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NDT Advance Access originally published online on September 19, 2007
Nephrology Dialysis Transplantation 2008 23(4):1211-1215; doi:10.1093/ndt/gfm583
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Familial pure proximal renal tubular acidosis—a clinical and genetic study

Ze’ev Katzir1, Dganit Dinour2, Haike Reznik-Wolf2, Andrea Nissenkorn3 and Eliezer Holtzman2

1 Nephrology and Hypertension Institute, Pediatric Nephrology Services, E. Wolfson Medical Center, Holon 2 Nephrology and Hypertension Institute, Chaim Sheba Medical Center, Tel Hashomer 3 Department of Pediatrics, E. Wolfson Medical Center, Holon, Israel

Ze’ev Katzir, Pediatric Nephrology Services, E. Wolfson Medical Center, Holon, Israel. E-mail: katzir{at}wolfson.health.gov.il



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. Inherited proximal renal tubular acidosis (pRTA) is commonly associated with more generalized proximal tubular dysfunctions and occasionally with other organ system defects. Inherited combined pRTA and distal RTA with osteopetrosis and pure pRTA associated with ocular abnormalities, a rare disease which has been recently described. Only one family with pure isolated pRTA has been reported so far and the genetic cause for this disease is unknown.

Objectives. We report a unique family with isolated pRTA. The aim of the project was to define the phenotype and to try to find the gene defect causing the disease.

Methods. Clinical and metabolic evaluation of all family members was performed and a family pedigree was constructed. DNA was extracted from blood samples of affected and unaffected family members. We amplified by PCR and sequenced the coding areas and splice-sites of the genes that contribute to HCO–3 reclamation in the proximal tubule. The genes studied were as follows: CA II, CA IV, CA XIV, NCB1, Na+/H+ exchanger (NHE)-3, NHE-8, the regulatory proteins of NHE3, NHRF1 and NHRF2 and the Cl–/HCO–3 exchanger, SLC26A6.

Results. The father and all four children had RTA with blood HCO–3 levels of 11–14 meq/l and urine pH of 5.3–5.4. Increased HCO–3 fractional excretion after bicarbonate loading to 40–60% confirmed the diagnosis pRTA. No other tubular dysfunction was found, and no organ system dysfunction was detected, besides short stature. No mutation was found in all candidate genes studied.

Conclusions. We presented a second family in the literature with familial isolated pure pRTA. The mode of inheritance is compatible with an autosomal dominant disease. Because of the small size of the family, wide genome search was not applicable and the gene candidate approach was chosen. Nine important candidate genes were extensively studied but the molecular basis of the disease was not yet found and genotyping nine important gene candidates were negative.



   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Renal tubular acidosis (RTA) is a clinical syndrome characterized by hyperchloraemic acidosis secondary to abnormalities of renal acidification [1]. Present views of the subject ascribe this metabolic defect to four distinct disorders of renal acidification: (i) distal RTA (dRTA or type I RTA), which is caused by a defect of H+ secretion in the distal tubule, and is characterized by the inability to maximally acidify the urine below pH 5.5 during systemic acidaemia [2], (ii) proximal RTA (pRTA or type II RTA), the result of the impairment of sodium bicarbonate reabsorption in the proximal tubules [3], (iii) combined proximal and distal RTA (type III RTA), in which a reduction in proximal tubular reclamation of filtered bicarbonate is combined with a disturbance in distal tubular mechanism of maximally acidifying the urine to compensate severe acidaemia [4] and (iv) hyperkalaemic RTA (type 4 RTA), a subnormal net acid excretion which is the result of very low rates of NH4 excretion. This type may occur as a result of aldosterone deficiency or tubular insensitivity to aldosterone in adults and children, with diabetes mellitus or obstructive uropathy [5].

Inherited pRTA is commonly associated with more generalized proximal tubular dysfunctions and occasionally with other organ system defects. Combined pRTA and dRTA with osteopetrosis and cerebral calcification is caused by mutations in the carbonic anhydrase (CA) II gene [6]. Pure pRTA associated with ocular abnormalities (cataracts, glaucoma and band keratopathy) is a rare disease, in which mutations in the sodium bicarbonate cotransporter (NBC1) gene have been recently described, including by our group [7,8].

