- Email: [email protected]
When proton pumps go sour: Urinary acidification and kidney stones
co m m e nt a r y
http://www.kidney-international.org © 2008 International Society of Nephrology
see original article on page 1151
When proton pumps go sour: Urinary acidification and kidney stones CA Wagner1 H+-ATPases mediate urinary acidification along the collecting duct, and mutations in their B1 and a4 subunits result in distal renal tubular acidosis. The pathomechanisms by which these mutations affect pump activity are only poorly understood. Common polymorphisms may impair pump activity and may link the pump to a higher risk for alkaline urine and the development of kidney stones. Kidney International (2008) 73, 1103–1105. doi:10.1038/ki.2008.137
Urinary acidification serves the major purpose of excreting acid stemming from daily metabolism — about 70 mmol per day. This process is mediated mainly by the action of electrogenic vacuolar-type H+-ATPases that convey the hydrolysis of adenosine triphosphate (ATP) to pumping protons across the plasma membrane.1,2 In the kidney, such H+ATPases are found in many epithelial cells lining the nephron and collecting duct system. In the proximal tubule, H+ATPases are involved in the first step of bicarbonate reabsorption from the urine as well as in the acidification of endocytic vesicles. In the collecting duct system (that is, the connecting tubule, cortical and outer medullary collecting duct, and initial inner medullary collecting duct), H+-ATPases are found mainly in intercalated cells, where these pumps can be localized in the luminal membrane of acid-secretory type A intercalated cells and in the basolateral and/or luminal side of type B and non-A/non-B intercalated cells, respectively (Figure 1). 1Institute of Physiology and Zurich Center for
Integrative Human Physiology, University of Zurich, Zurich, Switzerland Correspondence: CA Wagner, Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. E-mail: [email protected] Kidney International (2008) 73
H+-ATPases are multi-subunit protein complexes that consist in humans of at least 14 subunits with often multiple isoforms.2 The expression of these isoforms may be tissue or cell specific; in some cases multiple isoforms also are expressed in the same cell but with distinct subcellular localization. 3,4 It is assumed that specific isoforms are involved in targeting and regulation of different subsets of pumps, as indicated by experiments with yeast vacuolar-type H+-ATPases.1,2 The subunits of the proton pump are organized in two domains, a membrane-bound V 0 domain and a cytosolic V 1 domain. The V0 domain forms the proton translocation pore, whereas the V 1 domain binds and hydrolyzes ATP, which induces a conformational change in the V 0 domain conveyed through a connecting stalk structure. Proton pump activity can be regulated by several mechanisms: assembly and disassembly of the V1 and V0 domains, changes in the efficiency of the coupling of ATP to proton pumping, and trafficking into and retrieval from the membrane.1,2 Again, some of these regulatory mechanisms seem to be different between subunit isoforms. The biological importance of H + ATPases is underlined by the fact that genetic knockout of the ubiquitous proteolipid subunit ATP6V0C (c subunit) leads to early embryonic death
due to impaired nidation.5 In humans, at least three different genes have been found to be mutated in rare inherited diseases. Mutations of ATP6V0A3 (a3 subunit) cause infantile osteopetrosis;6,7 a renal phenotype of these patients and corresponding knockout mice has not been reported yet, despite the expression of this gene in the kidney. Mutations in ATP6V1B1 (B1 subunit) or ATP6V0A4 (a4 subunit) are responsible for syndromes of distal renal tubular acidosis (dRTA), which are often associated with progressive sensorineural deafness due to the extrarenal expression of these subunits in the inner ear.8,9 The B1 subunit, often (wrongly) referred to also as the kidney-specific subunit, is expressed mostly in the kidney (all types of intercalated cells), but also in the epididymis, lung, and inner ear. Similarly, the a4 isoform is highly expressed in the kidney (present in most epithelial cells along the nephron), the epididymis, and the inner ear. 10 Patients carrying homozygous mutations in the ATP6V1B1 and ATP6V0A4 genes often present early after birth with hypokalemic metabolic acidosis, an inappropriately alkaline urine, a high susceptibility to dehydration, hypercalciuria, and progressive hearing loss. Less severe cases may be identified later in life because of nephrocalcinosis or kidney stones. Alkaline urine pH and the occurrence of calcium kidney stones are closely linked in the general population, as is indicated by many studies in patients and animal models. 