecently was readdressed by Liedtke et al, who assayed urinaryoxalate by the oxalate-oxidase method together with two ionchromatographic methods in 10 normal volunteers before andafter ingestion of 2 g of vitamin C per day [21]. When fresh urineswere analyzed, they found no difference in urinary oxalate excre-tion from control.

Nephroiogy Forum: Calcium oxalate nephrolithiasis 1285

Thus a reasonable conclusion seems to be that the dietaryintake of ascorbic acid has little or no effect on the levels ofurinary oxalate in normal subjects. The only exception to thisconclusion is the study by Briggs, who administered a shortcourse of ascorbic acid (4 g/day) to 2 healthy men [221. Theascorbic acid promoted an extremely large increase in oxalateexcretion. The family members of one of these subjects alsowere investigated, and the same reaction to vitamin C occurredin the father. This observation raises the possibility that ratherthan an augmented intake, a derangement in ascorbate metab-olism might lead to hyperoxaluria. The possibility that anabnormality in ascorbate metabolism might result in hyperox-aluria in recurrent stone formers was investigated by Chalmerset al [23]. They studied 17 stone formers and 11 controls andfound that stone formers excreted significantly more oxalateand less ascorbate than did controls. They also loaded 5patients with ascorbate either by mouth (4 g) or intravenously(500 mg) and demonstrated a larger and more delayed oxalateexcretion only after ingestion of ascorbate. The authors con-cluded that malabsorption of ascorbate might lead to an in-creased conversion of ascorbate to oxalate in the gut. In a laterstudy, Cowley et a! compared the uptake of citrate and ascor-bate from the gut into the blood, presented evidence of impairedabsorption of hydroxycarboxylic acids in stone formers, andsuggested a defect in a common carrier that absorbs bothascorbate and citrate from the gut [24]. These studies leaveopen the question of a possible causal role of vitamin C intakein promoting hyperoxaluria but suggest that excessive intake ofcitrate might be a promoter of stone formation [251.

The other important precursor of oxalate, glyoxylate, ac-counts for 50% to 70% of urinary oxalate [26]. The majorsources of glyoxylic acid in humans are glycine, glycolic acid,and serine. Hydroxyproline also can be reversibly converted toglyoxylic acid by a vitamin B6-dependent step. Other possibleminor precursors of oxalate include gelatin, tryptophan, phen-ylalanine, tyrosine, threonine, aspartic acid, creatinine, andpurines [27]. The possibility that an increased dietary intake ofall these substances can cause hyperoxaluria raises the possi-bility, discussed in a recent Nephrology Forum presentation[28], that protein intake is a predisposing factor to stoneformation. To summarize, it seems unlikely that an increasedintake of oxalate or its precursors is responsible for stoneformation. Equally unlikely seems the chance of a derangementin endogenous oxalate synthesis. In fact, the indisputabledemonstration that hyperoxaluria in stone formers disappearsduring fasting argues against the possibility of a primary disor-der in oxalate metabolism [7, 29].

Does increased intestinal absorption of oxalate cause itsappearance in large amounts in the urine? This issue was firstaddressed by Hodgkinson, who showed an increased oxalate-to-creatinine ratio in the urine of 98 stone formers in compari-son with 67 normal controls [7]. Further, he showed that thisdifference disappeared after fasting. Because the dietary intakewas the same in both groups, he concluded that stone formershad a higher intestinal absorption of oxalate. Using '4C oxalateas a tracer, Marangella et al found a mild increase in intestinalabsorption of oxalate in patients with calcium oxalate nephro-lithiasis [301, which was more evident when the hypercalciuricstone formers' group was considered. However, applying thesame technique as Marangella et al, Tiselius and colleagues

found no significant difference between stone formers andcontrols [31]. Manoharan and coworkers, revisiting the problemof "mild hyperoxaluria" in renal stone disease, have confirmeda higher-than-normal urinary excretion of oxalate in stoneformers that disappeared after fasting [29]; this finding confirmsthe observation by Hodgkinson [7]. Although the data conflict,some of these studies do suggest the possibility of an increasedintestinal absorption of oxalate in calcium oxalate nephrolithi-asis.

Finally, a renal leak might produce hyperoxaluria. To prop-erly assess the possibility of a renal leak of oxalate, an accuratemeasurement of plasma oxalate is indispensable. Until recently,accurate measures of plasma oxalate were unavailable. In tworecent studies, however, both plasma and urinary oxalate wereassayed by highly accurate methods [29, 32]. In both studies,stone formers appeared to secrete more oxalate in the renaltubule. This increased excretion occurred despite plasma levelsof oxalate that were the same or even less than those incontrols. These observations support the view that a renal leakof oxalate due to a tubular handling defect is present in patientswith calcium oxalate nephrolithiasis, or at least in a subgroup ofthese patients. Further comparable studies are required toconfirm this hypothesis and establish its quantitative impor-tance.

Defective cellular oxalate transportIs a defect in cellular oxalate transport the fundamental

abnormality in calcium oxalate nephro!ithiasis? The commonfinding of mild hyperoxaluria, most probably resulting fromincreased intestinal oxalate absorption, exaggerated renal ox-alate loss, or both, in calcium oxalate stone formers raises thepossibility that defective cellular oxalate transport is the basicabnormality of the disease. However, adducing sufficient evi-dence for this hypothesis in humans is extremely difficult. Inprinciple, abnormalities in oxalate handling should reside bothin the gut and in the kidney, but cells from these locationscannot be investigated directly. Furthermore, no experimentalmodel of inherited calcium oxalate stone disease exists. Forthese reasons, any examination of this problem must focus oncells not as directly involved in oxalate transport as those in theintestine and kidney. Such an indirect approach to the study ofabnormalities in membrane ion transport is not new; it has beenapplied, for example, in the field of hypertension, in which theinvestigation of circulating cells such as platelets, red bloodcells, and leukocytes has suggested the possibility of an abnor-mality in membrane transport in vascular smooth muscle cells.

Our search for a cell model suitable for the study of oxalatetransport focused on the red blood cell. In 1975 Aubert andMotais demonstrated that the red blood cell is permeable toorganic acids and that its transport of dicarboxylic acids wascarrier-mediated [33]. In a later paper, mainly aimed at clarify-ing the mechanism by which sulfonamides inhibited aniontransport, Cousin and Motais evaluated the organic anionpermeability of red blood cells by studying the steady-state selfexchange of oxalate [341. The assay system we employed toevaluate red blood cell steady-state oxalate self exchange isderived from that described by Cousin and Motais [34].

In our first study on red blood cell oxalate self exchange in 28"idiopathic" renal stone formers, we found a higher-than-normal exchange rate in 78% of patients [35]. This fraction was


confirmed in a later study, which examined 98 patients [361. Todate we have examined a total of 217 renal stone formers, andthe abnormality in red blood cell oxalate self exchange has beenpresent in 68% (Fig. 1). However, this group is heterogeneousbecause it also includes those who had only single stones.