In 1977, Brenes et al. [9] described a family in which nine affected members from several generations presented a pure form of pRTA. The only physical finding in all affected members was low stature. This is the only family with pure isolated pRTA reported so far, and the genetic cause for this disease is not known.

We report a unique family with isolated pRTA, having undergone a comprehensive clinical and metabolic evaluation together with a molecular study, in order to find out the gene defect causing the disease and to define its phenotype.



   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Clinical and metabolic evaluation
A detailed medical and family history was obtained and a pedigree of the extended family was constructed (Figure 1). The proband, his parents and all three sibs were physically examined. Growth curves were drawn and bone age was estimated based on wrist and hand bone X-ray analysis according to Greulich and Pyle [10]. Laboratory investigation included blood gases, serum sodium, potassium, calcium, phosphor, urea and creatinine, amylase and thyroid function tests. Urine examination included pH, sodium, potassium, bicarbonate, calcium, phosphor, glucose and amino acids. An oral sodium bicarbonate loading test (3–8 mEq NaHCO3/Kg body weight/dose, according to individual response) was performed. Fractional excretion of bicarbonate was determined by collecting the urine sample under mineral oil and calculated by the formula: 100 x UHCO3 x PCr/PHCO3 x UCr [9].


Figure 1
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  		Fig. 1 Pedigree of the studied family.

 
Molecular analysis
To find the gene defect causing pRTA in our family, we searched for mutations in genes known or suspected to contribute to HCO–3 reclamation in the proximal tubule. We studied the genes for: carbonic anhydrase II (CA II), carbonic anhydrase IV (CA IV) and carbonic anhydrase XIV (CA XIV), Na+/HCO–3 co-transporter (kNBC1), Na+/H+ exchanger isoforms 2, 3 and 8 (NHE2, NHE3, NHE8) the regulatory proteins of NHE3, NHRF1 and NHRF2, and the Cl–/HCO–3 exchanger of the solute carrier 26 (SLC26) gene family, SLC26A6 [11–14,18].

Genomic DNA was extracted from blood samples of all available family members. We amplified by PCR the coding areas and splice-sites of the genes of interest, using intronic primers. Primers for SLC26A6 were as in Waldebber et al. [14]. Other primers and PCR conditions are available upon request. All PCR products were sequenced directly (ABI Prism 3100, Applied Biosystem).

Haplotype analysis
To rule out mutations in the promoter or in other regulatory sequences adjacent to the candidate genes, we performed haplotype analysis of the regions of each gene. Genotyping was performed at the Center for Genomic Technologies at the Hebrew University of Jerusalem, Israel. For each gene, we chose 3–4 microsatellite markers from the Genethon human linkage map (Applied Biosystems). PCR amplification of individual markers (fluorescence-dye-labelled forward primer and unlabelled reverse primer) was performed in PTC 225 DNA Engine (MJ) using 25–30 ng of genomic DNA, 6 pmoles of each primer, 1.5 mM MgCl2, 0.14 mM dNTPs, 1xPCR Gold buffer (15 mM Tris–HCl, pH 8.0, 50 mM KCl) and 0.4 unit of AmpliTaq Gold DNA polymerase (both from ABI) in a total volume of 10 µl.

PCR conditions were as follows; An initial 12 min denaturation at 95°C, 10 cycles of 15 sec at 94°C, 15 sec at 55°C and 30 sec at 72°C, 25 cycles of 15 sec at 89°C, 15 sec at 55°C and 30 sec at 72°C, 10 min at 72°C and hold for ever at 10°C.

After amplification, labelled PCR products were pooled, according to the panels’ lists, and 1–2 µl were sampled into in 9 µl of loading buffer (formamide with GENESCAN 400HD [ROX] size standard Applied Biosystems).

PCR product electrophoresis and detection were performed using the 3700 Automated DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sizing and genotyping were performed using GENESCAN and GENOTYPER softwares (Applied Biosystems).

Haplotypes were constructed for each gene and compared between family members. A gene was considered as excluded if the father and all four children did not share the same haplotype.



