Urinary L-lactate excretion is increased in renal Fanconi syndrome
Arumugavelu Thirumurugan1,
Andrew Thewles2,
Rodney D. Gilbert3,
Sally-Anne Hulton1,
David V. Milford1,
Christopher J. Lote2 and
C. Mark Taylor1
1 Department of Nephrology, Birmingham Children's Hospital, Birmingham, 2 Division of Medical Science, University of Birmingham, Birmingham and 3 Department of Paediatric Nephrology, Southampton General Hospital, Southampton, UK
Correspondence and offprint requests to: Dr C. Mark Taylor, Department of Nephrology, Birmingham Children's Hospital, Birmingham B4 6NH, UK. Email: cm.taylor{at}bch.nhs.uk
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Abstract
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Background. Measurement of L-lactate in body fluids is an established clinical tool to identify disorders of cellular respiration. However, there is very little known about the clinical value of urinary lactate measurements. We investigated urinary lactate excretion in children with renal Fanconi syndrome.
Methods. Freshly voided urine samples were obtained from children with Fanconi syndrome and controls both with and without renal disease. Urine lactate was estimated by conversion to pyruvate in the presence of lactate dehydrogenase and NAD. The NADH produced was measured photometrically. Urine lactate was factored for urine creatinine.
Results. Children with Fanconi syndrome had a significantly higher urine lactate/creatinine ratio [mean: 84 x 102 mmol/mmol; 95% confidence interval (CI): 40.8127.1 x 102 mmol/mmol] than healthy controls (mean: 1.3 x 102 mmol/mmol; CI: 1.11.5 x 102 mmol/ mmol) and those with a variety of renal diseases (mean: 3.1 x 102 mmol/mmol; CI: 1.84.5 x 102 mmol/mmol).
Conclusions. Urinary lactate is increased in Fanconi syndrome. The increase is likely to be due to reduced lactate co-transport in the proximal tubule. Urinary lactate/creatinine has clinical utility as a sensitive test of disordered proximal renal tubular function.
Keywords: Fanconi syndrome; lactate co-transport; proximal tubule disorders; urinary lactate cystinosis
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Introduction
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Fanconi syndrome (FS) is a generalized defect of proximal tubular reabsorption [1]. A unifying hypothesis to explain the generalized co-transport defect that characterizes the condition is that it is secondary to an impairment of primary sodium transport out of the cell. Primary sodium transport across the basolateral membrane requires NaK-ATPase and is the driving force for virtually all tubular recovery of electrolytes and non-electrolytes, such as glucose. Defects of tubular protein recovery also occur in FS.
Disorders of active transport, or of the necessary intracellular respiratory pathways that lead to ATP generation, can be expected to cause FS [2]. However, evidence for this in some of the specific forms of FS is incomplete. For example, in nephropathic cystinosis, the commonest cause of FS in childhood, the connection between failed lysosomal cystine metabolism and an active transport defect at the basolateral membrane is unclear. However, it may be relevant that cystine loading of tubular cells can experimentally reduce intracellular ATP [3].
More obvious support for the hypothesis can be found among the mitochondrial cytopathies, a group of disorders in which abnormalities of the enzyme complexes of the respiratory chain within the mitochondria lead to impaired energy production [47]. Under aerobic conditions, oxidative phosphorylation occurs within the inner membrane of the mitochondrion that harbours the respiratory chain. Glucose metabolism via the tricarboxylic acid cycle requires a steady supply of the hydrogen acceptors nicotinamide adenine dinucleotide (NAD) and flavine adenine dinucleotide (FAD) and is dependant on an intact mitochondrial respiratory chain to regenerate NAD from NADH and FAD from FADH [7]. Under anaerobic conditions, pyruvate, the end-product of glycolysis, can accept hydrogen from NADH, thus replenishing NAD stores, resulting in the production of lactate. This can help sustain glucose metabolism and energy production for some time. However, a shift towards anaerobic metabolism results in the accumulation of lactate. Lactate measurement in plasma or cerebrospinal fluid can be used to screen test for primary respiratory chain disorders affecting various organs in the body.
It has been proposed previously that urinary lactate excretion is increased in FS [8]. We therefore investigated this in children with FS and compared the results with those of healthy children and with those with other renal diseases. We considered that increased urinary excretion of lactate might occur for two possible reasons: one being that tubular recovery of filtered lactate may be reduced in FS and the other that there may be increased renal production.