Because family studies indicated an appreciable genetic con-tribution to the risk of having urolithiasis, we attempted toassess whether the abnormality in oxalate transport was genet-ically determined. We studied five families in which the abnor-mality in oxalate transport was detectable in one or moremembers and concluded that the abnormal red blood celloxalate self exchange follows the pattern of an autosomal,monogenic, dominant trait with complete penetrance and vari-able expressivity [36]. These data led us to surmise that agenetically transmitted cellular abnormality in oxalate transportmight be associated with calcium oxalate renal stone disease.Interestingly, the transmission of two other conditions associ-ated with renal stone disease, that is, familial hypercalciuria[37] and renal tubular acidosis [381, also has been considered anautosomal dominant trait. Although environmental factors con-found interpretation, a substantial genetic component in neph-rolithiasis is possible [39—45]. The model that fits best ismultifactorial, and a defect in cellular oxalate transport mightbe one such factor.

In the search for a closer link between the red blood cell

2 3

abnormality and stone formation, we looked for a possiblerelationship between the oxalate exchange rate and the 24-hoururinary oxalate excretion, but we were unable to demonstrateone, even with large numbers of patients (Fig. 2). However, fora calcium oxalate stone to form, oversaturation need not becontinuous; some peaks during the day should be sufficient.Indeed, in oxalate tolerance tests carried out in pairs of brothersliving together and eating the same diet, a significant increase inurinary oxalate could be demonstrated at 2 and 4 hours after theload in brothers with abnormal red blood cell oxalate transportwhen compared with their siblings with normal red blood celloxalate transport [36]. Thus, peaks of urinary oversaturationconsequent to more rapid absorption and excretion of dietaryoxalate remain possible.

Oxalate transport via band 3 protein. In an attempt to clarifythe red blood cell abnormality, we wanted to be sure that theabnormality was intrinsic to the cell. To this end, we incubatedred blood cells with high steady-state oxalate self exchange withnormal plasma and normal red blood cells with plasma ofpatients whose red blood cells showed an increased oxalate selfexchange; we found that these maneuvers did not significantlymodify the initial exchange rate (Fig. 3).

Given the results of Cousin and Motais' experiment [341, wesuspected the anion transporter, band 3 protein, as the culprit ofthe cellular abnormality, since these authors had suggested acompetition between oxalate and chloride/bicarbonate move-ments. Knowing that disulfonic stilbenes react almost exclu-sively (more than 95%) with band 3 in erythrocytes [46, 47], wetreated red blood cells with high transmembrane oxalate fluxeswith 5,5'-dithiobis (-2 nitrobenzoic acid) (DTNB) and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) and found

2—

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rnQaUa

o Ua a40 aDa 000 a a aao a a

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Fig. 2. Scattergram showing the relationship between steady-stateoxalate self exchange (K) in red blood cells and 24-hour urinaryoxalate. (For details see Ref. 36).

Fig. 1. Scattergram showing the '4C oxalate flux rate (K) in 217idiopathic calcium oxalate stone formers. The dotted line denotes theupper limit of normal (see Ref. 36).

Nephrology Forum: Calcium oxalate nephrolithiasis 1287

Fig. 3. A typical cross-incubation experiment is depicted. Red bloodcells from stone formers were incubated with compatible plasma fromnormal subjects and vice versa. Thereafter '4C oxalate self-exchangeswere determined. The ordinate depicts the transformation of '4Coxalate concentrations in the incubation medium (A) at defined time (T)(see Ref. 36).

that these agents not only corrected the high oxalate selfexchange, but almost abolished it [48]. We interpreted thisexperiment as evidence of a true movement of oxalate throughthe cell membrane occurring via band 3 protein.

It may be useful to recall here some properties of the band 3protein, which recently has been cloned [49, 50]; the corre-sponding gene is located on chromosome 17. Band 3 is a 911amino acid protein. Similar in structure to other anion exchang-ers, it is divided into 3 regions: a hydrophilic cytoplasmicdomain (residues 1-403), which interacts with a variety ofmembrane and cytoplasmic proteins; a hydrophobic transmem-brane domain (residues 404-882), which constitutes the aniontransporter and controls the rapid electroneutral sequentialexchange of chloride with bicarbonate; and an acidic C-terminalresidue (residues 883-911) of unknown function (Fig. 4). Theentire transmembrane portion of the molecule seems to benecessary for anion exchange. Amino acids that might beinvolved in anion exchanges include lysine, arginine, histidine,and glutamic acid. Although both intracellular and extracellulararginine are important in the anion exchange [51], possiblyserving as a critical binding site, proximal and distal lysines inor near the external opening of the channel are covalentlybound by the specific anion transport inhibitor DIDS [52].Finally, extracellular arginine and glutamate residues near theDIDS binding site seem to be necessary for anion transport [53,54].

What makes band 3 protein transport anions? One possibilitythat immediately comes to mind is that band 3 controls aniontransport by phosphorylation or dephosphorylation. Indeed,many red blood cell proteins are phosphorylated, and manymembrane and cytosolic protein kinases and several phos-phoprotein phosphatases are found in these cells. Human redblood cells contain two casein kinases, one membrane bound(type 1) and one in the cytosol (type 2); two cAMP-dependentkinases, again, one in the membrane (type 1), and one in thecytosol (type 2); a phospholipid-dependent protein kinase C; a

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• Control

0/0 Cross incubation

0 a

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domain 911COOH

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Fig. 4. Schematic description of band 3 protein in red blood cell, freelyderivedfrom Alper et al [941. The anion transporter is constituted by thehydrophobic transmembrane domain, residues 403-882.

30 60 90 120 Mm

N.0

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}— 0

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Fig. 5. A typical experiment after A TP depletion is described. Fullsymbols are the control values of the '4C oxalate self exchange in redblood cells from a stone former. Empty symbols refer to the same redblood cells depleted of ATP by incubation with 5 mM iodoacetamideand 5 mM inosine for 90 minutes at 37°C. (For a better understandingsee also Ref. 59).

calcium-calmodulin-dependent kinase; a membrane-bound ty-rosine kinase; and at least two more protein kinases thatcopurify respectively with protein 4,1 and 4.9 [55].

For phosphorylation to occur, the first step is the transfer ofa phosphate group of the donor ATP to an acceptor. WereasOned that if phosphorylation is important, reducing ATP inthe cell should result in a decrease of oxalate exchange. In fact,when we promoted red blood cell ATP depletion with iodoac-etamide, oxalate self exchange was almost abolished (Fig. 5).We also performed parallel experiments in which the kinetics ofband 3 phosphorylation were examined at different times afterenergy depletion (8, 16, 24 hours) and following the addition ofglucose and adenine to the incubation medium (4, 8, 16 hours).We saw a progressive decrease of band-3 phosphorylation, withan 85% recovery of the initial casein phosphorylation and adefinite loss of tyrosine phosphorylation (unpublished observa-tions). The assay of casein and tyrosine activities against


exogenous substrates confirmed the loss of activity of the latterenzymes. Furthermore, because DIDS exerts an effect onoxalate self exchange, we investigated the effect of DIDS (50jimol/red blood cell hematocrit of 25% for 30 mm at 30°C) on theband-3 phosphorylation rate. We found a 35% inhibition; mem-brane casein and tyrosine kinase activities against exogenoussubstrates did not change (unpublished observations). Theseexpriments suggest that the addition of DIDS promotes somemodifications in the protein that either interfere with phosphor-ylation of the intramembrane domain or are transmitted to theintracellular domain, where most phosphorylation occurs [561.