11,12 At least two mechanisms may favor the development of calcium stones in this setting: the lower solubility of calcium in alkaline urine, and an incipient or overt metabolic acidosis as part of a renal tubular acidosis syndrome. Forms of complete and incomplete renal tubular acidosis have been frequently detected in patients with a high (re)occurrence rate of calcium kidney stones. Thus, inappropriate urinary acidification along the collecting duct mediated by proton pumps could contribute to the development of calcium kidney stones, not only in patients with homozygous mutations 1103
co mmentar y
but also in a more general population. However, little is known about the functional consequences of proton pump subunit mutations detected in dRTA patients. Moreover, polymorphisms for some of these subunit isoforms have been described, but their functional relevance has not been investigated. The functional investigation of particular subunit isoforms is hampered by the fact that mammalian cells often express more than
one isoform of a given pump subunit and that different isoforms may substitute for at least some functions of the missing or defective isoform. We and others found that in a mouse model deficient in the B1 subunit (Atp6v1b1 knockout), residual proton pump activity and urinary acidification are preserved in the collecting duct.13–15 This finding was surprising at first glance, but we detected that the B2 isoform was partially substituting
Figure 1 | H+-ATPase expression along the collecting duct. Acid-secretory type A intercalated cells are localized along the collecting duct system from late distal convoluted tubule (DCT2) to the initial inner medullary collecting duct. Cytosolic carbonic anhydrase II (CA II) catalyzes the formation of bicarbonate and protons. Bicarbonate is released across the basolateral membrane into the pericapillary interstitium via chloride/bicarbonate exchanger(s), whereas protons are secreted into urine mainly by the vacuolar-type H+-ATPase (V-H+-ATPase) and to a lesser extent by H+/K+-ATPases. H+-ATPases are composed of two main sectors, the cytosolic V1 domain (depicted in orange) and the membrane-bound V0 domain (depicted in blue). ATP binds to the V1 domain, and its hydrolysis provides the energy to pump protons across the cell membrane-embedded V0 domain. Both domains are assembled from several subunits, often with many isoforms. Two isoforms of the B subunit exist, B1 (ATP6V1B1) and B2; the ‘a’ subunit exists in four isoforms (ATP6V0A1–4). Mutations in the B1 and a4 subunit isoforms, respectively, have been detected in patients with distal renal tubular acidosis due to the impaired proton secretion by proton pumps. 1104
for the loss of the B1 isoform. Interestingly, the B2 isoform cannot substitute for all functions, as is indicated by the fact that induction of metabolic acidosis in these animals results in severe dRTA and impaired urinary acidification, and no stimulation of H+-ATPase activity could be observed in intercalated cells from knockout animals. Similarly, angiotensin II stimulates H+-ATPase activity in outer medullary collecting duct intercalated cells from normal animals but not from mice lacking the B1 isoform. Thus, the B2 isoform can support some basal H+-ATPase activity in the plasma membrane, but the B1 isoform is required for the stimulation of pump activity, as it occurs in settings of acid loading. Fuster and colleagues16 (this issue) have overcome the problem of studying mutant B1 subunits in the background of cells expressing the B2 isoform by expressing wild-type and mutant human B1 isoforms in yeast cells that were engineered to lack all corresponding B isoforms. This elegant yeast complementation assay allowed them to examine the impact of B1 mutations detected in dRTA patients on assembly and function of the whole proton pump. Most mutations investigated disturbed assembly of the proton pump