Filtered citrate is reabsorbed along the renal proximal tubule by a transcellular process mediated by the apical membrane Na+-dicarboxylate cotransporter NaDC-1.1 After it is reabsorbed, citrate is metabolized in either the cytoplasm or the mitochondria to electroneutral end products, a process that consumes three H+ ions and is thus equivalent to the production of three HCO3− ions.2,3 The rate of luminal citrate uptake and its intracellular metabolism are determinants of citrate excretion.
Citrate has multiple physiologic roles.3 It protects from kidney stones by binding Ca2+ in a soluble form and inhibiting nucleation, growth, and aggregation of calcium oxalate crystals.4–6 In addition, citrate is a principal urinary base.3 Disturbances in acid-base status modulate citrate excretion, with an alkali load increasing and an acid load reducing citrate excretion.7,8
We previously shown that conditions that cause a proximal tubule intracellular acidosis are associated with hypocitraturia.9–11 The reduced citrate excretion is accompanied by increased NaDC-17,12,13 activity and increased renal citrate metabolism.10,14 The mechanisms mediating acid stimulation of NaDC-1 activity share several analogies with the acid stimulation of the apical Na+/H+ exchanger NHE3.15 Specifically, (1) conditions associated with intracellular acidosis increase both NaDC-19 and NHE3 activities,16 (2) both of the NaDC-1 and NHE3 adaptive responses are mediated by endothelin-1 (ET-1) through endothelin B (ETB) receptor,13,17 and (3) both acid and ET-1 regulation of NaDC-1 and NHE3 activities are mediated by protein trafficking to the apical membrane.13,18
These studies sought to elucidate the signaling pathway(s) mediating acid stimulation of NaDC-1 activity. OKP cells were used as a proximal tubule model to study transport in vitro, and Pyk2 wild-type and knockout mice were used for the in vivo studies. The studies show that acid stimulation of NaDC-1 activity requires the nonreceptor tyrosine kinase Pyk2 and its downstream effector c-Src and a parallel signaling pathway involving the Rapidly Accelerated Fibrosarcoma-kinase 1 (Raf1)/extracellular signal–regulated kinase 1,2 (ERK1,2)/p90 Ribosomal S6 Kinase (p90RSK) pathway and that both cascades are not downstream of ET-1/ETB signaling.
Methods
Materials and Supplies
All chemicals were obtained from Sigma Chemical (St. Louis, MO) except penicillin and streptomycin (Whittaker M.A. Bioproducts, Walkersville, MD); culture media (GIBCO BRL, Grand Island, NY); ET-1 (Peptide International, Louisville, KY); 14C-citrate (Amersham, Arlington Heights, IL); Lipofectamine, SP600125, and SB203580 (Invitrogen, San Diego, CA); and PD98059 and Raf1 inhibitor 1 (Alexis Biochemicals, San Diego, CA). Monoclonal antiphospho- and total p44/42MAPK (Thr202/Tyr204), monoclonal antiphospho- and total p90RSK (ser380), antiphospho- and total c-Raf (ser 338), and antiphospho-p38 MAP kinase (Thr180/Tyr182) were from Cell Signaling (Danvers, MA).
Cell Culture and Transfection
OKP cells, a cell culture model for the kidney proximal tubule epithelium, were passaged in high-glucose DMEM as described elsewhere.13 To overexpress NaDC-1 and ETB, OKP cells were transiently transfected with a pEGFP-C3 vector (Clontech Laboratories, Palo Alto, CA) with the Opossum-derived NaDC-1 tagged with the green fluorescent protein (GFP; GFP-oNaDC-1) and pMEhETB plasmids using Lipofectamine according to the manufacturer’s instructions given the low endogenous expression of both proteins as previously shown.19,20 Although overexpressing NaDC-1 and ETB may affect the experimental outcomes, the fact that the results from the in vitro studies were consistent with the results from the in vivo studies supports the data from the cell culture studies. Cells were cotransfected with pcDNA3.1/HisB/LacZ (vector only), pcDNA3.1/HisA/Pyk2K457A (dominant negative [DN] Pyk2), pcDNA3.1/HisA/Pyk2Y402F (mutant Pyk2; lacking the binding domain to c-Src), or pcDNA3/c-SrcK295 (DN c-Src). The generation of those constructs was described previously.21,22 Transfection efficiency was determined by measuring GFP fluorescence. The experiments were carried out if the efficiency of transfection was over 70%.