As a whole, these experiments suggest a link between aniontransport capability and the state of phosphorylation of band 3.The observation that thiazides and amiloride (which decreasethe exchange of oxalate) induce similar changes in the phos-phorylation rate of red blood cell membrane is in keeping withsuch an interpretation [57, 58].

Indeed, studies of the endogenous phosphorylation of redblood cell ghosts from stone formers with faster-than-normaloxalate self exchange revealed that the time-course profiles ofprotein 32P labeling exhibited a high phosphorylation both ofband 2 and band 3 in comparison with normal controls [591. Theincreased phosphorylation rate of band 3 observed in stoneformers might be explained by an alteration in the conforma-tional structure or by a topologic rearrangement in the membraneleading to the exposure of additional sites for phosphorylation,by an imbalance between protein kinase and phosphataseactivities, or by an excess of activators or a defect of inhibitorsof both enzymes. The first possibility seems to be ruled out bythe demonstration of an increased phosphorylation not only ofred blood cell band 3, but also of band 2 proteins. It is, in fact,difficult to support a defect in structure or arrangement of twoproteins simultaneously.

In investigating the remaining possibilities, we looked for oneor more substances known to be altered in nephrolithiasis, butones capable of interacting with protein kinase or proteinphosphatase. Glycosaminoglycans (GAGs), we thought, werethe best candidates.

Glycosaminoglycans are important in nephrolithiasis owingto their ability to oppose calcium oxalate crystallization in vitro[60—65]. To determine whether they have a similar action invivo, researchers have repeatedly assayed GAGs in stoneformers. Although researchers are not unanimous [66-69], theconsensus points to decreased urinary excretion of GAGs inpatients with renal stone disease [8, 70—75]. In nephrolithiasis,there is not only a quantitative defect, but also a qualitative one,because in stone formers the degree of sulfation of GAGs ishigher than normal [68, 761. Interestingly, the defect in GAGsynthesis in patients with renal stone disease does not seem tobe limited to the urinary tract; decreased production of GAGsby cultured skin fibroblasts also has been described [77]. On theother hand, GAGs are known to be potent inhibitors of manyprotein kinases, such as casein kinase [78—801, tyrosine kinase[811, and phospholipid-sensitive calcium-dependent protein ki-nase [821.

To assess the potential role of GAGs in determining theabnormality in steady-state oxalate self exchange, we carriedout some preliminary studies on red blood cells in vitro, andtested the effect of low-molecular-weight heparin, dermatansulfate, heparan sulfate, and chondroitin sulfate A and C on

both oxalate self exchange and band-3 phosphorylation. All thesubstances showed an inhibition of both oxalate self exchangeand band-3 phosphorylation, with the following order of mag-nitude: low-molecular-weight heparin > dermatan sulfate >heparan sulfate> chondroitin sulfate A > chondroitin sulfate C[83].

Next we assessed the effect of these agents in vivo. To thisend we evaluated GAG content, oxalate self exchange, andband-3 phosphorylation rate in a group of idiopathic calciumoxalate stone formers. The erythrocyte GAG content of stoneformers was lower than that in controls and, as expected,oxalate self exchange and band-3 phosphorylation rates werehigher. Furthermore, oxalate exchange and phosphorylationcorrelated inversely with GAG content [841.

According to these data, the following hypothesis seemslikely in our opinion: a reduced GAG content decreases theinhibition of the protein kinase(s) devoted to band-3 phosphor-ylation and leads to faster transmembrane oxalate transport.What remains to be done is an analysis of the possibility thatintestinal absorption and renal secretion of oxalate occur in asimilar fashion to that in red blood cells, that is, via band 3 orband-3-related proteins. Only if the answer is positive can acause-and-effect relationship between the red blood cell abnor-mality and the pathophysiology of renal stone disease beconfirmed.

Oxalate transport by the gut and localization of band-3-likeprotein along the gastrointestinal tract. Studies in vitro supportthe theoretical possibility that oxalate can be absorbed through-out the intestine [85]. In vivo, however, oxalate uptake occurswithin one to 8 hours after ingestion; thus, in normal subjectsthe proximal portion of the small bowel acts as a majorabsorptive site [86]. Most of the dietary oxalate is thought to bebound by intraluminal calcium in the small intestine and issubsequently excreted as insoluble calcium oxalate salts. Thisview assigns to intraluminal calcium ion concentration a pivotalrole in determining the amount of ingested oxalate absorbed anddepicts an inverse relationship between the quantity of ingestedcalcium and the amount of reabsorbed oxalate. According toPrenen et al [86], however, no difference exists in the absorp-tion kinetics of soluble oxalate and calcium oxalate.

Oxalate absorption by the gut has been considered for a longtime to be a passive, non-energy-dependent process [85, 871. Anet transport of oxalate was subsequently demonstrated in therat colon, however [88]. Furthermore, in the rat colon mucosa,the addition of disulfonic stilbenes inhibited both oxalate andchloride fluxes, and acetazolamide significantly reduced themucosal to serosal movements of both ions [88]. These findingssuggested that oxalate and chloride share a common transportpathway, and that chloride exchanges with bicarbonate. Thesestudies enable us to conclude, at least as far as the rat colon isconcerned, that an energy-dependent net oxalate absorptionoccurs that is sensitive to anion transport inhibitors and thatparallels chloride movements. Disulfonic-stilbene-sensitivechloride/bicarbonate exchangers also have been demonstratedin duodenum and jejunum [89—92]. Is there any evidence of thepresence of band 3 or band-3-related proteins along the gastro-intestinal tract? Polyclonal antibodies to purified mouse redblood cell band 3 protein bind to plasma membranes of gastricoxyntic cells [93]. Competitive studies revealed that the epitopeshared between gastric antigen and red blood cell band 3 was

Nephrology Forum: Calcium oxalate nephro/ithiasis 1289

restricted to the -COOH terminal domain of the protein, whichis known to contain the catalytic site for anion exchanges [93].Alper et al, using Northern blot hybridization with mRNA frommany murine epithelial cells, showed that a probe encoding astretch of the red blood cell band 3 membrane spanning domaindetected, under low stringency conditions, an mRNA fromcolon [941. Another attempt at identifying and characterizingcDNAs encoding band-3-related proteins and determining theirmRNA distribution in the gut has been made in the rat [95]. Twoproteins related to the intramembranous domain of band 3 wereisolated. Northern blot hybridization analysis disclosed anmRNA of apparently 4.4 kb in the stomach as well as in thesmall and large intestines. In the rat duodenal brush-border-membrane vesicles, chloride/bicarbonate exchange was stimu-lated by cAMP and was inhibited by specific cAMP-dependentprotein kinase inhibitors [96]. These findings suggest that acti-vation of the exchange requires a cAMP-dependent proteinkinase with phosphorylation of some element of the transportpathway. Evidence for protein kinase C as a regulator ofintestinal electrolyte transport also has been presented. In fact,protein kinese C promotes the phosphorylation of specificproteins of microvillous membrane of enterocytes [971, and itsactivators, phorbol ester and diacylglycerol, stimulate electro-genic anion secretion in intestine [98].