   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The proband is the youngest child of non-consanguineous parents of Jewish-Syrian origin. He was born after an uneventful pregnancy. Birth weight was 2.5 kg and height 45 cm (4th percentile). Perinatal course had been uneventful. A glandular hypospadias of 2 cm deviation was noticed, with no additional malformations. Since the first week of life, he suffered from poor appetite and episodic vomiting. At the age of 2 weeks, persistent metabolic acidosis, resistant to bicarbonate ≤ 15 meq/kg/day was found. Biochemical evaluation revealed: blood (arterial) pH 7.13, HCO–3 13.9 meq/l, PCO2 23 mmHg, PO2 87 mmHg, BE –5.3, Cl 115 meq/l, Na 137 meq/l, K 3.4 meq/l, Ca 9.4 mg/dl, P 5.8 mg/dl, urea 18 mg/dl, creatinine 0.6 mg/dl. Urine pH 5.4, Na 108 meq/l, K 22 meq/l, Cl 176 meq/l, HCO–3 43 meq/l, urine ‘anion-gap’ = –89. Laboratory results following an oral sodium bicarbonate load were as follows: Blood (arterial) pH 7.33, HCO–3 18.0 meq/l, K 3.9 meq/l, creatinine 0.5 mg/dl; Urine pH 6.8, HCO–3 97, PCO2 39 mmHg, PO2 85 mmHg creatinine 12 mg/dl. Fractional excretion of bicarbonate = 22%

The diagnosis of pRTA was made, and maintenance bicarbonate treatment was initiated. A daily dose of 15–20 meq was required to achieve serum bicarbonate level of 20 meq/l.

The father and all three sibs (two girls and a boy) had short stature with delayed bone age, normal thyroid functions (Table 1) and metabolic acidosis (Table 2). Family history disclosed that four additional relatives (all females, from the father's side) had short stature (Figure 1). Unfortunately, they were not available for investigation. Metabolic evaluation of the father and all four children showed normal serum electrolytes and normal renal and pancreatic functions (Table 2). Low baseline urine pH and negative urine anion gap excluded dRTA (Table 3). Typical increase in fractional excretion of bicarbonate, in response to sodium bicarbonate load, confirmed the diagnosis of pRTA in all affected family members (Table 4). None of them had evidence of Fanconi syndrome or hypercalciuria (Table 5). Ophthalmologist examination, including slit-lamp examination and ocular pressure measurement, did not reveal any pathology.


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  		Table 1 Baseline biochemical data of the members of the family with PRTA. There are no abnormalities in renal functions, calcium phosphorus or magnesium metabolism. Normal serum amylase rules out pancreatic involvement

 

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  		Table 2 Measurements of bicarbonate fractional excretion of the affected family following oral bicarbonate load, showing the typical pattern of a proximal tubular defect of bicarbonate reabsorption

 

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  		Table 3 The results of urinary anion-gap calculations [(Na+ + K+) – (Cl– + HCO–3)] of the affected family members. Negative ‘gaps’ indicate intact distal tubular cells acidifying capacity

 

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  		Table 4 Weight, height and bone age of the family. The younger family members had less prominent delay. Thyroid function tests are normal in all family members

 

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  		Table 5 Measurements of urine excretion of glucose, amino acids and calcium, tubular reabsorption of phosphorus (TRP). The results are incompatible with Fanconi syndrome, and there is no hypercalciuria

 
Table 6 presents the response to bicarbonate treatment. The younger family members had higher alkaline requirements than the older patients. Figure 2A and B outline the growth curves of the four affected children since the initiation of bicarbonate therapy in the older children. Height gain was most accelerated in the youngest brother, in whom treatment had been maintained since the first month of life.


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  		Table 6 Daily bicarbonate requirement, in order to achieve plasma bicarbonate concentration ≥18.5 meq/l, of the father and each of the children

 

Figure 2
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  		Fig. 2 Growth curves of the two girls (A) and two boys (B) with pRTA, along 4 years from the initiation of bicarbonate therapy to all of them (the three older children started the therapy 2 years after their youngest brother). Blue line, bigger sister; black line, younger sister; green line, bigger brother; red line, younger brother.

 
No mutation was found in the coding regions and intron–exon boundaries of the genes for CA II, CA IV, CA XIV, kNCB1, NHE3, NHE8, NHRF1, NHRF2 and SLC26A6 amplified from genomic DNA of family members with pRTA.

Haplotype analysis excluded linkage to the genes for: CA II, CA XIV, kNCB1, NHE3, NHE8, NHRF2 and SLC26A6. Linkage to NHE2 was also excluded. The genes for CA IV and NHRFR1 could not be excluded, since all the affected family members shared the same haplotype in the region of these genes (data not shown).