The roles of ERK1,2, JNK, and p38 were tested by using inhibitors PD98059, SP600125, and SB203580, respectively. DN p90RSK was provided by the University of Rochester School of Medicine (Rochester, NY).
For experimentation, OKP cells were exposed to control (pH 7.4) versus acidic media (pH 6.8) for 6 hours or 10−8 M ET-1 for 35 minutes versus 0.1% acetic acid.
[14C]-Citrate Uptake
[14C]-citrate uptake was performed as described elsewhere.13 The cells were then lysed, protein content was measured by Bradford assay, and [14C]-citrate content was measured with the scintillation counter.
Immunoblot
OKP cells were harvested in Tris⋅Cl/SDS sample buffer. Protein was size fractionated by SDS-PAGE on minigels, electrophoretically transferred to nitrocellulose, and probed with the appropriate primary and secondary antibodies. Signals were visualized by Enhanced Chemiluminescence (Sigma Chemical).
Animal Studies
Animal studies were conducted in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Four-month-old sex-matched wild-type (Pyk2+/+) and knockout (Pyk2−/−) mice were used. An acute acid load was induced by gavage with 0.1 ml NH4Cl (0.3 M)/10 g body wt versus water. Fifteen minutes after gavage, the mice were anesthetized; a sample of arterial blood was taken for blood gas analysis (Heska i-STAT Portable Clinical Analyzer, Loveland, CO), and the kidneys were rapidly harvested. Cortex was dissected for immunoblotting. All of the studies involving live animals were approved by the Institutional Animal Care and Use Committee and performed as approved.
Metabolic Cage Studies
Animals were housed in metabolic cages permitting urine collection and measurement of water and chow consumed. Metabolic acidosis was induced by providing drinking water containing 0.3 M NH4Cl for 7 days; control mice received plain drinking water. After anesthetizing the mice with isoflurane by inhalation (Baxter, Deerfield, IL), the kidneys were harvested and kept in PBS on ice until the cortex was removed. Arterial blood gas was measured using a Heska i-STAT Portable Clinical Analyzer.
BBMV Isolation and Measurement of NaDC-1 Transport Activity
NaDC-1 activity was assayed as [14C]-citrate uptake in BBMV as described previously.13 Briefly, dissected kidney cortex from six kidneys was immersed in ice-cold buffer containing 300 mM mannitol, 5 mM ethylene glycol tetra-acetic acid, 18 mM HEPES, and 0.1 mM PMSF titrated to pH 7.5 with Tris and homogenized with a Polytron homogenizer (Brinkmann Instruments, Westburg, NY). BBMV was isolated using the Mg-ethylene glycol tetra-acetic acid aggregation method.11 BBMV fraction was loaded with intravesicular buffer (200 mM Mannitol, 50 mM KCl, and 16 mM HEPES titrated to pH 7.5 with Tris); 45 ml extracellular buffer (100 mM NaCl/100 mM Choline Cl, 50 mM KCl, and 16 mM HEPES titrated to pH 7.5 with Tris) containing 0.1 mM [14C]-citrate was added to 15 ml of BBMV (100–110 mg protein) at room temperature. After 5 seconds, the reaction was stopped with ice-cold stop solution (135 mM NaCl, 10 mM Na2 succinate, and 16 mM HEPES titrated to pH 7.5 with Tris), the sample was filtered through a 0.65-mm filter (Millipore Corp., Bedford, MA), the filter was put into scintillation fluid, and [14C]-citrate was counted using a scintillation counter (Packard Tri-Carb 2900TR, Downers Grove, IL). [14C]-citrate uptake was carried out in triplicate. Uptake is reported as picomoles of citrate per 1 mg of protein per 5 seconds.
Data Analyses
Data are expressed as means±SEM. ANOVA or t test, as appropriate, was used to establish statistical significance. Quantification of immunoblot band intensity was performed using ImageJ. Differences were considered to be significant at P values <0.05.