In sum, current evidence suggests the possibility of activeoxalate uptake by the small intestine, which occurs through achloride/bicarbonate exchanger and which is inhibited by di-sulfonic stilbenes. Evidence also indicates that band-3-relatedproteins are present along the gastrointestinal tract; theseproteins, although not encoded by the same gene as red bloodcell band 3 protein, are likely the product of very similar genes.Furthermore, it is likely that the transporter must be phosphor-ylated to be active.

Renal handling of oxalate and localization of band-3-likeprotein along the nephron. Many transport systems that displaysome functional similarities with red blood cell band 3 proteinare expressed in urinary epithelia. If we posit a transportsystem as band 3 related according to an anion-exchangemechanism or to stilbene disulfonate sensitivity, we couldconclude that band-3-related anion transporters exist all alongthe renal tubule [94]. Following an immunologic approach andemploying antibodies raised against red blood cell band 3,immunoreactivity was detected in a variety of cell types [99,1001. However, the antisera initially used were not very specific[101, 1021 and probably recognized only epitopes restricted tothe cytoplasmic domain of band 3, which is not involved inanion transport. Subsequent studies with more purified antibod-ies, or monoclonal antibodies raised against the intramembra-nous domain of band 3, have shown specific fluorescence onlyalong the basolateral membrane of alpha intercalated cells ofthe collecting duct [103—106]. The application of molecularbiologic techniques has enabled this field to take a big stepforward. Red blood cell band 3 from many species has beencloned [49, 50, 94, 107—1091, as has kidney band 3 from humansand experimental animals [110—112]. It appears from thesestudies that red blood cell band 3 differs little from kidney band3, except for the intracytoplasmic domain, which is truncated inthe renal protein. At the moment, a protein encoded by thesame gene as red blood cell band 3 appears to exist only in the

basolateral membrane of alpha intercalated cells of the collect-ing duct of the kidney.

Oxalate is freely filtered by the glomerulus, as suggested bythe absence of protein binding of '4C oxalate. Micropuncturetechniques have been widely applied in the study of oxalateconcentration along the nephron. Endogenous filtrate collec-tions in the proximal convoluted tubule have shown highoxalate concentrations in the tubular fluid as compared withinulin; these findings indicate a net tubular secretion of oxalate[113—115]. Net tubular secretion of oxalate in this nephronsegment has been confirmed in isolated and perfused rabbitrenal tubules [1161. In distal tubule and final urines, the oxalate-to-creatinine ratio was very close to that of the proximal tubule,excluding net secretion of oxalate beyond the proximal convo-lution [113—115]. The fractional excretion of oxalate in the finalurines was between 1.09 and 1.28, meaning that approximately10% to 30% of urinary oxalate in normal conditions derivesfrom net tubular secretion.

Active secretion of organic anions in the proximal tubulerequires uptake from the contraluminal site across the mem-brane into the cell and luminal secretion through the brush-border membrane. As far as proximal tubule contraluminalanion transport is concerned, three main systems have beencharacterized: (1) an exchange system for sulfate and oxalate,(2) a cotransport system for sodium and dicarboxylates, and (3)an exchange system for hydrophobic anions and long-chainfatty acids [117, 118]. This list might be extended to five giventhe evidence that urate and PAH are transported on a separateexchanger, and that a stilbene-sensitive, sodium-independentchloride/bicarbonate exchanger also has been identified [1191.Oxalate transport occurs only through the sulfate, oxalate,bicarbonate exchanger [1 17, 118], which shares in common withthe red blood cell anion exchanger the ability to transport bothsulfate and oxalate, and to be inhibited by the same compounds,such as disulfonic stilbenes, which inhibit red blood cell band 3protein [120, 1211. To create a driving force for sulfate exit viathe contraluminal membrane, sulfate must first accumulate inthe cell. This happens by virtue of sodium-coupled sulfatetransport in the luminal membrane. Because the first step inoxalate secretion is an exchange with sulfate at the contralumi-nal cell side, this exchange is indirectly driven via the sodium-sulfate cotransport system in the luminal membrane.

How can oxalate leave the cell to be secreted into the tubularlumen? In the rabbit renal microvillus membrane, a chloride(formate)/oxalate exchanger has been described [122]. Theexchange is electrogenic, pointing to the movement of onemonovalent anion (chloride or formate) in exchange for adivalent anion (oxalate), and it is inhibited by DIDS and, to alesser extent, by furosemide. A hypothetical way for oxalate tocross the tubular cell can now be outlined (Fig. 6). Oxalate istaken up from blood at the contraluminal side in exchange withsulfate, and it enters the luminal fluid in exchange with chloride.Sulfate, in turn, enters the cell with sodium.

As we look for a link between the abnormality found in redblood cells and a possible renal leak of oxalate, we cannotneglect two certainties: (1) an anion transporter encoded by thesame gene as band 3 is located in the basolateral membrane ofthe alpha intercalated cell of the collecting tubule, but it doesnot transport oxalate; and (2) the proximal tubular cell containsat least two anion transporters involved in transcellular oxalate


movements that share with band 3 protein all its functionalproperties but are structurally different from it.

Let us consider point one first. Alpha intercalated cells areproton-secreting cells in which a fall in intracellular pH isthought to switch on a hydrogen translocating ATPase insertedin the luminal membrane. The hydroxide formed as a result ofthe proton extrusion reacts with carbon dioxide to form bicar-bonate in a reaction catalyzed by carbonic anhydrase. Thegenerated bicarbonate then leaves the cell via the basolateralmembrane anion transporter in exchange with chloride, whichleaves the cell through a chloride channel [123]. An indirectattempt at establishing the relative importance of the sodium!hydrogen antiporter and the chloride/bicarbonate transporter inregulating intracellular pH has been made in LLC-PK1 cells[124]. When stimulated by an acid load of about 0.3 pH unit, thechloride/bicarbonate exchanger could mediate acid-base fluxesgreater than those mediated by the sodium!hydrogen ex-changer.

If such a mechanism is in place, and a basolateral anionexchanger works at a higher than normal rate, the result couldbe an increased secretion of hydrogen with a consequential fallin urinary pH. Do we have proof that urinary pH is lower inpatients who form pure calcium oxalate stones? The answer isyes. Robertson et al reported data on urinary pH in normalsubjects and calcium oxalate stone formers in the UnitedKingdom, Saudi Arabia, and United Arab Emirates; in all threecountries, urinary pH was lower in the calcium oxalate stoneformers [125].

Let us now recall what occurs in red blood cells, where theabnormality seems to be related to a defect in the phosphory-lation of band 3, and which probably occurs not so much

because of a structural defect of the protein, but rather becauseof an excess of activity of the specific protein kinase(s). Thenext logical question is whether there is any evidence ofphosphorylation of alpha intercalated cell band-3-like protein.Indeed, some indirect evidence does exist. Proton secretion byrabbit medullary collecting duct is stimulated by cAMP ana-logues and prostaglandin E2 [1261. It is reasonable to suspectthat at least the action of cAMP requires protein phosphoryla-tion, but the fact that band-3-like protein is the specific sub-strate of this supposed phosphorylation is still open to doubt. Infact, the chloride that enters the cell in exchange with bicar-bonate must exit via a specific chloride channel, and diphenyl-amine-2-carboxylate, which is a specific chloride channelblocker [127], can stop chloride/bicarbonate exchange [1281.Because cAMP activates chloride channels [129], the increasein chloride/bicarbonate exchange could be secondary to anincreased chloride conductance. A direct effect of diphenyl-amine-2-carboxylate on the chloride/bicarbonate exchangeralso has been reported, however [128].