   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we present a family that resembles very much the description of Dr Luis G. Brenes et al. [9], by having a characteristic familial appearance of pure pRTA with short stature as the only peculiar clinical finding, inherited as an autosomal dominant trait.

A variety of mechanisms had been suggested for the impaired ability of the proximal tubule to reabsorb and restore bicarbonate, and they all stem from disturbances in activity of one or more of the ion-exchangers, co-transporters or CA. The proximal tubule reclaims 80–90% of the filtered bicarbonate, using its polarized epithelial cells (Figure 3), which have the ability to secrete H+ across their apical membrane into the tubular lumen via the NHE3 [15], and according to the recent report by Goyal et al. [12], also via the NHE8 Na+/H+ exchanger isoform. These exchanger isoforms are regulated by multiple agents through a variety of acute as well as chronic mechanisms. Currently, two homologous proteins associating with NHH3 have been described: Na+/H+ exchanger regulatory factors (NHERF) 1 (also known as ezrin binding protein 50, or EBP50) and 2 (also known as E3KARP/TKA1). Both of them have a crucial function in a signalling complex, mediating the regulation of Na+/H+ exchange [16,17]. This exchange is driven by a sodium concentration gradient that is maintained by the basolateral Na+–K+ ATPase activity, which shows a compensatory over activation in case of impaired NHE3 function. This is probably the causing mechanism of low serum potassium in pRTA patients.


Figure 3
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  		Fig. 3 Major sites of proximal tubular cell acid–base handling, according to which we searched for candidate genes mutations.

 
Filtered HCO–3 combines with H+ to form carbonic acid (H2CO3), which quickly dissociates to form CO2 and H2O [1]. This latter step is catalysed by two luminal (CA), which have been identified to be expressed on the brush border membrane of the renal proximal tubule cells: CA IV and CA XIV [18]. CO2 in the glomerular ultrafiltrate diffuses into the proximal tubular cells, where hydroxylation occurs to form HCO–3 in the presence of soluble cytosol CA II [11]. Three intracellular HCO–3 and one Na+ are co-transported into blood by the kidney type Na+/HCO–3 co-transporter (kNBC1) presents in proximal tubular basolateral membranes.

An additional contribution to the acidifying capacity of the proximal tubule cells might be the activity of Cl–/HCO3 exchanger (encoded by SLC26A6 gene), which had been identified to be expressed on the brush border membrane [18,20].

We considered all the aforementioned enzymes and transporters as possible candidates for causing pRTA in our family. However, no mutation was found in any of these candidate genes. Although we did not sequence all the introns and promoter regions, we could exclude these regions for most of the genes by haplotypes analysis. Therefore, we assume that another protein, either a new transporter or, more likely, an enzyme or a regulatory factor, is responsible for the disease. We chose the candidate gene approach because of the small size of the family, making a wide genome search not applicable. If other families exhibiting autosomal-dominant isolated pure pRTA are found, the positional cloning of an unpredicted causative gene might be feasible. A renal biopsy with gene expression study could have been of benefit, but we did not receive an agreement for the procedure from the parents.

Furthermore, this report emphasizes the importance of family history investigation and comprehensive metabolic evaluation for early diagnosis. Of note is the positive influence of prompt diagnosis and treatment on the growth and development of the two younger boys.

A follow-up on two brothers from the Costa Rican family of Brenes et al., 23 years after the first study, revealed persistent metabolic acidosis and, as a result, reduced bone stores of HCO–3/CO2–3 which led to on going reduction in growth, including that of bone. The authors of this follow-up study mentioned that the poor outcome stems from difficulties in maintaining very large and multiple daily doses of alkali require sustaining normal serum bicarbonate level in the face of marked impairment of renal tubular HCO–3 reabsorption [9,21].

In summary, a second family in the literature with familial isolated pure pRTA is described. The mode of inheritance is compatible with an autosomal dominant disease. Prompt diagnosis and treatment was beneficial for the younger children in this family, having an efficacious influence on their growth and development. Despite extensive studies using the gene candidate approach, the molecular basis of the disease could not be determined.

Conflict of interest statement. None declared.

(See related article by P. Gross et al. Proximal RTA: Are all the charts completed yet? Nephrol Dial Transplant 2008; 23: 1101–1102.)



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 21. 5.06
Accepted in revised form: 31. 7.07


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