Results
Pyk2 Is Required for Acid Stimulation of NaDC-1 In Vitro
We previously showed that Pyk2 is activated by media acidification and that it is required for acid stimulation of NHE3 activity.21 To determine whether Pyk2 also plays a role in acid stimulation of NaDC-1 activity, an opossum kidney cell model of the proximal tubule OKP cells was used. Cells were transfected with an inactive Pyk2 that inhibits wild-type Pyk2 (DN pyk2K457A). In the vector-transfected cells, media acidification increased NaDC-1 activity by 84% (P<0.001; n=6) (Figure 1A), whereas in cells expressing DN pyk2K457A, NaDC-1 activity was inhibited (P<0.04; n=6). To test the role of Pyk2 on ET-1–dependent increases in NaDC-1 activity, the effect of ET-1 was examined in the presence and absence of a functional Pyk2. Figure 1B shows that, in the presence of either a functional Pyk2 or DN construct, ET-1 was still able to increase NaDC-1 activity. These findings indicate that Pyk2 is required for acid stimulation of NaDC-1 but that its position in the signaling cascade is not downstream of ET-1/ETB.
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Figure 1.: Acid but not endothelin-1 (ET-1) stimulation of NaDC-1 activity requires the Pyk2-c-Src signaling pathway. OKP cells were transiently cotransfected with NaDC-1 and endothelin B (ETB). In addition, (A and B) dominant negative (DN) Pyk2K457A, (C and D) c-SRCK295M, (E and F) mutant Pyk2Y402F, or the vector alone was cotransfected. Cells were exposed to control (pH 7.4) or acidic (pH 6.8) media for 6 hours (left) and 0.1% acetic acid (vehicle for ET-1) versus ET-1 for 35 minutes (right). NaDC-1 activity was measured as Na-dependent 14C-citrate uptake and expressed as picomoles of citrate per 1 mg of protein per 5 minutes. (A and B) Transfection with Pyk2K457A totally prevented acid stimulation of NaDC-1 activity (n=6) but not ET-1 stimulation of NaDC-1 activity (n=9). (C and D) Expression of c-srcK295M prevented acid (n=9) stimulation of NaDC-1 activity but not ET-1 stimulation of NaDC-1 activity (n=9). (E and F) Pyk2Y402F blocked acid but not ET-1 stimulation of NaDC-1 activity (n=9). P values were calculated using ANOVA.
c-Src Is Required for Acid Stimulation of NaDC-1 In Vitro
Previous studies have shown that acid increases c-Src activity in vitro and in vivo and that c-Src mediates the Pyk2-dependent acid stimulation of NHE3.22–25 To determine whether c-Src is a mediator of the Pyk2-dependent acid stimulation of NaDC-1, cells were transfected with DN c-SrcK295M. In the presence of c-SrcK295M, acid stimulation of NaDC-1 activity was totally blocked (P=0.33; n=9) (Figure 1C). In contrast, c-Src inhibition did not prevent ET-1 stimulation of NaDC-1 activity (n=8) (Figure 1D). Therefore, like Pyk2, c-Src is not downstream of ET-1/ETB.
c-Src Binding to Pyk2 Is Required for Pyk2/c-Src–Mediated Acid Stimulation of NaDC-1 Activity In Vitro
OKP cells were transfected with mutant pyk2Y402F, a construct encoding a protein that lacks the ability of Pyk2 to bind and activate c-Src. In control conditions, media acidification led to a 53% increase in NaDC-1 activity (P<0.004; n=9), whereas in pyk2Y402F transfected cells, acid incubation had no effect on NaDC-1 activity (P=0.15; n=9) (Figure 1E). In contrast, ET-1 was still able to increase NaDC-1 activity in both the control cells (P<0.002; n=9) and cells expressing pyk2Y402F (P<0.05; n=9) (Figure 1F).
Pyk2 Is Required for NaDC-1 Stimulation by Acid In Vivo
To confirm that Pyk2 signaling is required for acid stimulation of NaDC-1 in vivo, studies were performed in Pyk2+/+ and Pyk2−/− mice. Pyk2−/− mice were normal in appearance, and their body weights were slightly higher than their wild-type littermates; however, food and water intakes were not different between the two genotypes. Basal renal function, plasma electrolytes levels, and acid-base balance were also the same comparing Pyk2+/+ with Pyk2−/− mice (Supplemental Table 1).