domains. Some mutants showed apparently normal pump assembly but did not rescue the reduced growth of complemented yeast strains at alkaline pH, indicating that these mutants impaired pump activity despite normal assembly. Coexpression of wild-type and mutant B1 isoforms did not impair proton pump assembly or function, in agreement with the autosomal recessive inheritance of this disease. Most interestingly, two alleles were investigated, the Thr30Ile and Glu161Lys alleles, that had been previously considered common polymorphisms. The Thr30Ile did not show any defects and can indeed be viewed as a polymorphism, whereas the Glu161Lys showed strongly impaired proton pump activity despite normal assembly. Some of these results are in contrast to previous investigations testing the impact of B1 and a4 isoform mutations.17,18 These studies had also found evidence for impaired pump assembly Kidney International (2008) 73
co m m e nt a r y
and function but suggested that mutant B1 protein could exert a dominantnegative effect on the assembly and trafficking of normal pumps when coexpressed. Some of the discrepancies may be due to the fact that mutant isoforms were highly overexpressed and that a rat collecting duct cell line was used that expressed both normal B1 and B2 isoforms. 18 It will not be easy to resolve such discrepancies as long as we do not have good cell models at hand that resemble human intercalated cells. Furthermore, all models rely on the (over)expression of wildtype or mutant proteins, which may alter stoichiometries between isoforms and alleles. Unfortunately, very little is known about expression of H+-ATPase subunits in the human kidney, as most studies were performed in rodent models, and no data are available on the situation in kidneys from patients with ATP6V1B1 mutations. The story resembles data obtained on the expression of wild-type and mutant AE1 proteins. Mutations in chloride/ anion exchanger AE1 (SLC4A1) cause autosomal dominant dRTA,19,20 as this transporter is expressed on the basolateral side of intercalated cells of the kidney. Expression of mutants in renal cell lines indicated that in some instances loss of polarized expression due to trafficking of otherwise functional transporters to the luminal side with possible bicarbonate wasting into urine would underlie the disease.21,22 Among these mutants is the S613F mutant, which is found on the apical membrane in overexpressing cell lines. However, investigation of a rare biopsy from a dRTA patient with this mutation did not show any detectable luminal expression of the mutant but rather showed intracellular or even absent staining. 23 Thus, data from overexpression systems should be taken with great caution. What are the alternatives? The generation of humanized mouse models that do not lack the gene of interest but express the mutant gene recapitulating human mutations may be an additional useful tool, although not suited for high-throughput screening of mutations. Tissues or cells from patients would be another Kidney International (2008) 73
attractive way; however, although the attempts to raise good renal cell lines are numerous, little progress has been made. Yeast cell lines as used by Fuster et al.16 are another alternative, as, in the case of proton pumps, much is known about the biology of yeast vacuolar-type ATPases, genetic models lacking subunits of the yeast pump, and easy complementation assays are at hand. The work of Fuster and colleagues16 goes beyond investigating the direct impact of B1 mutations on pump assembly and function. The authors suggest that alleles viewed as polymorphisms of proton pump subunits may actually be genuine mutations impairing urinary acidification. In view of the link between reduced urinary acidification and calcium stone formation, it may be quite worthwhile to take a closer look at such polymorphisms found in these patients. Even though full-blown dRTA is caused only in the presence of two mutated alleles, it may be enough to carry one dysfunctional allele to reduce urinary acidification to an extent sufficient to increase the risk of kidney stone formation. ACKNOWLEDGMENTS Work in the laboratory of the author has been supported by the Swiss National Science Foundation and the 6th EU Framework project EuReGene.
8.
9.
10.
11. 12.
13.
14.
15.
16.
17.
18.
REFERENCES 1. 2. 3.
4.
5.
6.
7.
