Renal Fanconi syndrome with ultrastructural defects in lysinuric protein intolerance

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1 JIMD Short Report #052 (2007) Online DOI /s SHORT REPORT Renal Fanconi syndrome with ultrastructural defects in lysinuric protein intolerance M. A. Benninga & M. Lilien & T. J. de Koning & M. Duran & F. G. A. Versteegh & R. Goldschmeding & B. T. Poll-The Received: 8 August 2006 /Submitted in revised form: 6 March 2007 /Accepted: 7 March 2007 # SSIEM and Springer 2007 Summary Renal Fanconi syndrome developed rapidly in a 3-year-old Moroccan girl with established lysinuric protein intolerance. She was hospitalized because of lowered consciousness, uncoordinated movements and hepatosplenomegaly after a febrile period. Laboratory investigations revealed plasma ammonia 270 mmol/l (normal <70 mmol/l), ferritin 159 mmol/l (normal 2 59 Communicating editor: Mike Gibson Competing interests: None declared References to electronic databases: Lysinuric protein intolerance (LPI): OMIM M. A. Benninga Academic Medical Center, Department of Paediatric Gastroenterology and Nutrition, Emma Children_s Hospital, Amsterdam, The Netherlands M. Lilien Department of Nephrology, University Children_s Hospital Wilhelmina Kinderziekenhuis, Utrecht, The Netherlands T. J. de Koning Department of Metabolic Diseases, University Children_s Hospital Wilhelmina Kinderziekenhuis, Utrecht, The Netherlands M. Duran : B. T. Poll-The (*) Academic Medical Center, Department of Metabolic Diseases and Pediatrics, Emma Children_s Hospital, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands B.T.PollThe@amc.uva.nl F. G. A. Versteegh Groene Hart Ziekenhuis, Department of Paediatrics, Gouda, The Netherlands R. Goldschmeding University Medical Center Utrecht, Department of Pathology, Utrecht, The Netherlands mmol/l), LDH 1180 U/L (normal U/L). LPI was diagnosed based on the findings of reduced plasma ornithine, arginine and lysine, and an increased level of glutamine. Urinary orotic acid (645 mmol/mmol creatinine; normal <3.6) was strongly increased. A defect in the SLC7A7 amino acid transporter was established (homozygous c.726g>a mutation). Detailed renal function tests including an acid challenge test, bicarbonate loading, and tubular maximal reabsorption of glucose showed complex tubular dysfunction. No evidence of respiratory chain defects was found in muscle or kidney tissue. No morphological abnormalities were demonstrated in the mitochondria. Ultrastructural analysis of proximal tubular cells showed vacuolization and sloughing of the apical brush border. Renal involvement in LPI has only been described in a few reports; however, no detailed studies of the renal acidification mechanism were performed. Our patient had evidence of a full-blown Fanconi syndrome. Surprisingly, a metabolic acidosis was found with a moderately increased serum anion gap combined with repeatedly normal plasma organic acid values. This finding is in contrast with the diagnosis of renal tubular acidosis. Patients with hyperlysinaemia have a similar heavy load on the renal tubules; they never develop a renal Fanconi syndrome. Therefore, we consider the intratubular accumulation of lysine an unlikely candidate for the development of the renal Fanconi syndrome. Abbreviations GFR glomerular filtration rate LDH lactate dehydrogenase LPI lysinuric protein intolerance RTA renal tubular acidosis

2 2 JIMD Short Report #052 (2007) Online Introduction Lysinuric protein intolerance (LPI) (OMIM ) is an autosomal recessively inherited multiorgan disorder in which the renal and intestinal transport of the cationic amino acids lysine, arginine and ornithine is defective (Perheentupa and Visakorpi 1965). The molecular defect resides in mutations of the SLC7A7 gene, encoding for the y + LAT-1 amino acid transporter (Torrents et al 1999). The basolateral transport defect is expressed in the epithelial cells of the intestine and the kidney tubules, hepatocytes and skin fibroblasts (Rajantie et al 1980; Simell and Perheentupa 1974). The reduced intestinal absorption and tubular reabsorption of these cationic amino acids lead to a depletion of their tissue pools and, as a consequence, to impaired functioning of the hepatic urea cycle with hyperammonaemic episodes. Patients may show aversion to protein-rich foods, failure to thrive, hepatosplenomegaly, muscle hypotonia and osteoporosis (Carpenter et al 1985). Furthermore, seizures and mental retardation as a late consequence of the hyperammonaemic episodes have been described (DiRocco et al 1993). Thus far, abnormalities have been reported of the respiratory, renal, immunological and