To summarize, evidence exists of an exchanger encoded bythe same gene as band 3 protein in the basolateral membrane ofcollecting duct proton-secreting cells, probably requiring phos-phorylation for its activation; and, calcium oxalate renal stoneformers have a lower-than-normal urinary pH. One couldconclude that, leaving the oxalate aside, a link between the redblood cell abnormality and renal stone formation might berelated to the formation of more acidic urines, in which theprecipitation of calcium oxalate salts is facilitated.

Now let us return to the proximal tubule, the site of oxalatesecretion. We know that here at least two transporters areinvolved in oxalate movement. These transporters share almostall the functional properties of red blood cell band 3 protein but,despite functional similarities, are not the same molecule asband 3. It is appropriate to ask this question: are these anionexchangers encoded by genes closely related to the one of band3? In human genomes, band 3 is encoded by a single-copynuclear gene, the expression of which is limited to erythroidcells and to alpha intercalated cells of the collecting duct of thekidney. However, the observation of homologous transcriptsand the identification of immunologic cross-reacting peptides inmany non-erythroid cells raises the possibility that anion ex-change in non-erythroid cells might be mediated by closelyrelated molecules. It is true that, using Northern blots of mousekidney poly (A) selected RNA under high stringency condi-tions, a probe encoding a stretch of the red blood cell band 3membrane spanning domain detects only a 4.2 kb transcript.But under low stringency conditions, two major renal mRNAsand four minor species can be identified [94]. The mRNAtranscripts were identified by probes from the domain of band 3performing the anion exchange, so it is possible that thesetranscripts encode proteins functionally similar to the chloride/bicarbonate exchanger of red blood cells. In keeping with sucha possibility is the demonstration that a monoclonal antibodyagainst the membrane domain of band 3 protein binds to a 43 kDprotein with an amino acid composition very similar to redblood cell band 3 localized in rabbit renal tubular brush-bordermembrane [130]. Furthermore, complementary DNAs encodingtwo proteins related to red blood cell band 3 have been isolatedfrom rat stomach, brain, and kidney [951. These proteins exhibitan amino acid homology to band 3 of 50% and 52%, respec-

Brush-bordermembrane

Proximal tubuleBasolateralmembrane

Sulfate

HC03oxalate

Fig. 6. Schematic depiction of the supposed mechanism of oxalatesecretion by the proximal renal tubule. Oxalate enters the cell acrossthe basolateral membrane in exchange for sulfate and leaves the cellacross the brush-border membrane in exchange for chloride. Sulfate, inturn, enters the cell across the brush-border membrane with sodium.


tively. Interestingly, the C-terminal hydrophobic region ofthese proteins has a higher amino acid homology with the anionexchanger (64% and 69%, respectively), and very similar hy-dropathic profile, suggesting the same transmembrane organi-zation.

Although the problem of the structural similarities as well asthat of the genetic control of true band 3 and band-3-likeproteins is far from being resolved, these studies raise thepossibility that if something is wrong in red blood cell band 3,the same might be true in other anion exchangers, like thosedevoted to oxalate transport in the proximal tubule.

What I have reviewed until now establishes only the reason-ableness of a causal connection between the red blood cellabnormality and defective handling of oxalate by the kidney andthe gut. We recently tried to confirm the validity of thishypothesis with an experiment that must be considered prelim-inary. We reasoned that if we could correct the red blood cellabnormality, we could lower oxalate excretion. To this end, wetreated a group of stone formers with a mixture of GAGs (70%low-molecular-weight heparin, and 30% dermatan sulfate). Weknow from previous pharmacokinetic studies that 55% of anadministered dose is absorbed and 25% is excreted unmodifiedin the urine. A 15-day treatment yielded a fall in both red bloodcell oxalate self exchange and band-3 phosphorylation, butmore important was our observation that these modifications inred blood cells were paralleled by a significant decrease inurinary oxalate excretion [131]. Furthermore, the '4C oxalate-to-creatinine ratio was changed from 1 .4 before treatment to 1.2after treatment, meaning that oxalate handling was affectedsimilarly in red blood cells and renal tubular cells.

This is not a definitive answer, but it does foresee a promisingfuture, Although much work is still to be done, the availabilityof molecular genetic techniques surely enables us to clarify themolecular structure of oxalate transporter(s) and the mecha-nism(s) by which their activity is controlled. We don't knowwhether anion exchangers other than band 3 require phosphor-ylation for activation. However, the chance to examine thispossibility is not far away. In fact, an anion transport has beeninduced in oocytes of Xenopus laevis by expression of a mouseerythroid band 3 protein encoding a cRNA [132]. It is reason-able to predict the induction in the same way of other band-3-like proteins; it then might be possible to control the mecha-nisms that initiate the exchange.

Questions and answers

DR. GIUSEPPE ROMBOLA (Division of Nephrology, Niguarda-Ca' Granda Hospital, Milan, Italy): With respect to the phys-iologic function of the red blood cell band 3, one major role ofthis exchanger could be the participation in the regulation ofintracellular pH (pHi). Olsnes et al recently showed that theactivity of a DIDS-inhibitable anion antiport in green monkeyand fetal hamster renal cells (a similar if not identical exchangeras band 3) is strictly regulated by pHi [133]. High levelsstimulate the exit of bicarbonate from cells, and low levelsinhibit it [1331. Thus one might expect that increased red bloodcell oxalate self exchange in calcium stone formers either mightresult from a primary disturbance of intracellular acid-basebalance (that is, intracellular alkalosis) or, alternatively, mightproduce intracellular acidosis due to exaggerated base loss. Werecently presented preliminary data on pHi (using the fluores-

cent probe BCECF) in peripheral lymphocytes from hypercal-ciuric calcium stone formers and controls [134]. Resting pHiwas not different from normal, but when the cells were exposedto a sodium-free medium, pHi decreased further than in controlpatients. We suggest that an exaggerated base loss from thecells is a primary event that results in pHi changes only if Hexit (by Na/H antiport) has been concurrently turned off. Didyou characterize the red blood cell anion exchanger in calciumstone formers by kinetic studies? This could enable you todifferentiate better between increased activity, that is, Vmax,and increased affinity, that is, Km.

DR. BORSATTI: Leukocytes possess a band-3-like protein, soyour data might fit well with a primary dysfunction of thisexchanger. We did not perform kinetic studies in red blood cellsof stone formers.

DR. MAuRIzIo SURIAN (Chief, Dialysis Unit, Maggiore Hos-pital, Lodi-Milan): What relationship might exist between thered blood cell abnormality in oxalate self exchange and theabnormality in calcium-magnesium ATPase described by Bian-chi et al [135]?