Nishi T, Forgac M. The vacuolar (H+)-ATPases: nature’s most versatile proton pumps. Nat Rev Mol Cell Biol 2002; 3: 94–103. Wagner CA, Finberg KE, Breton S et al. Renal vacuolar H+-ATPase. Physiol Rev 2004; 84: 1263–1314. Schulz N, Dave MH, Stehberger PA et al. Differential localization of vacuolar H+-ATPases containing a1, a2, a3, or a4 (ATP6V0A1-4) subunit isoforms along the nephron. Cell Physiol Biochem 2007; 20: 109–120. Hurtado-Lorenzo A, Skinner M, El Annan J et al. V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. Nat Cell Biol 2006; 8: 124–136. Inoue H, Noumi T, Nagata M et al. Targeted disruption of the gene encoding the proteolipid subunit of mouse vacuolar H+-ATPase leads to early embryonic lethality. Biochim Biophys Acta 1999; 1413: 130–138. Kornak U, Schulz A, Friedrich W et al. Mutations in the a3 subunit of the vacuolar H+-ATPase cause infantile malignant osteopetrosis. Hum Mol Genet 2000; 9: 2059–2063. Frattini A, Orchard PJ, Sobacchi C et al. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal
19.
20.
21.
22.
23.
recessive osteopetrosis. Nat Genet 2000; 25: 343–346. Karet FE, Finberg KE, Nelson RD et al. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet 1999; 21: 84–90. Smith AN, Skaug J, Choate KA et al. Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing. Nat Genet 2000; 26: 71–75. Stehberger P, Schulz N, Finberg KE et al. Localization and regulation of the ATP6V0A4 (a4) vacuolar H+-ATPase subunit defective in an inherited form of distal renal tubular acidosis. J Am Soc Nephrol 2003; 14: 3027–3038. Buckalew VM Jr. Nephrolithiasis in renal tubular acidosis. J Urol 1989; 141: 731–737. Tessitore N, Ortalda V, Fabris A et al. Renal acidification defects in patients with recurrent calcium nephrolithiasis. Nephron 1985; 41: 325–332. Finberg KE, Wagner CA, Bailey MA et al. The B1 subunit of the H+ATPase is required for maximal urinary acidification. Proc Nat Acad Sci USA 2005; 102: 13616–13621. Paunescu TG, Russo LM, Da Silva N et al. Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice. Am J Physiol Renal Physiol 2007; 293: F1915–F1926. Rothenberger F, Velic A, Stehberger PA et al. Angiotensin II stimulates vacuolar H+-ATPase activity in renal acid-secretory intercalated cells from the outer medullary collecting duct. J Am Soc Nephrol 2007; 18: 2085–2093. Fuster DG, Zhang J, Xie X-S, Moe OW. The vacuolar-ATPase B1 subunit in distal tubular acidosis: novel mutations and mechanisms for dysfunction. Kidney Int 2008; 73: 1151–1158. Ochotny N, Van Vliet A, Chan N et al. Effects of human a3 and a4 mutations that result in osteopetrosis and distal renal tubular acidosis on yeast V-ATPase expression and activity. J Biol Chem 2006; 281: 26102–26111. Yang Q, Li G, Singh SK et al. Vacuolar H+-ATPase B1 subunit mutations that cause inherited distal renal tubular acidosis affect proton pump assembly and trafficking in inner medullary collecting duct cells. J Am Soc Nephrol 2006; 17: 1858–1866. Karet FE, Gainza FJ, Gyory AZ et al. Mutations in the chloride-bicarbonate exchanger gene AE1 cause autosomal dominant but not autosomal recessive distal renal tubular acidosis. Proc Natl Acad Sci USA 1998; 95: 6337–6342. Bruce LJ, Cope DL, Jones GK et al. Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene. J Clin Invest 1997; 100: 1693–1707. Devonald MA, Smith AN, Poon JP et al. Nonpolarized targeting of AE1 causes autosomal dominant distal renal tubular acidosis. Nat Genet 2003; 33: 125–127. Toye AM, Banting G, Tanner MJ. Regions of human kidney anion exchanger 1 (kAE1) required for basolateral targeting of kAE1 in polarised kidney cells: mis-targeting explains dominant renal tubular acidosis (dRTA). J Cell Sci 2004; 117: 1399–1410. Walsh S, Turner CM, Toye A et al. Immunohistochemical comparison of a case of inherited distal renal tubular acidosis (with a unique AE1 mutation) with an acquired case secondary to autoimmune disease. Nephrol Dial Transplant 2007; 22: 807–812.