For experimentation, mice had free access to either regular drinking water (control) or NH4Cl solution. After 7 days of acid feeding, both Pyk2+/+ and Pyk2−/− mice showed a significant decrease of blood pH and bicarbonate levels. Although plasma bicarbonate levels were not different between the genotypes, Pyk2−/− mice had a more acidic blood pH (Figure 2, A–C). As shown in Figure 2D, citrate uptake was significantly increased by acid in Pyk2+/+ mice but not in Pyk2−/− mice. Thus, both in vitro and in vivo models show that Pyk2 is required for acid stimulation of NaDC-1 activity.
Figure 2.: Pyk2 abrogation in vivo totally blocks acid stimulation of NaDC-1 activity and partially reduces acid-induced hypocitraturia. (A–C) Blood gas parameters of Pyk2+/+ and Pyk2−/− mice after 7 days of normal water (control) or water containing 0.3 mol/L NH4Cl (acid) for 7 days (n=6 for all groups). (D) Citrate uptake was measured as described in Methods in brush border membrane vesicles prepared from renal cortex of Pyk2+/+ and Pyk2−/− mice (n=6 per group). (E) Urine citrate was measured in spot urine samples at baseline and after 2, 3, 5, and 7 days of NH4Cl loading, and it was expressed as the citrate-to-creatinine ratio. ***P<0.001 compared with control.
However, Pyk2−/− mice had lower urine citrate-to-creatinine ratios compared with the wild type at basal; after 5 and 7 days of NH4Cl loading, in both Pyk2+/+ and Pyk2−/− mice, citrate excretion was significantly reduced compared with baseline, but acid-induced hypocitraturia was significantly less severe in Pyk2−/− mice (Figure 2E).
ERK1,2, but Not JNK and p38, Is Required for Acid Stimulation of NaDC-1 In Vitro
ERK1,2 activity is increased by media acidification in OKP cells, whereas JNK activity is reduced.22Figure 3, A and B shows that the ERK1,2 inhibitor, PD98059, totally blocked acid but not ET-1 stimulation of NaDC-1 activity, suggesting that ERK1,2 is required for acid activation of NaDC-1 but not located downstream of ET-1/ETB in the acid-activated signaling cascade. In contrast, neither SP600125 nor SB203580 (JNK and p38 inhibitors, respectively) had any effect on acid stimulation of NaDC-1 activity (Figure 3, C and D).
Figure 3.: Extracellular signal–regulated kinase 1,2 (ERK1,2) is required for acid but not endothelin-1 (ET-1) stimulation of NaDC-1 activity. (A) PD98059 totally prevented acid stimulation of NaDC-1 activity (n=9) but (B) had no effect on ET-1 stimulation of NaDC-1 activity (n=9). (C and D) Neither SP600125 (JNK inhibitor; n=9) nor SB203580 (p38 inhibitor; n=9) blocked acid stimulation of NaDC-1 activity. P values were calculated using ANOVA.
ERK1,2 Is Activated by Media Acidification in a Time-Dependent Manner In Vitro
To better elucidate the role of ERK1,2 in the acid-activated signaling pathway, ERK1,2 activation was assayed after media acidification at several time points (Figure 4A). After acid addition to the media, ERK1,2 phosphorylation peaked between 3 and 5 minutes and then decreased. (To determine the sensitivity of ERK1,2 phosphorylation to media acidification, OKP cells were exposed to different pH values ranging from 7.4 to 6.3, and ERK1,2 phosphorylation was assayed 3–5 minutes later.) As shown in Figure 4B, ERK1,2 phosphorylation was increased in 3–5 minutes, even when the pH of the medium was decreased by just 0.1 pH units (7.4–7.3).
Figure 4.: Extracellular signal–regulated kinase 1,2 (ERK1,2) phosphorylation by media acidification is Pyk2/c-Src-independent. (A) Time-dependent phosphorylation of ERK1,2. (B) Effect of degree of media acidification on phospho–ERK1,2-to-total ERK1,2 ratio at 3 minutes (n=9). (C) ERK1,2 phosphorylation induced by media acidification in OKP cells transfected with dominant negative (DN) Pyk2 (Pyk2K457A), mutant Pyk2 (Pyk2Y402F), or dominant negative c-Src (c-srcK295M) versus empty vector (n=9). P values were calculated using ANOVA. *P<0.05 compared with control; **P<0.01 compared with control; ***P<0.001 compared with control.