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Subjects and methods
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Patients were recruited from the nephrology outpatient clinics and in-patient wards at the Birmingham Children's Hospital for measurement of random urine lactate creatinine ratio (L/C). Informed consent was obtained from parent and/or child, as appropriate. Ethical approval for the study was obtained from the Birmingham Children's Hospital Ethics Committee and the South Birmingham Local Research Ethics Committee.
Patients included 10 children with FS. The age range of these children was from 9 months to 18 years. Seven of these had nephropathic cystinosis confirmed by leukocyte cystine measurement, three cases coming from a single pedigree. One child in whom the diagnosis had been made antenatally had L/C measured sequentially in the first year of life. In one child with cystinosis, L/C was measured before and after cadaveric transplantation. FS in one child was secondary to Wilson's disease. In this case there was abnormal liver function, plasma lactate was elevated at 3.6 mmol/l and there was mild reversible renal impairment at the time of sampling. In the two remaining cases, extensive investigation failed to reveal the cause of FS. One was a teenage girl with a syndrome comprising cerebellar hypoplasia, oculoplegia, visual impairment and myopathy. The other was a 7-year-old boy with developmental delay, microcephaly and growth retardation, whose FS presented in infancy. He also had unexplained neonatal hepatitis that appeared to resolve and an intraventricular haemorrhage requiring ventriculoperitoneal shunting. Up to and including the time of investigation, plasma lactate concentrations were normal, but he has subsequently had mild elevation, the plasma lactate being 3.6 mmol/l without intercurrent illness. A renal biopsy in infancy showed interstitial fibrosis. Both these cases had slowly progressive renal impairment. All FS cases were receiving oral electrolyte replacement, with the exception of the youngest child with cystinosis who had not developed electrolyte depletion at that stage. All cystinosis patients were treated with cysteamine. A total of 26 urine samples was obtained in the FS group (Table 1).
A second group comprised 23 children with various kidney disorders other than FS. Diagnoses are given in Table 2. Twenty-nine urine samples were obtained in this group.
Urine was also collected from 31 healthy controls. These were children aged between 2 and 15 years attending ENT and plastic surgery outpatient departments for minor conditions, such as recurrent otitis media, cutaneous vascular malformations and minor trauma.
Samples were obtained from five children with other disorders. These included three children with phenylketonuria, one child with unexplained encephalopathy and one child with idiopathic cardiomyopathy. None of these children had overt renal disease. The diagnosis in the latter two was unknown and their plasma lactate concentrations were 3.2 and 1.0 mmol/l, respectively (normal: < 2 mmol/l).
All urine samples were negative for blood, leukocyte esterase and nitrite on strip reagent testing (Ames, Slough, UK). They were immediately frozen after collection and analysed within 2 h of thawing. The L-lactate in urine was measured by enzymatic conversion of L-lactate to pyruvate with concomitant conversion of NAD to NADH, the increase in absorbance at 340 nm being proportional to NADH formation. Lactic acid and glycine buffer (pH 9.2) were obtained from Sigma-Aldrich Co. (St Louis, Missouri, USA) NAD (solid; 2 mg/ml) and lactate dehydrogenase (15 kU/ml; 50 U/ml) were from ICN Pharmaceuticals Ltd (Basingstoke, UK). The experimental protocol consisted of preparing reagents freshly for each assay by dissolving NAD and lactate dehydrogenase in glycine buffer (0.6 mol/l) at concentrations of 5 mg/ml and 50 U/ml, respectively. Urine samples were assayed undiluted or suitably diluted if the lactate concentration exceeded 0.3 mmol/l. Duplicate urine samples (0.15 ml) or standards or water were combined with 0.07 ml of reagent (or glycine buffer for sample blanks). Thus, the effective lactate dehydrogenase concentration was 15.9 U/ml. In addition, sample blanks were incubated with glycine buffer containing no NAD or lactate dehydrogenase. Following incubation (30 min at 37°C), sample absorbance was measured at 340 nm (plate reader). Zero lactate (water) was subtracted from urine and standard values and sample blank values were subtracted from sample values to give absorbance due to lactate. Urine lactate concentration was calculated from the standard value using the formula:
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In order to confirm the linearity of the assay, urine samples were spiked with L-lactate to give mean final concentrations of 0.248, 0.716 and 2.065 mmol/l. The measured increase in lactate concentration was 97.4±3.4%, 100.1±3.6% and 100.9±3.4% of the calculated increase (n = 8 per group). Urinary lactate was factored for urinary creatinine (mmol lactate per mmol creatinine). The intra- and interassay coefficients of variation were 2.45% (n = 8) and 4.76% (n = 5), respectively.