1105
http://www.kidney-international.org © 2008 International Society of Nephrology
see original article on page 1151
When proton pumps go sour: Urinary acidification and kidney stones CA Wagner1 H+-ATPases mediate urinary acidification along the collecting duct, and mutations in their B1 and a4 subunits result in distal renal tubular acidosis. The pathomechanisms by which these mutations affect pump activity are only poorly understood. Common polymorphisms may impair pump activity and may link the pump to a higher risk for alkaline urine and the development of kidney stones. Kidney International (2008) 73, 1103–1105. doi:10.1038/ki.2008.137
Urinary acidification serves the major purpose of excreting acid stemming from daily metabolism — about 70 mmol per day. This process is mediated mainly by the action of electrogenic vacuolar-type H+-ATPases that convey the hydrolysis of adenosine triphosphate (ATP) to pumping protons across the plasma membrane.1,2 In the kidney, such H+ATPases are found in many epithelial cells lining the nephron and collecting duct system. In the proximal tubule, H+ATPases are involved in the first step of bicarbonate reabsorption from the urine as well as in the acidification of endocytic vesicles. In the collecting duct system (that is, the connecting tubule, cortical and outer medullary collecting duct, and initial inner medullary collecting duct), H+-ATPases are found mainly in intercalated cells, where these pumps can be localized in the luminal membrane of acid-secretory type A intercalated cells and in the basolateral and/or luminal side of type B and non-A/non-B intercalated cells, respectively (Figure 1). 1Institute of Physiology and Zurich Center for
Integrative Human Physiology, University of Zurich, Zurich, Switzerland Correspondence: CA Wagner, Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. E-mail: [email protected] Kidney International (2008) 73
H+-ATPases are multi-subunit protein complexes that consist in humans of at least 14 subunits with often multiple isoforms.2 The expression of these isoforms may be tissue or cell specific; in some cases multiple isoforms also are expressed in the same cell but with distinct subcellular localization. 3,4 It is assumed that specific isoforms are involved in targeting and regulation of different subsets of pumps, as indicated by experiments with yeast vacuolar-type H+-ATPases.1,2 The subunits of the proton pump are organized in two domains, a membrane-bound V 0 domain and a cytosolic V 1 domain. The V0 domain forms the proton translocation pore, whereas the V 1 domain binds and hydrolyzes ATP, which induces a conformational change in the V 0 domain conveyed through a connecting stalk structure. Proton pump activity can be regulated by several mechanisms: assembly and disassembly of the V1 and V0 domains, changes in the efficiency of the coupling of ATP to proton pumping, and trafficking into and retrieval from the membrane.1,2 Again, some of these regulatory mechanisms seem to be different between subunit isoforms. The biological importance of H + ATPases is underlined by the fact that genetic knockout of the ubiquitous proteolipid subunit ATP6V0C (c subunit) leads to early embryonic death
due to impaired nidation.5 In humans, at least three different genes have been found to be mutated in rare inherited diseases. Mutations of ATP6V0A3 (a3 subunit) cause infantile osteopetrosis;6,7 a renal phenotype of these patients and corresponding knockout mice has not been reported yet, despite the expression of this gene in the kidney. Mutations in ATP6V1B1 (B1 subunit) or ATP6V0A4 (a4 subunit) are responsible for syndromes of distal renal tubular acidosis (dRTA), which are often associated with progressive sensorineural deafness due to the extrarenal expression of these subunits in the inner ear.8,9 The B1 subunit, often (wrongly) referred to also as the kidney-specific subunit, is expressed mostly in the kidney (all types of intercalated cells), but also in the epididymis, lung, and inner ear. Similarly, the a4 isoform is highly expressed in the kidney (present in most epithelial cells along the nephron), the epididymis, and the inner ear. 10 Patients carrying homozygous mutations in the ATP6V1B1 and ATP6V0A4 genes often present early after birth with hypokalemic metabolic acidosis, an inappropriately alkaline urine, a high susceptibility to dehydration, hypercalciuria, and progressive hearing loss. Less severe cases may be identified later in life because of nephrocalcinosis or kidney stones. Alkaline urine pH and the occurrence of calcium kidney stones are closely linked in the general population, as is indicated by many studies in patients and animal models. 