ERK1,2 Phosphorylation by Media Acidification Does Not Require the Pyk2/c-Src Pathway In Vitro and In Vivo
To determine if the Pyk2/c-Src pathway plays a role in acid-induced ERK1,2 activation, ERK1,2 phosphorylation was assayed in OKP cells transfected with DN Pyk2K457A, DN Pyk2Y402F, or DN c-SrcK295M versus empty vector (control) (Figure 4C). In none of the transfected cells was acid-induced ERK1,2 phosphorylation blocked, showing that Pyk2 and c-Src are not required for acid activation of ERK1,2 in vitro.
To address if the Pyk2/c-Src signaling is required in vivo for acid stimulation of ERK1,2 activity, Pyk2+/+ and Pyk2−/− mice were gavaged with water (control) or an NH4Cl solution. After acid loading, Pyk2−/− and wild-type mice had similar decreases in plasma bicarbonate and pH (Figure 5, A–C). Increased ERK1,2 phosphorylation in renal cortex harvested from both wild-type and Pyk2−/− mice was evident 15 minutes after NH4Cl loading (Figure 5, D and E). Thus, as in the in vitro setting, in vivo acid-mediating ERK1,2 phosphorylation is independent of the Pyk2/c-Src signaling pathway.
Figure 5.: Pyk2 abrogation in vivo is without effect on extracellular signal–regulated kinase 1,2 (ERK1,2) phosphorylation by acid. (A–C) Blood gas parameters in Pyk2+/+ and Pyk2−/− mice 15 minutes after gavage with water (control) versus 0.3 M NH4Cl. (D and E) Immunoblotting showing phospho–ERK1,2-to-total ERK1,2 ratio in Pyk2+/+ and Pyk2−/− mice (n=9 for group). P values were calculated using ANOVA. **P<0.01 compared with control; ***P < 0.001 compared with control.
Raf1 Is Activated by Media Acidification In Vitro
Raf1 is a frequent regulator of ERK1,2 activity. Raf1 phosphorylation at ser338 (a marker of its activation) was assayed in a time-dependent manner after media acidification. Similar to acid-induced ERK1,2 phosphorylation, acid-induced Raf1 phosphorylation peaked around 3–5 minutes, returning to the baseline around 30 minutes and showing a second peak between 1 and 2 hours (Figure 6, A and B).
Figure 6.: The signaling pathway activated by acid includes Rapidly Accelerated Fibrosarcoma-kinase 1 (Raf1)/extracellular signal–regulated kinase 1,2 (ERK1,2)/p90 Ribosomal S6 Kinase (p90RSK). Time course of (A) Raf1, (B) ERK1,2, and (C) p90RSK phosphorylation after media acidification (n=9). P values were calculated using ANOVA. *P<0.05 compared with control.
p90RSK Is Activated after Media Acidification In Vitro, and ERK1,2 and p90RSK Phosphorylation Is Blocked by PD98059 and an Raf1 Inhibitor
p90RSK is a known downstream effector of ERK1,2 that is activated by phosphorylation and then migrates to the nucleus to activate transcription factors.26 As shown in Figure 6C, media acidification increased phosphor-p90RSK abundance within 3–5 minutes.
The acid-induced increases in ERK1,2 and p90RSK phosphorylation were both blocked by an Raf1 inhibitor and PD98059 (Figure 7). Taken together, these data suggest that the acid-activated “ERK” signaling pathway involves Raf1, ERK1,2, and p90RSK in that order.
Figure 7.: Extracellular signal–regulated kinase 1,2 (ERK1,2) and p90 Ribosomal S6 Kinase (p90RSK) phosphorylation by acid require Rapidly Accelerated Fibrosarcoma-kinase 1 (Raf1)/ERK1,2 signaling. (A and B) ERK1,2 and (C and D) p90RSK phosphorylation by media acidification at 3–5 minutes is totally blocked in the presence of (A and C) PD98059 and (B and D) Raf1 inhibitor (n=9). P values were calculated using ANOVA. *P<0.05 compared with control; **P<0.01 compared with control; # P>0.05.