DR. B0RsATTI: Calcium-magnesium ATPase activity dependson intracellular calcium, and it is calmodulin dependent. It ispossible that the protein kinase or one of the protein kinasesassociated with the phosphorylation of band 3 is calciumdependent. Indeed, trifluoperazine, which is considered to be acalmodulin antagonist, lowers oxalate self exchange in redblood cells of stone formers [136]. So intracellular calciummight be the link. We need to learn more about the one or morefactors that control oxalate handling by the proximal renaltubular cell. I know that Drs. Cab' and Wandzilak are workingon oxalate transport by LLC-PK1 cells, and probably they canadd something more.

DR. LORENZO CAL0' (Institute of Internal Medicine, Divisionof Nephrology, University of Padova, Padova): Our prelimi-nary data show that diacylglycerol increases oxalate uptake byLLC-PK1 cells, while the reverse happens with heparin.

DR. ANTONIA FABBRIS (Division of Nephrology, UniversityHospital, Verona): Recently your group showed that sulodex-ide, an animal extractive GAG composed of 70% low-molecu-lar-weight heparin and 30% dermatan sulfate, modulated mem-brane kinase activities [83, 131] and reduced oxaluria [1311. Inour experience, sulodexide increases the urinary excretion ofGAGs. Do you have any information about the mechanismsunderlying the effects of GAG on intestinal absorption or inrenal handling of oxalate?

DR. B0RSATTI: Acute intravenous administration of GAGslowers oxalate renal clearance, which is evidence of a directeffect of these substances on the kidney. However, if GAGswere only active on the kidney, they could not exert anylong-lasting effect on urinary oxalate, because the steady-stateinput and output must be the same. A two-week oral treatmentwith GAGs, a time interval at which a new steady state mostprobably was achieved, promotes a significant fall in urinaryoxalate; thus we have indirect evidence that GAGs' effectsextend beyond the kidney. Because oxalate input to the systemrelies on intestinal absorption and on endogenous production,we presume that GAGs also must be active at one of thesesteps.

DR. GIUSEPPE VEzzoLl (Division of Nephrology, San Ra-faele University Hospital, Milan): Many cell functions are


regulated by phosphorylation processes and many protein ki-nases are present in cells. Among them, casein kinases phos-phorylate band 3 and spectrin, whereas GAGs inhibit theiractivity and the activity of other protein kinases. So it ispossible that GAG reduction could affect some enzyme func-tions or the cytoskeleton organization in calcium oxalate stoneformers. Do you have evidence for or against this hypothesis?

DR. BORSATTI: The cytoplasmic domain of red blood cellband 3 protein binds ankyrin, band 4.1 and 4.2 proteins,glycolytic enzymes, and hemoglobin. Furthermore, the redblood cell band 3 cytoplasmic domain is actively phosphory-lated by several kinases, including a tyrosine kinase, a cAMP-independent kinase, a protein kinase C, and a calcium-stimu-lated protein kinase. The binding site for glycolytic enzymesand hemoglobin is lost in non-erythroid band-3-like protein,whereas that for ankyrin is preserved. In fact, band-3-likeprotein has been found co-localized with an immunoreactiveform of ankyrin and spectrin in the basolateral membranes ofcollecting duct intercalated cells [103]. I would not be surprisedto find that a malfunction in the cytoskeleton organization ofcells responsible for oxalate metabolism exists. The problem ishow to identify such a defect. As far as the erythrocyte isconcerned, we have not looked for abnormalities in glycolyticenzymes or in cytoskeleton organization.

DR. VJNCENZO CALDERARO (Institute of Internal Medicineand Nephrology, Federico II University, Napoli): I think wemust be cautious in interpreting your data obtained with DIDSin concentrations as high as l0 to 102 M. In other systems,such as the large intestine, apical chloride-bicarbonate ex-change is inhibited by concentrations of DIDS on the order of 5to 10 p.M. With these high doses, you might disrupt themembrane.

DR. BORSATTI: The experiment you refer to has been carriedout in red blood cell ghosts to evaluate DIDS' effect on band 3protein phosphorylation, and not to challenge oxalate transport.When we looked for a DIDS effect on red blood cell oxalatetransport, we used a 5 p.M concentration.

DR. GIUSEPPE MAscHIo (Chief and Professor of Nephrology,Division of Nephrology, University Hospital, Verona): Youshowed that at least three pharmacologic agents (amiloride,hydrochlorothiazide, and GAGs) can interfere with band 3phosphorylation and promote a fall in oxalate flux rate in redblood cells. Which of these drugs is most specific to theenzymatic system? Are you aware of any other agents that canact at a cellular level in patients with recurrent calcium neph-rolithiasis?

DR. BORSATTI: I am not sure it is important that we knowwhich of the three substances you mentioned is the mostspecific inhibitor of red blood cell band 3 phosphorylation.Rather, I think it is important that we know which of them ismost effective in lowering urinary oxalate. To this end, GAGsare far more powerful than amiloride and hydrochlorothiazide.In answer to your second question, nifedipine also lowers bothred blood cell steady-state self exchange and urinary output ofoxalate [137].

DR. LORIS BORGHI (Institute of Internal Medicine, UniversityHospital, Parma): When you measured red blood cell oxalateflux, you used a 10 mM sodium oxalate solution. In normalplasma the concentration of oxalate is about 2 p.M, and thus inyour experiments red blood cells are incubated in a medium

with a concentration of oxalate about 500 times the physiologicconcentration. I wonder whether this unphysiologic concentra-tion of oxalate alters cellular function and, in particular, thepermeability of the cellular membrane to oxalate.

DR. BORSATTI: To charge red blood cells we used the samesolution as did Cousin and Motais. A high concentration ofoxalate is necessary to appreciate the self exchange, because at0°C the exchange rate of chloride is 500 times higher than thatof oxalate. We did not observe any significant modification ofred blood cell shape or volume, and there was no hemolysis.Even if cellular functions are altered or if an increased passivepassage of oxalate occurs through the membrane in theseexperiments, we still find a clear-cut difference between stoneformers and controls in oxalate handling by red blood cells.

DR. GIORGTO BAzZATO (Chief, Division of Nephrology, Urn-berto I Hospital, Mestre-Venezia): Given the similarities amongred blood cells, gut cells, and proximal and distal tubular cells,are there similarities with gastric cells? If so, did you find anyincrease of gastric proton secretion in patients with the abnor-mal red blood cell oxalate self exchange?

DR. BORSATTI: Monoclonal antibodies raised against redblood cell band 3 protein cross-react with gastric oxintic cells,so these cells do have a red blood cell band-3-related protein.Your idea of looking for an abnormality in proton secretion inthe stomach in patients with an accelerated red blood celloxalate self exchange is excellent. We did not perform such astudy.

DR. BAZZATO: Have you studied the effect of proton-block-ing agents on urinary oxalate excretion?

DR. BORSATTI: No.DR. NATALE DE SANTO (Chi ef and Professor of Nephrology,

Division of Pediatric Nephrology, Federico II University,Napoli): You suggested that urinary pH might be relevant incalcium oxalate stone formation. Because ascorbic acid lowerspH, do you think it is a further possible mechanism by whichvitamin C favors calcium oxalate nephrolithiasis? Can the samemechanism be invoked for high protein intake?