haematological systems (DiRocco et al 1993; Nagata et al 1987; Parenti et al 1995; Parto et al 1994). We describe the clinical course and the biochemical and pathological findings in a patient with lysinuric protein intolerance and severe renal pathology. Case report A girl was born to consanguineous (first-cousin), healthy Moroccan parents after a normal pregnancy and delivery. Birth weight was 2870 g. At 2 years and 6 months, failure to thrive and mildly delayed psychomotor development were apparent. At the age of 3 years she was hospitalized because of lowered consciousness, uncoordinated movements and hepatosplenomegaly after a febrile period. Her length was 80.5 cm (4 cm below the 3rd centile) and her weight was 10 kg (10th centile). Routine CSF analysis and an EEG showed no abnormalities. Initial laboratory investigations in blood revealed Hb 5.4 mmol/l, Ht 0.32 L/L, platelet count /L and white blood cell count /L. Capillary ph was 7.33, base excess was j6.0 meq/l and bicarbonate was 18 mmol/l. Lactate was 2.4 mmol/l (normal <2 mmol/l), plasma ammonia was 270 mmol/l (normal <70 mmol/l), ferritin was 159 mmol/l (normal 2 59 mmol/l), transaminases were normal and LDH was 1180U/L (normal U/L). LPI was diagnosed based on the findings of reduced plasma ornithine, arginine and lysine, and an increased level of glutamine (Table1). Urine amino acid analysis revealed increased excretion of lysine, arginine and ornithine (Table1). Urinary orotic acid (645 mmol/mmol creatinine, normal <3.6) was strongly increased. Subsequently she was put on a protein-restricted diet (1.5 g/kg per day) and L-citrulline (400 mg/kg per day). The final proof of a defect in the SLC7A7 amino acid transporter was given by the finding of a homozygous c.726g>a mutation in the patient_s DNA (Mykkänen et al 2000). This mutation predicts premature termination of the protein at amino acid 242. In the following 9 months her clinical condition remained stable and the plasma ammonia levels were in the normal range. Then, over a 3-month period at the age of 3.9 years, a persistent metabolic acidosis (capillary ph 7.33, PCO 2 29mmHg, base excess j9.7 meq/l and bicarbonate 15 mmol/l) with an elevated anion gap 20.4 (normal <15) was observed. Plasma organic acids were normal. Furthermore, renal investigations revealed all laboratory signs of Fanconi syndrome, with glucosuria, generalized aminoaciduria and phosphaturia. Analysis of organic acids in the urine showed elevated levels of lactic acid (200 mmol/ mmol creatinine, normal <100), 2-ketoglutaric acid (860 mmol/mmol creatinine, normal <70) and citric acid (390 mmol/mmol creatinine, normal <180). In addition, low plasma concentrations of free carnitine (12 mmol/l, normal 25 60) and total carnitine (18 mmol/l, normal 30 65) were found. No abnormal acylcarnitines were found in the plasma by fast-atombombardment mass spectrometry. Renal function tests were performed after parental informed consent had been obtained. In order to explain the combination of a renal Fanconi syndrome and elevated levels of organic acids in the urine, a muscle biopsy and a renal biopsy were performed to probe the existence of a respiratory chain defect. Renal investigations Renal function tests were performed over a 3-day period. Two needles were placed into peripheral veins, one for the infusion of the test substances and one for the blood sampling. Urine was collected via an indwelling catheter. The glomerular filtration rate was evaluated by the creatinine clearance. The renal threshold phosphate concentration (TmPi/GFR) was calculated using the

3 JIMD Short Report #052 (2007) Online 3 Table 1 Amino acids in plasma and urine in the patient with lysinuric protein intolerance in the initial diagnostic phase at the age of 3 years and after the development of the renal Fanconi syndrome, 9 months later Amino acid Plasma concentration (mmol/l) Urine concentration (mmol/mol creatinine) Patient Controls Patient 3 years Patient 3 years 9 months Controls Taurine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Citrulline Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine n.d Lysine Histidine Arginine n.d., not determined. nomogram of Walton and Bijvoet (Walton and Bijvoet 1975). Acid challenge test. Urinary acidification and acid excretion were assessed after i.v. administration of ammonium chloride (75 meq/m 2 body surface). Blood samples were analysed for creatinine, ph and bicarbonate and timed urine samples for volume, creatinine, ph, titratable acid and ammonia. Bicarbonate loading. Sodium bicarbonate 3 mmol/kg was administered orally to obtain maximally alkalinized urine. During this period and during the