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Results
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The mean urinary L/C for each subject are shown in Figure 1 and summarized in Table 3. Patients with FS have a 40-fold increase in L/C compared with normal children. There appeared to be no difference between the L/C of cystinosis patients and the three FS cases with other aetiologies. A clear separation between the FS group and children with other renal diseases, including renal impairment, was also seen.

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Fig. 1. Urinary L/C in the three study groups: healthy controls, FS and controls with various renal diseases other than FS.
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One child with cystinosis observed from birth had L/C close to normal (range: 2.05.6 x 102 mmol/mmol) before reaching a ratio typical of other cases (98.4 x 102 mmol/mmol) at 5 months of age (Figure 2). The rise in L/C preceded other biochemical evidence of FS in this case. These near normal results were included in the FS group and account for the low outlying mean result for this case.

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Fig. 2. Sequential measurements of urinary L/C in a child with nephropathic cystinosis followed from birth.
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In the group with renal disorders there were two outliers: one a patient with Bardet Biedl syndrome (L/C: 12.5 x 102 mmol/mmol) and another with renal dysplasia (L/C: 11.3 x 102 mmol/mmol), neither of whom had reduced glomerular filtration rate (GFR). Two children had undergone cadaveric renal transplantation at the time of the study. In both of these the possibility of a primary respiratory chain disorder was considered, but in neither case was it proven. Patient 20 presented with sensorineural deafness, hypoparathyroidism and advanced renal failure. His sibling had sensorineural deafness only. A screening test for the more common mutations in mitochondrial DNA did not identify an abnormality. Patient 31 presented with cleft plate, pseudohypoparathyroidism, primary hypothyroidism, cataracts and advanced renal failure. Her native kidney renal biopsy showed extensive glomerular sclerosis. Although her plasma lactate was normal at the time of investigation, on other occasions she had elevated concentrations up to 5.6 mmol/l. Mitochondrial DNA studies were not undertaken. In both these transplanted cases, urinary L/C was in the mid-range for the group. Moreover, in a child with cystinosis not included in the above, who had a successful kidney transplant, the L/C was similar to the non-Fanconi renal controls at 3.0 and 4.7.
In the five children with other disorders, the three children with phenylketonuria had a L/C of 0.95 x 102, 1.5 x 102 and 4.3 x 102 mmol/mmol. The child with unexplained encephalopathy had a L/C of 18.1 x 102 mmol/mmol and the child with primary dilated cardiomyopathy had a L/C of 117.6 x 102 mmol/mmol. Neither of these cases had been fully investigated for disorders of cellular respiration.
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Discussion
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Our investigation shows that the urinary excretion of lactate is markedly and consistently increased in FS patients. FS is rare both in children and adults and out of a long list of possible causes, nephropathic cystinosis is by far the most prevalent in paediatric practice. The numbers in this study are, therefore, small and the patient group is dominated by cystinosis. However, we show a clear separation between the urinary L/C of Fanconi patients and normal children and to a lesser extent those with other renal diseases.
In nephropathic cystinosis, the primary problem is a failure to export cystine from lysosomes. How this leads to FS is not clear. It has been shown that cystine loading inhibits active transport in proximal tubules in microculture studies [9]. Coor et al. [3] showed that cystine-loaded proximal tubular epithelial cells exhibit ATP depletion that is of a similar severity to 10 min of anoxia. Adding exogenous ATP to cystine-loaded cells partly restored the volume absorption observed in their model. However, NaK-ATPase activity per se was not decreased by cystine loading. In a similar model, the sodium entry gradient into tubular cells and sodium-coupled transport of other solutes across the epithelial membrane was decreased [10]. Large mitochondria have been observed in proximal tubular cells of a mouse model of cystinosis [11] as well as in renal biopsies from patients [12,13]. The Ctns / mouse shows a high level of cystine accumulation in the kidney but does not develop tubulopathy, suggesting a rescue pathway that prevents ATP depletion [11].
Nephropathic cystinosis is not clinically apparent in the first 6 months of life and yet we observed a rise in the L/C in an infant with cystinosis before the appearance of the typical features of the syndrome (glycosuria, amino-aciduria, phosphate and bicarbonate wasting).
The cause of the increased excretion of lactate in FS is not immediately clear. From the normal ranges of lactate and creatinine in plasma and urine, it can be deduced that the filtered lactate in normal individuals is extensively reabsorbed or metabolized. For example, an individual with a plasma lactate of 12 mmol/l, a plasma creatinine of 100 µmol/l and a GFR of 100 ml/min/1.73 m2 would have a urine lactate creatinine in the order of 10002000 x 102 mmol/mmol if the filtered lactate were not reabsorbed. This is an order of magnitude greater than observed in FS and two orders of magnitude greater than normal. In a disease state in which tubular recovery is impaired, there is at least the potential for a very high urinary excretion of lactate.