DR. BORSATTI: In studies using the gel crystallizationmethod, calcium oxalate crystal growth and pH were inverselycorrelated [138]; at high pH, the crystallization of calciumoxalate dihydrate, which is less stable, seems to prevail overthat of calcium oxalate monohydrate [139]. So a high ascorbateintake, as well as a high-protein diet, might facilitate calciumoxalate precipitation. What I do not know is how much vitaminC has to be ingested before urinary pH decreases. Maybe Dr.Williams knows more about this issue.

DR. HIBBARD E. WILLIAMS (Professor of Medicine, Schoolof Medicine, University of California, Davis, California, USA):We have studied the effect of oral ascorbate on urine composi-tion, and specifically on urinary oxalate. We collected urines inconcentrated hydrochloric acid to be sure that there was littlenonenzymatic conversion of ascorbate to oxalate, so I cannotanswer Dr. De Santo's question. We gave as much as 10 gldayto several normal individuals. Our preliminary conclusion from15 individuals is that the urinary excretion of oxalate is notaffected significantly by the ingestion of as much as 5 g ofascorbic acid per day. At a vitamin C intake of 10 glday, a smallnumber of patients have a slight increase. Perhaps a fewpatients, possibly those with some abnormalities in the gastro-intestinal tract, do respond abnormally, but in most normal


individuals, moderately large quantities of ascorbate do notaffect urinary oxalate. Let me switch to another substance,namely, sulfate. Do you have any evidence of an abnormality inthe handling of sulfate in stone formers? If so, what role mightsuch an abnormality play in stone disease?

DR. BORSATTI: Studies on urinary sulfate in renal stonedisease are scanty. Urinary sulfate output seems to depend ondietary intake of animal protein, and a fairly good positivecorrelation between sulfate and GAG excretion appears to bethe rule [1401. A positive correlation between sulfate andcalcium in the urine also has been reported [141], but I am notaware of any relationship found with oxalate. As I have shown,however, oxalate uptake from peritubular fluid occurs in ex-change with sulfate, which means that an increased secretion ofoxalate should be paralleled by an increased absorption ofsulfate. We have no data yet on tubular sulfate handling in stoneformers with high red blood cell oxalate self exchange.

DR. MARTINO MARANGELLA (Director, Renal Stone Labora-tory, Mauriziano Hospital, Torino): You have pointed out thatoxalate secretion by the proximal tubule might be affected bysulfate and that GAGs' concentration and their degree ofsulfation can affect the oxalate self exchange in red blood cells.Moreover, you have suggested that a low urinary pH can occurin patients with pure calcium oxalate stones, and that sulfate,GAGs, the degree of GAG sulfation, and urine pH all areaffected by animal proteins [142]. Have you studied the possibleinfluence of animal protein intake on the red blood cell selfexchange? Is there a possible role for animal protein intake inyour hypothesis?

DR. BoRsArrI: Animal protein intake does not affect thesteady-state oxalate self exchange in red blood cells. A highanimal protein intake increases both sulfate and GAG excre-tion. The first step in moving oxalate from peritubular fluid tothe lumen is an uptake in exchange with sulfate; the moresulfate that is reabsorbed, the more oxalate is secreted. By thismechanism, a diet high in animal protein could stimulateurinary oxalate excretion. However, we have evidence thatexogenous GAG administration lowers urinary oxalate, and thesame result should be obtained following an increase in theurinary excretion of GAGs as occurs with high animal proteinintake. To summarize, sulfate and GAGs should have opposingeffects on oxalate renal handling.

DR. GlAcoMo CoLussi (Division of Nephrology, Niguarda-Ca' Granda Hospital, Milan): You have hypothesized that,based on structural identity between the band 3 protein residingin red blood cells and alpha intercalated cells of the collectingtubule, increased oxalate self exchange in erythrocytes could beassociated with increased C1/HC03 exchange in the collect-ing tubule; thus, one would expect an increased acidificationcapacity (that is, proton secretion) of the distal tubules, andindeed you have suggested that a lower urinary pH in calciumoxalate stone formers might support this possibility. However,a lower urine pH might reflect diminished buffer availability(that is, ammonia and/or phosphate) rather than increasedproton secretion. In fact, when proton secretion in calciumstone formers was investigated by acid loading or by themeasurement of pCO2 in alkaline urines, a high incidence (up to40%) of acidification defects (defined as incomplete type-i renaltubular acidosis) has been observed [143, 1441. How can you fitthese observations with your hypothesis?

DR. BORSATTI: Concerning urinary pH in patients with purecalcium oxalate stones, I have quoted data presented by Rob-ertson et al [127]. We should appreciate that the prototypicalstone formed by patients with renal tubular acidosis containscalcium phosphate, not calcium oxalate. Yet you are correct inasserting that an incomplete distal renal tubular acidosis hasbeen observed in a high proportion of calcium stone formers.The problem is: were they pure calcium oxalate stone formers?Putting aside this consideration, I can conceive a defect inexcreting a proton load in patients suppposed to have analpha-intercalated cell band 3 protein working at a faster rate.Just a small difference between actual rate and maximal possi-ble rate might reduce the quantity of proton secreted after aload.

DR. RENATA CAUDARELLA (Associate Professor of Meta-bolic Diseases, Institute of Internal Medicine, University Hos-pital, Bologna): You observed an increased rate of red bloodcell oxalate self-exchange in approximately 68% of patients, butyou could not find any relationship between the oxalate self-exchange rate and the 24-hour urinary oxalate excretion. Howdo you explain this observation? Might it be accounted for byan overlap of several genetic and environmental factors, such asdietary intake of proteins?

DR. BORSATTI: I do not believe that a defect in cellularhandling of oxalate is the single mechanism responsible forstone formation. I do believe we have enough data to supportthe view that many calcium oxalate stone formers have a defectin oxalate transport at a cellular level, and that is all. It is likelythat other genetic or environmental factors contribute to thedevelopment of nephrolithiasis, and a diet rich in animal proteinmight be one of these factors. Today, blaming a high-proteindiet for stone formation is fashionable, but a definitive linkbetween dietary protein intake and nephrolithiasis is far fromestablished [28].

DR. JEROME P. KASSIRER (Associate Physician-in-Chief, De-partment of Medicine, New England Medical Center, Boston,Massachusetts, USA): A fundamental premise of your argu-ment depends on exaggerated urinary oxalate excretion inpatients with calcium oxalate stone formation. Yet publisheddata show considerable variability in oxalate excretion in suchpatients, and in many total urinary oxalate is normal. You arguethat even when total excretion is normal, diurnal variations inexcretion may yield high urinary oxalate concentrations duringpart of the day. How convincing is the evidence for an in-creased excretion of oxalate at some time during the day? Dosuch patterns correlate with abnormalities in red blood celloxalate transport?