Acid Stimulation of NaDC-1 Activity Requires Raf1/ERK1,2/p90RSK Signaling In Vitro
To confirm that the Raf1/ERK1,2/p90RSK signaling pathway plays a role in acid stimulation of NaDC-1, NaDC-1 activation by media acidification was measured in (1) the presence of Raf1 inhibitor 1 versus vehicle (DMSO) and (2) cells transfected with DN p90RSK versus empty vector. The Raf1 inhibitor partially blocked acid stimulation of NaDC-1 activity (Figure 8A), whereas the lack of functional p90RSK protein totally blocked acid stimulation of NaDC-1 activity (Figure 8B).
Figure 8.: NaDC-1 stimulation by acid requires Rapidly Accelerated Fibrosarcoma-kinase 1 (Raf1) and p90 Ribosomal S6 Kinase (p90RSK). (A) Acid stimulation of NaDC-1 activity is partially prevented by Raf1 inhibitor (n=6). (B) Cells transfected with dominant negative (DN) p90RSK do not show acid-induced stimulation of NaDC-1 activity (n=9). P values were calculated using ANOVA.
Discussion
The rate of renal proximal tubule citrate reabsorption and its intracellular metabolism are determinants of urinary citrate excretion. Both processes are regulated by systemic acid-base status, and they are stimulated in vivo by chronic metabolic acidosis.27,28 Since citrate is a base equivalent, increasing citrate reabsorption and metabolism serves as an adaptive response to facilitating acid excretion. However, the resulting hypocitraturia increases the risk for lithogenesis.29 Understanding how the proximal tubule modulates these processes is critical from both biologic and clinical standpoints.
Luminal citrate uptake is mediated by the Na-dicarboxylate cotransporter NaDC-1.30 We previously showed that chronic metabolic acidosis increases NaDC-1 activity through the ET-1/ETB signaling pathway.13 These studies show that acid activates parallel signaling pathways that are both required for the stimulation of NaDC-1 activity; one pathway involves the Pyk2-c-Src, and the other involves the c-Raf/ERK1,2/p90RSK signaling pathway.
The proximal tubule plays a pivotal role in the maintenance of systemic acid-base balance.15 It reabsorbs approximately 80% of the filtered bicarbonate, a process mostly dependent on the apical membrane Na+/H+ antiporter (NHE3) and the basolateral membrane Na-3HCO3− cotransporter.31 The proximal tubule is also the main site of ammonia production, the primary buffer for excreting daily metabolic acid loads,32 and the sole intrarenal site of citrate reabsorption and metabolism. In addition, the proximal tubule modulates phosphate reabsorption, which also affects net acid excretion.15 In our model, a common acid-activated signaling pathway increases the activity of two key proximal tubule transporters involved in maintaining acid-base balance: NHE3 and NaDC-1.
We and others have shown that ET-1 expression is increased in OKP cells exposed to media acidification and the kidney after an acid load33,34 and that ET-1/ETB signaling mediates NHE3 regulation by acid both in vivo and in vitro.17,35
In OKP cells, the protein kinases Pyk2 and c-Src are rapidly activated by acid (within 30 and 60 seconds, respectively), and decreases in pH activate Pyk2 in a cellfree system, suggesting that Pyk2 is directly regulated by proton concentration.21 Inhibiting both Pyk2 and c-Src activity blocks acid- but not ET-1–dependent stimulation of NHE3 activity, suggesting that these proteins are required in the acid-induced signaling pathway regulating NHE3 activity but are not located downstream of ET-1/ETB in the signaling cascade.22 Media acidification also increases mRNA abundance of the immediate early genes, c-fos and c-jun, but their position in the acid-activated signaling cascade is not yet known.36 ERK1,2 is a known regulator of these genes, and we have shown that acid-induced phosphorylation (and activation) of ERK1,2 is required for acid stimulation of NHE3 activity via a mechanism that is independent of the Pyk2/c-Src signaling cascade.23 Taken together, these findings suggest that acid stimulation of NHE3 requires involvement of at least two parallel pathways: Pyk2/c-Src and ERK1,2.