11,12 At least two mechanisms may favor the development of calcium stones in this setting: the lower solubility of calcium in alkaline urine, and an incipient or overt metabolic acidosis as part of a renal tubular acidosis syndrome. Forms of complete and incomplete renal tubular acidosis have been frequently detected in patients with a high (re)occurrence rate of calcium kidney stones. Thus, inappropriate urinary acidification along the collecting duct mediated by proton pumps could contribute to the development of calcium kidney stones, not only in patients with homozygous mutations 1103
co mmentar y
but also in a more general population. However, little is known about the functional consequences of proton pump subunit mutations detected in dRTA patients. Moreover, polymorphisms for some of these subunit isoforms have been described, but their functional relevance has not been investigated. The functional investigation of particular subunit isoforms is hampered by the fact that mammalian cells often express more than
one isoform of a given pump subunit and that different isoforms may substitute for at least some functions of the missing or defective isoform. We and others found that in a mouse model deficient in the B1 subunit (Atp6v1b1 knockout), residual proton pump activity and urinary acidification are preserved in the collecting duct.13–15 This finding was surprising at first glance, but we detected that the B2 isoform was partially substituting
Figure 1 | H+-ATPase expression along the collecting duct. Acid-secretory type A intercalated cells are localized along the collecting duct system from late distal convoluted tubule (DCT2) to the initial inner medullary collecting duct. Cytosolic carbonic anhydrase II (CA II) catalyzes the formation of bicarbonate and protons. Bicarbonate is released across the basolateral membrane into the pericapillary interstitium via chloride/bicarbonate exchanger(s), whereas protons are secreted into urine mainly by the vacuolar-type H+-ATPase (V-H+-ATPase) and to a lesser extent by H+/K+-ATPases. H+-ATPases are composed of two main sectors, the cytosolic V1 domain (depicted in orange) and the membrane-bound V0 domain (depicted in blue). ATP binds to the V1 domain, and its hydrolysis provides the energy to pump protons across the cell membrane-embedded V0 domain. Both domains are assembled from several subunits, often with many isoforms. Two isoforms of the B subunit exist, B1 (ATP6V1B1) and B2; the ‘a’ subunit exists in four isoforms (ATP6V0A1–4). Mutations in the B1 and a4 subunit isoforms, respectively, have been detected in patients with distal renal tubular acidosis due to the impaired proton secretion by proton pumps. 1104
for the loss of the B1 isoform. Interestingly, the B2 isoform cannot substitute for all functions, as is indicated by the fact that induction of metabolic acidosis in these animals results in severe dRTA and impaired urinary acidification, and no stimulation of H+-ATPase activity could be observed in intercalated cells from knockout animals. Similarly, angiotensin II stimulates H+-ATPase activity in outer medullary collecting duct intercalated cells from normal animals but not from mice lacking the B1 isoform. Thus, the B2 isoform can support some basal H+-ATPase activity in the plasma membrane, but the B1 isoform is required for the stimulation of pump activity, as it occurs in settings of acid loading. Fuster and colleagues16 (this issue) have overcome the problem of studying mutant B1 subunits in the background of cells expressing the B2 isoform by expressing wild-type and mutant human B1 isoforms in yeast cells that were engineered to lack all corresponding B isoforms. This elegant yeast complementation assay allowed them to examine the impact of B1 mutations detected in dRTA patients on assembly and function of the whole proton pump. Most mutations investigated disturbed assembly of the proton pump domains. Some mutants showed apparently