following hour, urine samples were collected in 30 min periods, and capillary blood samples were obtained at 1 and 2 h after the start of the bicarbonate intake. In all urine and blood samples, ph, PCO 2, creatinine and bicarbonate concentration were determined. Distal tubular hydrogen ion secretion was assessed by calculation of the difference between urinary and blood PCO 2, (U B) PCO 2, after maximal alkalinization of the urine was obtained. Simultaneous measurements of the clearance of creatinine and bicarbonate allowed calculation of tubular reabsorption and fractional excretion of bicarbonate. Tubular maximal reabsorption of glucose was calculated after intravenous administration of 300 ml glucose 25% per 1.73 m 2 as a priming dose, followed by 180 ml glucose 20% per 1.73 m 2 per h maintenance infusion. Two timed urine samples were analysed for volume, creatinine and glucose. During urine collection, three blood samples were analysed for creatinine and glucose. The assay of the activities of the various complexes of the mitochondrial respiratory chain in muscle and kidney was done by established spectrophotometric methods. Results The glomerular filtration rate was 97 ml/min per 1.73 m 2 (normal ), urine anion gap was 72, urine osmol gap was 23.4 mosm/kg, renal threshold phosphate concentration was 0.7 mmol/l (normal >2.2), fraction-

4 4 JIMD Short Report #052 (2007) Online Fig. 1 Left panel shows irregular and partially absent brush border in the patient with LPI. The panel on the right shows the regular brush border architecture in a patient of the same age (biopsy taken in remission phase of idiopathic nephritic syndrome). Note also that in this normal specimen the villi are longer than in the patient with LPI al carnitine excretion was 24% (normal <19%) and fractional total carnitine excretion was 19.5% (normal <13%). Acid challenge test. During the acid challenge test, serum bicarbonate decreased to 12 mmol/l; minimal urine ph achieved during the test was Glomerular filtration rate during the test was 95 ml/min per 1.73 m 2, titratable acid secretion was 78 meq/min per 1.73 m 2 (normal >62), ammonium excretion was 43 meq/min per 1.73 m 2 (normal >57), hydrogen secretion was 121 meq/min per 1.73 m 2 (normal >119) and ammonium excretion per 100 ml glomerular filtrate was 46 meq/ 100 ml (normal >68). Bicarbonate loading. During bicarbonate loading it was not possible to raise serum bicarbonate concentration above 17 mmol/l. The fractional excretion of bicarbonate under these conditions was 34% (normal <20), however. The (U B) PCO 2 was 73 mmhg. Tubular maximal reabsorption of glucose (TmG). After an overnight fast, blood glucose was 4.6 mmol/l. Urine glucose at that time was 3.7 mmol/l. Therefore, the renal glucose threshold must be lower than 4.6 mmol/ L (normal >10). TmG measured during induced hyperglycaemia was 127 mg/min per 1.73 m 2 (normal >170). A renal biopsy demonstrated focal vacuolization in the proximal tubuli in both cortex and medulla. Electron microscopy showed many proximal tubuli with sloughing of the brush border (Fig. 1). No morphological abnormalities were demonstrated in the mitochondria. No evidence of immune deposits was shown by immunofluorescence. Furthermore, normal activities were found of complex I and IV of the respiratory chain. Echosonography of the kidney showed mild hyperechogenicity of the medulla. This finding is compatible with beginning nephrocalcinosis. Muscle biopsy of the vastus lateral muscle demonstrated no abnormalities of the pyruvate dehydrogenase complex or respiratory chain with normal histology and histochemistry of skeletal muscle. Discussion Lysinuric protein intolerance is a rare genetic disorder associated with episodes of hyperammonaemia upon ingestion of protein. Its diagnosis may be prompted by the finding of isolated elevations of serum ferritin and

5 JIMD Short Report #052 (2007) Online 5 LDH, as observed in our patient. Renal involvement in LPI has been described in only a few reports (DiRocco et al 1993; Parenti et al 1995). One patient showed chronic renal failure due to chronic tubulo-interstitial disease with focal glomerulosclerosis, and another patient presented urinary abnormalities associated with renal tubular acidosis. Additionally, Parenti and colleagues described one patient with findings consistent with a Fanconi syndrome; however, no detailed studies of the renal acidification mechanism were performed (Parenti et al 1995). Our patient had evidence of a full-blown Fanconi syndrome. Surprisingly, a metabolic acidosis was found with a moderately increased serum anion gap combined with repeatedly normal plasma organic