Several parallelisms exist between acid stimulation of NaDC-1 and NHE3 activities. As with acid stimulation of NHE3 activity, acid-induced NaDC-1 activity is mediated by ET-1/ETB signaling in vitro and in vivo.13 This study shows that Pyk2/c-Src and ERK1,2 pathways mediate acid stimulation of NaDC-1 activity (Figure 9) and that neither pathway is located downstream of ET-1/ETB in the signaling cascade. Despite Pyk2 inhibition totally blocking acid stimulation of NaDC-1 in vitro and in vivo, overall renal function and systemic acid-base homeostasis were not significantly different between wild-type and deficient Pyk2 mice. Given the physiologic importance of maintaining systemic acid-base balance, it is likely that acid loading activates biologic mechanisms that compensate for the absence of a functional Pyk2 in the Pyk2−/− mice; these mice had a lower urine citrate excretion in basal condition, and after acid loading, the magnitude of hypocitraturia was less than in Pyk2+/+ mice. Although it is not immediately clear why Pyk2−/− mice showed less urinary citrate excretion at baseline, these results suggest that urine citrate excretion is regulated at several levels, and acid stimulation of NaDC-1 activity is only one component of this process.
Figure 9.: Acid stimulation of NaDC-1 activity is mediated by both Pyk2/c-Src and Raf/ERK1,2/p90RSK signalling. Black arrows depict mechanisms that have been shown in previous studies and this study to be involved in acid stimulation of NaDC-1 activity. Signaling intermediates are placed in the order experimental confirmed. Gray arrows indicate the possible signaling pathways involved in acid stimulation of NaDC-1 activity but have not been confirmed. The times noted on the right side of the graph are the peak activation times after exposure to acid. ERK1,2, extracellular signal–regulated kinase 1,2; ET-1, endothelin-1; ETB, endothelin B; p90RSK, p90 Ribosomal S6 Kinase; Raf1, Rapidly Accelerated Fibrosarcoma-kinase 1.
Similar to acid stimulation of NHE3 activity, PD98059, an ERK1,2 inhibitor, totally blocks acid but not ET-1 stimulation of NaDC-1 activity. Whether simultaneous inhibition of Pyk2 and ERK1,2 pathways would have an additive effect on acid stimulation of NaDC-1 activity remains a question, although inhibition of either pathway totally prevents NaDC-1 activation. Nevertheless, this study shows that both pathways are required for acid stimulation of NaDC-1 activity. A possible explanation for requiring both pathways when each pathway alone totally blocks the acid effect is that, as it is known in other biologic systems, multiple and/or redundant pathways mediate the regulation of processes that are critical to systemic homeostasis, such as maintaining acid-base balance.37
Acid sensing is critical in all cells that possess the ability to defend themselves against an acid load. The renal epithelia play a key role in ensuring that systemic acid-base balance and intracellular pH remain normal. The signaling pathways that mediate these functions are of fundamental biologic significance, and understanding the molecular pathophysiology of hypocitraturia is critical to understanding the biochemical basis by which changes in acid-base balance affect the risk for urinary stones. This study shows that the signaling pathways mediating acid regulation of NaDC-1 activity share several commonalities with acid regulation of NHE3 activity. Acid stimulation of both transporter activities is mediated by and requires a functional Pyk2-c-Src pathway and a parallel functional ERK1,2 pathway. These studies also show that neither the Pyk2/c-Src nor ERK1,2 pathways are required for ET-1/ETB stimulation of NaDC-1 activity. Although these findings do not rule out a role for either or both Pyk2/c-Src and ERK1,2 in ET-1 stimulation of NaDC-1 activity, they do show that the position of the Pyk2/c-Src and ERK1,2 pathways is not downstream of ET-1/ETB in the signaling cascade.
In conclusion, these studies underscore the fact that, in modern Western lifestyle, where dietary habits result in a daily metabolic acid load, the maintenance of a stable systemic acid-base balance puts the kidney in a position of improving one situation (maintaining systemic acid-base balance), while worsening another (increasing the risk of kidney stone formation). Thus, these studies contribute to our understanding of how the kidney responds to an acid load and triggers rectification of the consequences of an acid load.
Disclosures
None.
We thank Dr. Orson Moe and Dr. Giovambattista Capasso for their careful reading of the manuscript and Lonnette Diggs for the technical assistance.
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-39298.
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