normal pump assembly but did not rescue the reduced growth of complemented yeast strains at alkaline pH, indicating that these mutants impaired pump activity despite normal assembly. Coexpression of wild-type and mutant B1 isoforms did not impair proton pump assembly or function, in agreement with the autosomal recessive inheritance of this disease. Most interestingly, two alleles were investigated, the Thr30Ile and Glu161Lys alleles, that had been previously considered common polymorphisms. The Thr30Ile did not show any defects and can indeed be viewed as a polymorphism, whereas the Glu161Lys showed strongly impaired proton pump activity despite normal assembly. Some of these results are in contrast to previous investigations testing the impact of B1 and a4 isoform mutations.17,18 These studies had also found evidence for impaired pump assembly Kidney International (2008) 73
co m m e nt a r y
and function but suggested that mutant B1 protein could exert a dominantnegative effect on the assembly and trafficking of normal pumps when coexpressed. Some of the discrepancies may be due to the fact that mutant isoforms were highly overexpressed and that a rat collecting duct cell line was used that expressed both normal B1 and B2 isoforms. 18 It will not be easy to resolve such discrepancies as long as we do not have good cell models at hand that resemble human intercalated cells. Furthermore, all models rely on the (over)expression of wildtype or mutant proteins, which may alter stoichiometries between isoforms and alleles. Unfortunately, very little is known about expression of H+-ATPase subunits in the human kidney, as most studies were performed in rodent models, and no data are available on the situation in kidneys from patients with ATP6V1B1 mutations. The story resembles data obtained on the expression of wild-type and mutant AE1 proteins. Mutations in chloride/ anion exchanger AE1 (SLC4A1) cause autosomal dominant dRTA,19,20 as this transporter is expressed on the basolateral side of intercalated cells of the kidney. Expression of mutants in renal cell lines indicated that in some instances loss of polarized expression due to trafficking of otherwise functional transporters to the luminal side with possible bicarbonate wasting into urine would underlie the disease.21,22 Among these mutants is the S613F mutant, which is found on the apical membrane in overexpressing cell lines. However, investigation of a rare biopsy from a dRTA patient with this mutation did not show any detectable luminal expression of the mutant but rather showed intracellular or even absent staining. 23 Thus, data from overexpression systems should be taken with great caution. What are the alternatives? The generation of humanized mouse models that do not lack the gene of interest but express the mutant gene recapitulating human mutations may be an additional useful tool, although not suited for high-throughput screening of mutations. Tissues or cells from patients would be another Kidney International (2008) 73
attractive way; however, although the attempts to raise good renal cell lines are numerous, little progress has been made. Yeast cell lines as used by Fuster et al.16 are another alternative, as, in the case of proton pumps, much is known about the biology of yeast vacuolar-type ATPases, genetic models lacking subunits of the yeast pump, and easy complementation assays are at hand. The work of Fuster and colleagues16 goes beyond investigating the direct impact of B1 mutations on pump assembly and function. The authors suggest that alleles viewed as polymorphisms of proton pump subunits may actually be genuine mutations impairing urinary acidification. In view of the link between reduced urinary acidification and calcium stone formation, it may be quite worthwhile to take a closer look at such polymorphisms found in these patients. Even though full-blown dRTA is caused only in the presence of two mutated alleles, it may be enough to carry one dysfunctional allele to reduce urinary acidification to an extent sufficient to increase the risk of kidney stone formation. ACKNOWLEDGMENTS Work in the laboratory of the author has been supported by the Swiss National Science Foundation and the 6th EU Framework project EuReGene.
8.
9.
10.
11. 12.
13.
14.
15.
16.
17.
18.
REFERENCES 1. 2. 3.
4.
5.
6.
7.