acid values. This finding is in contrast with the diagnosis of renal tubular acidosis. Proximal tubular acidosis is associated with a high urinary citrate excretion, i.e. above 180 mmol/mmol creatinine, due to diminished proximal reabsorption of filtered citrate (Scharer and Manz 1985). The present patient also had a high citrate excretion. The presence of these biochemical abnormalities prompted us to examine the renal acidification mechanism in detail. The glomerular filtration rate was normal. During the acid challenge, minimal achieved urine ph was not indicative of distal tubular acidosis. Furthermore, we found a normal excretion of titratable acid and total hydrogen excretion. However, the ammonium excretion was diminished. This finding is consistent with proximal tubular acidosis, as renal ammonia production is a feature of the proximal tubule. High fractional bicarbonate excretion is also compatible with proximal tubular acidosis. Furthermore, a high urine to blood PCO 2 difference excludes distal tubular acidosis. Besides a proximal tubular acidosis, other signs of a complex tubular disorder were found, i.e. diminished renal reabsorption of phosphate (Walton and Bijvoet 1975), glucose and amino acids and carnitine. The majority of cases of proximal renal tubular acidosis fit into the category of generalized tubule dysfunction with the Fanconi syndrome. This defect can be due either to backleak of solutes via the paracellular pathway or to a generalized disorder of the apical membrane and its transporters, a disorder of the basolateral Na +,K + -ATPase, or a metabolic disorder that lowers ATP concentration. Electron microscopy of renal tissue showed severe abnormalities in the ultrastructure of proximal tubular apical membranes. We suggest that this is the basic disorder causing proximal tubular dysfunction, either through a diminished resorptive area and decreased availability of transport proteins or through increased backleak of solutes from the proximal tubular cell. The cause of the ultrastructural changes remains to be determined; possible mechanisms are a direct toxic effect of retained metabolites on the cell membrane or a generalized disorder in cellular energy metabolism (DiRocco et al 1993). Lo and colleagues showed in a case report that the oral intake of L-lysine was associated with the development of a renal Fanconi syndrome (Lo et al 1996). Interestingly, the ultrastructural abnormalities in the proximal tubular cells of their patient were similar to those found in this child. It has been recognized that the infusion of L-lysine inhibits proximal tubular protein reabsorption and reduces bicarbonate reabsorption in the rat proximal tubule (Chan and Kurtzman 1982). The administration of L-lysine at high doses can lead to acute renal failure with proximal tubular necrosis, cast formation and obstruction (Racusen et al 1985). Therefore, a possible mechanism in this patient might be the accumulation of intracellular lysine in the proximal tubule because of diminished basolateral transport, thereby causing a cytotoxic effect. Patients with hyperlysinaemia due to 2-aminoadipic semialdehyde synthetase deficiency have a similar heavy load on the renal tubular y + LAT-1 system. However, they never develop a renal Fanconi syndrome (Slingerland et al 2004). Therefore, we consider the intratubular accumulation of lysine an unlikely candidate for the development of the renal Fanconi syndrome. Biochemical analysis of muscle and renal tissue showed no signs of a respiratory chain defect in this patient. The renal studies, however, had to be limited to the analyses of complexes I and IV of the mitochondrial respiratory chain. Malfunctioning of other parts of the respiratory chain may well have contributed to the renal pathology. Other causes of renal Fanconi syndrome such as vitamin D deficiency were excluded. It is known that in LPI there is a specific defect of dibasic amino acid transport, located at the basolateral membrane of epithelial cells. However this defect does not cause disturbances in other basolateral transport systems, such as Na +,K + -ATPase. It is therefore not likely that it would be the cause of the Fanconi syndrome. The low plasma free carnitine observed in this patient may be considered a consequence of excessive renal loss of this substance as a part of the renal Fanconi syndrome. Similar observations have been made in patients with cystinosis. Reports on carnitine status in LPI are scarce: Korman and colleagues (2002) reported moderately decreased carnitine levels in two patients; they attributed this to the lack of lysine, which is one of the biosynthetic precursors.