DR. BORSATTI: The appearance of peaks of urinary oversat-uration with calcium oxalate sometime during the day is the ruleeven in normal subjects. The urinary oxalate level fluctuatesduring the day both in stone formers and in normal subjects.The excretion is highest 2 or 3 hours after a meal, and thispostprandial increase is more pronounced in stone formers[145]. We did carry out an oxalate tolerance test in five pairs ofbrothers. One of each brother had an abnormal red blood celloxalate self exchange, but all had normal excretions of oxalate.We found that the increase in urinary oxalate was significantlyhigher at 2 and 4 hours after the oral oxalate load in the brotherswith the red blood cell abnormality [36].

Da. ANDREA TASCA (Institute of Urology, University Hospi-


tal, Padova): To follow up Dr. Kassirer's question, how manypatients with an abnormal red blood cell oxalate self exchangewere hyperoxaluric? Have you observed hyperoxaluric stoneformers with a normal red blood cell self exchange?

DR. BORSATTI: As far as 24-hour urinary oxalate is con-cerned, about 30% of patients with an abnormal red blood celloxalate self exchange were hyperoxaluric, as well as 9% ofpatients with a normal red blood cell oxalate self exchange.Whereas it appears easy to propose oversaturation peaks duringthe day as a cause of stone formation in patients with abnormalred blood cell self exchange and normal 24-hour urinary ox-alate, the observation that some patients with a normal selfexchange are hyperoxaluric is intriguing. As I suggested earlier,I believe that an abnormality in oxalate handling at a cellularlevel is not a conditio sine qua non for hyperoxaluria to takeplace. What brings hyperoxaluria about in patients with normalred blood cell oxalate self exchange? Although I have no proof,I suspect that a high dietary oxalate intake or even a subtleabnormality in endogenous oxalate synthesis might be in-volved.

DR. SURIAN: If postprandial episodes of oversaturation ofcalcium oxalate have a crucial role in stone formation ratherthan the state of saturation in a 24-hour urine collection, do youthink that the determination of urinary oxalate after a standardoxalate meal could be a better diagnostic tool than the measure-ment of oxalate in a 24-hour urine collection?

DR. BORSATTI: Most oxalate uptake from the gut occurswithin 8 hours after ingestion, and to date nobody has demon-strated a difference in urinary oxalate between stone formersand normal subjects during fasting, If a defect in oxalatehandling by stone formers is to be identified, it should be moreappropriate to evaluate urinary oxalate during the 8 hoursfollowing a standard oxalate-rich meal.

DR. TASCA: Approximately one-third of hyperoxaluric pa-tients have other urinary abnormalities such as hypercalciuriaand hyperuricosuria. Is there a connection in these patientsbetween red blood cell oxalate self exchange alterations andthese abnormalities?

DR. BORSATTI: Hypercalciuria was present in 27% and hyper-uricosuria in 16% of our patients with an abnormal red bloodcell oxalate self exchange. I cannot exclude that cellular han-dling of oxalate, urate, and calcium are in some way intercon-nected. One can construct an elaborate hypothesis based onseveral observations. First, for oxalate and urate to cross thecell wall, both need an exchanger, which probably must beactivated to work. Second, calcium is an extremely importantintracellular messenger. Thus we could speculate that an in-crease in free intracellular calcium is the primary defect, whichleads to activation of both oxalate and urate exchanger.

Da. FRANCESCO PAOLO SCHENA (Chief and Professor ofNephrology, Division of Nephrology, University Hospital,Ban): Did you find any correlation between sodium and oxalateexcretion in the urine? My question is based on the notion thatexcretion of oxalate could be related to that of sodium. If so, thequantity of sodium in the diet might be an important factor.

DR. BORSATTI: We did not look for a relationship betweensodium and oxalate in stone formers, and I do not even knowwhether such a correlation exists in a normal population. NataleDe Santo recently carried out a careful epidemiologic investi-

gation on urinary constituents in the young population of avillage near Naples, and he might be able to provide some data.

DR. DE SANTO: Weconducted an epidemiologic investigationon urinary oxalate excretion in all the young population ofCimitile, a tiny village near Naples. We studied 220 childrenaged 3 to 16 years. Twenty-four-hour oxalate excretion was32.2 16,8 mg (mean SD) or 1.0 0.6 mg/kg body weight.Oxaluria was not related to gender, but it correlated positivelywith age (r = 0.37; P < 0.001), height (r = 0.42; P <0.001) andbody-mass index (r = 0.360; P < 0.001). No correlation wasfound with weight (r = 0.126; not significant). As far as otherurinary constituents were concerned, we found a positivecorrelation with calcium (P < 0.005), phosphate (P < 0.005),magnesium (P < 0.001), creatinine (P < 0.008), potassium (P <0.002) and sodium (P <0.002). The clinical significance of thesecorrelations is yet to be assessed.

DR. SCHENA: in your studies you have demonstrated genetictransmission of the red blood cell defect. Does gender deter-mine stone formation?

DR. BORSATTI: From the family study we performed, it wasclear that many females who had the abnormality of red bloodcell oxalate self exchange never formed stones; on the contrary,almost all males with the red blood cell abnormality were stoneformers. Because male sex hormones increase the activity ofenzymes involved with oxalosynthesis, basal urinary excretionof oxalate in males might be higher, and when peaks ofhyperoxaluria—possibly due either to intestinal hyperabsorp-tion or renal oversecretion or both—occur, they could have alarger impact on urinary oversaturation with calcium oxalate.

DR. ANGELA D'ANGELO (Associate Professor of Nephrol-ogy, Institute of Internal Medicine, University Hospital, Pa-dova): Recurrence of hypercalciuria in patients with renalstones is not unusual after successful parathyroidectomy. Insome of these patients, hypercalciuria seems to be due tointestinal calcium hyperabsorption. Do you think that mildhyperoxaluria might be linked to this metabolic abnormality inthe case you presented?

DR. BORSATTI: The patient I described became normocalci-uric after parathyroidectomy and was still so during the secondobservation (262 mg/day). Nevertheless, there must be some-thing common in intestinal absorption of calcium and oxalate.This view is strengthened by the demonstration by Marangellaet al that a statistically significant increase in intestinal oxalateabsorption after an oral oxalate load was evident only inpatients who were hypercalciuric [301. Furthermore, vitamin Dadministration increases dietary oxalate absorption in normalsubjects [146]. The common interpretation is that vitamin D, byincreasing calcium absorption, results in less binding of calciumto oxalate in the intestine and makes more free oxalate availablefor absorption. What we do not know is whether vitamin D hasa direct effect on oxalate handling by the cell.

DR. PAOLO MORACCHIELLO (Division of Nephrology, Urn-berto I General Hospital, Mestre-Venezia): Do you have dataon red blood cell oxalate self exchange in patients with primaryhyperparathyroidism before and after parathyroidectomy?

DR. B0RsATrI: As a rule, oxalate red blood cell self exchangeis normal in primary hyperparathyroidism 136], and it remainsunchanged after parathyroidectomy.


Reprint requests to Dr. A. Borsatti, Division of Nephrology, Instituteof Internal Medicine, University Hospital, via Giustiniani, 2, 35128,Padova, Italy

Acknowledgments

The author is grateful to Dr. Giovanni Gambaro for help in thepreparation of the manuscript, and to Prof. Hibbard E. Williams forcritically reading it.

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Calcium oxalate nephrolithiasis: Defective oxalate transport · 1284 Nephrology Forum: Calcium...

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