Nishi T, Forgac M. The vacuolar (H+)-ATPases: nature’s most versatile proton pumps. Nat Rev Mol Cell Biol 2002; 3: 94–103. Wagner CA, Finberg KE, Breton S et al. Renal vacuolar H+-ATPase. Physiol Rev 2004; 84: 1263–1314. Schulz N, Dave MH, Stehberger PA et al. Differential localization of vacuolar H+-ATPases containing a1, a2, a3, or a4 (ATP6V0A1-4) subunit isoforms along the nephron. Cell Physiol Biochem 2007; 20: 109–120. Hurtado-Lorenzo A, Skinner M, El Annan J et al. V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. Nat Cell Biol 2006; 8: 124–136. Inoue H, Noumi T, Nagata M et al. Targeted disruption of the gene encoding the proteolipid subunit of mouse vacuolar H+-ATPase leads to early embryonic lethality. Biochim Biophys Acta 1999; 1413: 130–138. Kornak U, Schulz A, Friedrich W et al. Mutations in the a3 subunit of the vacuolar H+-ATPase cause infantile malignant osteopetrosis. Hum Mol Genet 2000; 9: 2059–2063. Frattini A, Orchard PJ, Sobacchi C et al. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal
19.
20.
21.
22.
23.
recessive osteopetrosis. Nat Genet 2000; 25: 343–346. Karet FE, Finberg KE, Nelson RD et al. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet 1999; 21: 84–90. Smith AN, Skaug J, Choate KA et al. Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing. Nat Genet 2000; 26: 71–75. Stehberger P, Schulz N, Finberg KE et al. Localization and regulation of the ATP6V0A4 (a4) vacuolar H+-ATPase subunit defective in an inherited form of distal renal tubular acidosis. J Am Soc Nephrol 2003; 14: 3027–3038. Buckalew VM Jr. Nephrolithiasis in renal tubular acidosis. J Urol 1989; 141: 731–737. Tessitore N, Ortalda V, Fabris A et al. Renal acidification defects in patients with recurrent calcium nephrolithiasis. Nephron 1985; 41: 325–332. Finberg KE, Wagner CA, Bailey MA et al. The B1 subunit of the H+ATPase is required for maximal urinary acidification. Proc Nat Acad Sci USA 2005; 102: 13616–13621. Paunescu TG, Russo LM, Da Silva N et al. Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice. Am J Physiol Renal Physiol 2007; 293: F1915–F1926. Rothenberger F, Velic A, Stehberger PA et al. Angiotensin II stimulates vacuolar H+-ATPase activity in renal acid-secretory intercalated cells from the outer medullary collecting duct. J Am Soc Nephrol 2007; 18: 2085–2093. Fuster DG, Zhang J, Xie X-S, Moe OW. The vacuolar-ATPase B1 subunit in distal tubular acidosis: novel mutations and mechanisms for dysfunction. Kidney Int 2008; 73: 1151–1158. Ochotny N, Van Vliet A, Chan N et al. Effects of human a3 and a4 mutations that result in osteopetrosis and distal renal tubular acidosis on yeast V-ATPase expression and activity. J Biol Chem 2006; 281: 26102–26111. Yang Q, Li G, Singh SK et al. Vacuolar H+-ATPase B1 subunit mutations that cause inherited distal renal tubular acidosis affect proton pump assembly and trafficking in inner medullary collecting duct cells. J Am Soc Nephrol 2006; 17: 1858–1866. Karet FE, Gainza FJ, Gyory AZ et al. Mutations in the chloride-bicarbonate exchanger gene AE1 cause autosomal dominant but not autosomal recessive distal renal tubular acidosis. Proc Natl Acad Sci USA 1998; 95: 6337–6342. Bruce LJ, Cope DL, Jones GK et al. Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene. J Clin Invest 1997; 100: 1693–1707. Devonald MA, Smith AN, Poon JP et al. Nonpolarized targeting of AE1 causes autosomal dominant distal renal tubular acidosis. Nat Genet 2003; 33: 125–127. Toye AM, Banting G, Tanner MJ. Regions of human kidney anion exchanger 1 (kAE1) required for basolateral targeting of kAE1 in polarised kidney cells: mis-targeting explains dominant renal tubular acidosis (dRTA). J Cell Sci 2004; 117: 1399–1410. Walsh S, Turner CM, Toye A et al. Immunohistochemical comparison of a case of inherited distal renal tubular acidosis (with a unique AE1 mutation) with an acquired case secondary to autoimmune disease. Nephrol Dial Transplant 2007; 22: 807–812.
1105