It is generally agreed that lactate is extensively recovered from the filtrate in the proximal tubule. More than 50 years ago, Craig [14] observed that lactate is normally avidly cleared from the plasma to a non-urine compartment, or more likely metabolized. We confirmed this in a preliminary study in rats, in which intravenous lactate infusion elicited only a small increase in the plasma lactate concentration and no significant change in the urinary lactate excretion (C. J. Lote, personal communication). There is considerable reserve capacity, as it was not possible within the practical constraints of the experiment to achieve the tubular maximum for lactate recovery.
There is very little published information on the mechanism of lactate recovery in the nephron. We think it is likely that it is handled in a similar way to other small organic anions and that recovery is directly linked to Na+/H+ co-transport (Figure 3). The filtered lactate anion associates with H+ and enters the proximal tubular cell as uncharged lactic acid by facilitated diffusion through a uniporter system. This mimics the formate/formic acid exchange known to occur at the apical membrane, which depends on chloride co-transport. Given that both sodium and chloride co-transport systems are defective in FS, a failure to absorb luminal lactate might simply be a reflection of this and, therefore, an integral part of FS.

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Fig. 3. Mechanisms of lactate transport and metabolism in renal tubular cells. Lactate reabsorption is linked to sodium reabsorption, probably by Na+/H+ and Cl/organic anion (COO) exchange. Basolateral efflux of lactate, whether reabsorbed from the lumen or generated within the cell, occurs via a MCT in association with H+. Apical lactate transport may also involve a MCT (see text).
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Lactate metabolism in the renal medulla is different. It has long been known that while the concentration of lactate in the cortex is similar to that in plasma, higher concentrations are found in the medulla and the increase is related to the urine concentrating capacity [15]. The medulla is relatively hypoxic and energy metabolism relies on glycolysis with the consequent production of lactate. Theoretical modelling suggests that lactate accumulates so as to contribute to the total osmolar load of the medullary interstitium [16]. Efflux of lactate from medullary tubular epithelial cells appears to occur through monocarboxylate transporters (MCT) located in the basolateral membrane [17,18]. In the rat, for example, MCT2 is localized specifically to this site in the medullary thick ascending limb of the loop of Henle. The mechanism for basolateral transport of lactate in cortical tubular cells is not clear, but is also likely to involve a MCT.
A unifying hypothesis for the pathogenesis of FS is that there is a defect in active transport of sodium at the basolateral membrane due to a defect of Na/K-ATPase. This, in turn, might be linked to diseases of cellular respiration, such as the mitochondrial cytopathies, in which ATP generation is impaired and intracellular lactate may accumulate. This raises the possibility that if FS is caused by disordered cellular respiration leading to an increased production of lactate in tubular cells, lactate would compete with formate for apical export by the chloride/formate co-transporter. In this scenario, the increased urinary lactate might reflect increased renal lactate generation. In theory, both reduced reabsorption and increased renal production might occur together.
Touati et al. [19] reported increased urinary L/C in some, but not all, children with known respiratory chain disorders. This suggests it is the expression of the disorder in the kidney itself that determines whether or not there is increased lactate excretion. Urinary L/C appears to be a very sensitive marker, as evidenced by the increase in L/C prior to the development of FS in cystinosis. It is, therefore, likely that lactate excretion is increased in disorders of cellular respiration affecting the kidney that are not severe enough to cause overt FS. We suspect that our cases of idiopathic cardiomyopathy and encephalopathy may have an underlying cellular respiratory disorder and that the raised lactate excretion in these cases is clinically meaningful rather than a false positive. An explanation for the elevated L/C in the two children with structural renal disease (patients 18 and 32) is less clear.
In summary, we cannot be sure whether the increased urinary lactate in FS reflects failed absorption as part of the co-transport defect of the syndrome or increased renal cellular production of lactate due to a cellular respiration problem or both. Given that there are reliable laboratory models of FS, it would be possible to explore this detail further. Nevertheless, there is reason to think that the measurement of urinary lactate in humans may have clinical utility and prove to be a subtle and inexpensive test of proximal tubular dysfunction.
Conflict of interest statement. None declared.
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Received for publication: 5. 9.03
Accepted in revised form: 13. 2.04