6 6 JIMD Short Report #052 (2007) Online In conclusion, this case demonstrates a rapid development of a Fanconi syndrome in a child with LPI, with no evidence of respiratory chain defects. The existence and possible cytotoxic effect of intracellular lysine accumulation in proximal tubular cells deserve further attention. Acknowledgement The authors thank Prof. Dr J.M.F. Trijbels, University Medical Center St Radboud, Nijmegen, The Netherlands for his studies on the mitochondrial respiratory chain in kidney tissue. References Carpenter TO, Levy HL, Holtrop ME, Shih VE, Anast CS (1985) Lysinuric protein intolerance presenting as childhood osteoporosis. N Engl J Med 312: Chan YL, Kurtzman NA (1982) Effects of lysine on bicarbonate and fluid absorption in the rat proximal tubule. Am J Physiol 242: F DiRocco M, Garibotto G, Rossi GA, et al (1993) Role of haematological, pulmonary and renal complications in the long-term prognosis of patients with lysinuric protein intolerance. Eur J Pediatr 152: Korman SH, Raas-Rothschild A, Elpeleg O, Gutman A (2002) Hypocarnitinemia in lysinuric protein intolerance. Mol Genet Metab 76: Lo JC, Chertow GM, Rennke H, Seifter J (1996) Fanconi_s syndrome and tubulointerstitial nephritis in association with L-lysine ingestion. Am J Kidney Dis 28: Mykkänen J, Torrents D, Pineda M, et al (2000) Functional analysis of novel mutations in y + LAT-1 amino acid transporter gene causing lysinuric protein intolerance (LPI). Hum Mol Genet 9: Nagata M, Kawamura G, Kono S, Koda N, Yamaguchi S, Aoki K (1987) Immunological abnormalities in a patient with lysinuric protein intolerance. Eur J Pediatr 146: Parenti G, Sebastio G, Strisciuglio P, et al (1995) Lysinuric protein intolerance characterized by bone marrow abnormalities and severe clinical course. J Pediatr 126: Parto K, Kallajoki M, Aho H, Simell O (1994) Pulmonary alveolar proteinosis and glomerulonephritis in lysinuric protein intolerance. Hum Pathol 25: Perheentupa J, Visakorpi J (1965) Protein intolerance with deficient transport of basic amino acids: another inborn error of metabolism. Lancet 2: Racusen LC, Finn WF, Whelton A, Solez K (1985) Mechanisms of lysine-induced acute renal failure in rats. Kidney Int 27: Rajantie J, Simell O, Perheentupa J (1980) Basolateralmembrane transport defect for lysine in lysinuric protein intolerance. Lancet 1: Scharer K, Manz F (1985) Renal handling of citrate in children with various kidney disorders. Int J Pediatr Nephrol 6: Simell O, Perheentupa J (1974) Renal handling of diamino acids in lysinuric protein intolerance. J Clin Invest 54: Slingerland RJ, Ruiter JPN, Blau N, et al (2004) Hyperlysinaemia type I: effects on urea-cycle and methylation reactions. J Inherit Metab Dis 27: 55. Torrents D, Mykkanen J, Pineda M, et al (1999) Identification of SLC7A7, encoding y + LAT-1, as the lysinuric protein intolerance gene. Nat Genet 21: Walton RJ, Bijvoet OL (1975) Nomogram for derivation of renal threshold phosphate concentration. Lancet 16: