Acid-base regulation in the renal proximal tubules: using novel pH sensors to maintain homeostasis

Abstract

The kidneys play a critical role in precisely regulating the composition of the plasma to maintain homeostasis. To achieve this, the kidneys must be able to accurately determine or “sense” the concentration of a wide variety of substances and to make adjustments accordingly. Kidneys face a key challenge in the arena of pH balance, as there is a particularly narrow range over which plasma pH varies in a healthy subject (7.35–7.45) and this pH must constantly be protected against a variety of onslaughts (changes in diet, activity, and even elevation). The proximal tubule, the first segment to come into contact with the forming urine, plays an important role in helping the kidneys to maintain pH homeostasis. Recent studies have identified a number of novel proximal tubule proteins and signaling pathways that work to sense changes in pH and subsequently modulate renal pH regulation. In this review, we will highlight the role of novel players in acid-base homeostasis in the proximal tubule.

INTRODUCTION

For cells, tissues, and organs to function normally, blood pH levels must be tightly regulated within an extremely narrow range. In fact, blood pH values outside the range of 7.35–7.45 can cause significant pathophysiologies, including death. This is all the more impressive when one considers that blood pH must be protected against a variety of dietary intakes and other challenges. To achieve this, the kidneys and the lungs work in tandem to regulate levels of bicarbonate (${\text{HCO}}_3^{-}$) and carbon dioxide (CO₂), respectively. In this review, we will focus on the role of the kidney in this process and, in particular, the role of the renal proximal tubule. We will first briefly describe the role of the proximal tubule in acid-base regulation and then highlight the roles of novel interacting proteins that modulate these processes.

ROLE OF THE PROXIMAL TUBULE IN ACID-BASE REGULATION

The kidney plays a key role in reabsorbing and producing bicarbonate and excreting H⁺, thereby promoting acid-base homeostasis (6, 13). Bicarbonate is freely filtered at the glomerulus, but ~80% of this is reabsorbed by the proximal tubule, with the remainder being reabsorbed in the distal nephron such that the final urine is nearly free of bicarbonate (13). This process is coupled to Na⁺ reabsorption primarily through the actions of two transporters, Na⁺/H⁺ exchanger 3 (NHE3) on the apical membrane and electrogenic sodium bicarbonate cotransporter 1-A (NBCe1-A; SLC4A4) on the basolateral membrane (Fig. 1). In addition, a recent study has highlighted a role for electroneutral sodium bicarbonate cotransporter 2 (NBCn2) in helping to mediate bicarbonate reabsorption (5). As alluded to above, in addition to recovering bicarbonate from the forming urine, the proximal tubule also generates new bicarbonate (6). In addition, the kidney must excrete H⁺ ions. Excreted H⁺ ions are bound to either ammonia (NH₃) or compounds such as phosphate (“titratable acids”). The proximal tubule is thought to be the primary site of ammoniagenesis, and the production of ammonia accounts for approximately one-half to two-thirds of net acid excretion. A recent study illustrated the essential role of the NBCe1 transporter in proximal tubule ammoniagenesis, in addition to its well-known function as a bicarbonate transporter (7). NBCe1 deletion causes metabolic acidosis, which normally results in increased ammonia excretion in the urine. However, NBCe1 knockout (KO) mice exhibited decreased ammonia excretion in their urine despite metabolic acidosis. Further analysis showed that expression of proteins involved in proximal tubule ammonia generation was decreased (phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamate dehydrogenase) and a protein involved in ammonia recycling was increased (glutamine synthetase), which would be counterintuitive to the proximal tubule response expected during metabolic acidosis if ammoniagenesis were intact. Thus, these data demonstrate the uniquely important role of the proximal tubule in regulating acid-base transport, with regard to both bicarbonate reabsorption and ammoniagenesis.

Fig. 1.

Schematic illustration of acid-base handling in the renal proximal tubules. H⁺ ions secreted by the proximal tubules via Na⁺/H⁺ exchanger 3 (NHE3) combine with filtered bicarbonate to form H₂CO₃. Through the action of carbonic anhydrase (CA) IV and II, the filtered bicarbonate gets reabsorbed back into the blood via electrogenic sodium bicarbonate cotransporter 1 (NBCe1) on the basolateral side. Upon stimulation, several proteins such as calcium-sensing receptor (CaSR), proline-rich tyrosine kinase 2 (Pyk2), and G protein-coupled receptor family C group 5 member C (Gprc5c) can influence NHE3 activity, thereby modulating acid-base homeostasis. TWIK-related acid-sensitive K⁺ channel 2 (TASK2) is essential for maintaining basolateral membrane potential, and its absence affects bicarbonate reabsorption in the proximal tubules.

The regulatory systems in the kidney, and in the proximal tubule, must be able to robustly respond and adjust to a variety of common physiological and pathophysiological challenges, including the acid load that comes with eating a diet rich in animal protein, high altitude-induced respiratory alkalosis, changes in acid-base balance during exercise, and gastrointestinal distress resulting in diarrhea. To protect blood pH against these various “everyday” challenges (not to mention conditions such as chronic obstructive pulmonary disease), mechanisms exist to help regulate and fine-tune acid-base transport processes. Recently, several novel proteins have been identified that play roles in modulating proximal tubule acid-base transporters. In the following sections of this review, we will discuss the various roles of proteins that have been shown to modulate renal proximal tubule acid-base handling.

TWIK-RELATED ACID-SENSITIVE K⁺ CHANNEL 2

TWIK-related acid-sensitive K⁺ channel 2 (TASK2) belongs to a large family of K⁺ channels characterized by two pore-forming and four transmembrane domains (21). TASK2 is highly expressed in the kidneys with minor expression also seen in the liver and trachea (26). TASK2 has been localized in the kidney using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining as a surrogate for localization; using this method, TASK2 has been found in the renal proximal tubules and papillary collecting ducts (26). Patch-clamp recordings of primary proximal tubule cells from TASK2 wild-type (WT) and KO animals showed that TASK2 channels are activated by alkaline extracellular pH (26). On the basis of the sensitivity of the K⁺ outward current to bicarbonate blocker (DIDS) and highly buffered bath solution it was proposed that TASK2 channels localize on the basolateral membrane in proximal tubules (26).

In vivo studies support an alkaline pH-dependent role of TASK2 channels: TASK2 KO mice exhibit reduced water and sodium reabsorption by the kidneys when challenged with an alkali load challenge but are indistinguishable from WT in baseline conditions. The blood pH and bicarbonate levels of TASK2 KO mice were found to be reduced at baseline, whereas the urinary pH and bicarbonate levels were increased, indicating that TASK2 deletion in the kidney causes bicarbonate loss in the urine, leading to metabolic acidosis (26). TASK2 KO mice also exhibit significantly reduced mean arterial blood pressure compared with WT mice with a slightly reduced, but not statistically significant, glomerular filtration rate as measured by inulin clearance (26). It should be noted that these studies were performed in whole animal KO mice and that TASK2 channels are expressed in a variety of tissues; however, studies using primary cultures of nephron segments from TASK2 WT and KO mice provide compelling evidence that the proximal tubule plays a key role in driving this phenotype. It has been proposed (26) that reabsorbed bicarbonate ions exit proximal tubule cells basolaterally via NBCe1 and that the resultant rise in pH activates TASK2 channels. This allows for an efflux of K⁺ ions accumulated by Na⁺/K⁺-ATPase, thus repolarizing the basolateral membrane to set up the driving force for Na⁺ and bicarbonate reabsorption via NBCe1.

PROLINE-RICH TYROSINE KINASE 2

As alluded to above, NHE3 plays a key role in proximal tubule acid-base handling. The protons secreted by NHE3 are critical to facilitate bicarbonate reabsorption, as well as ammonia secretion. Therefore, proteins that regulate NHE3 activity are particularly important modulators of proximal tubule acid-base handling. One such protein is proline-rich tyrosine kinase 2, or Pyk2.

Pyk2 has been shown to be a key player in mediating the increase in NHE3 activity following acid stimulation (11). It had been previously known that NHE3 activity was stimulated by changes in intracellular pH and that this regulation was associated with an initial increase in apical membrane abundance in opossum kidney clone P (OKP) cells (followed by a subsequent increase in whole cell protein abundance; 27). In 2004, Li et al. reported that Pyk2 activation is essential for this process (11). Pyk2 is activated (as indicated by increased phosphorylation) by acid incubation, with a peak activation occurring at 30 s. Of note, this activation (autophosphorylation) occurred even in a cell-free system, indicating that Pyk2 itself is responding directly to the change in pH. Consistent with this, disrupting Pyk2 with either dominant-negative pyk2 or siRNA for pyk2 prevented acid-induced NHE3 activation. In addition to the role of Pyk2 in modulating NHE3 function, the same group has implicated endothelin-1/endothelin receptor type B as a modulator of NHE3 activity (9, 10, 15), although they believe the Pyk2 activation to be upstream of endothelin receptor type B activation (16).

Finally, of note, Pyk2 has also been shown to regulate H⁺-ATPase activity in the outer medullary collecting duct (4). Thus, although beyond the scope of this proximal tubule-centric review, Pyk2 may act to help coordinate a response to an acid challenge across multiple nephron segments.

CALCIUM-SENSING RECEPTOR

The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR), a class that also includes metabotropic glutamate receptors, GABA receptors, and taste receptors. Initially, the CaSR was hypothesized to be expressed only in the thick ascending limb; however, subsequent localization studies by in situ hybridization (23) and immunohistochemistry (22) showed that CaSR is expressed all along the nephron, where it is involved in parathyroid hormone-mediated regulation of phosphate excretion. Interestingly, CaSR membrane localization varies depending on the nephron segment. It localizes apically in the proximal tubule and collecting duct, but it is detected basolaterally in the thick ascending limb of Henle’s loop. In vitro studies on human embryonic kidney 293 (HEK293) cells stably expressing CaSR showed that it is sensitive to extracellular pH, with alkaline pH enhancing the response of the receptor to calcium and magnesium ions. CaSR stimulation using calcimimetic R-568 increased proximal tubule NHE3 activity as measured by BCECF dye in both in vitro microperfusion studies in mice and in vivo micropuncture studies in rats (2). CaSR-deficient animals showed no apparent change in NHE3, suggesting that CaSR activation by pH or calcium increases NHE3 activity in the proximal tubules. Thus, CaSR influences a mediator of sodium reabsorption (NHE3). In addition, increased NHE3 activity by CaSR acidifies the lumen pH, which has been hypothesized to ionize calcium and favor calcium reabsorption in the distal regions of the nephron. Furthermore, CaSR activation has been shown to block parathyroid hormone-mediated inhibition of phosphate reabsorption, thereby preventing renal Ca²⁺-phosphate precipitation and calcium nephrolithiasis (20).

GPCR FAMILY C GROUP 5 MEMBER C

GPCR family C group 5 member C (Gprc5c), like the CaSR, is a member of the class C metabotropic GPCRs (24). Using a TaqMan GPCR array card, we initially identified Gprc5c as being highly expressed in the murine kidney (17). Recently, we investigated further to identify the localization and physiological role of Gprc5c in the kidney (18). As determined by both immunohistochemistry and X-gal staining, Gprc5c localizes specifically to the apical membrane of renal proximal tubules of mice. Furthermore, we confirmed that Gprc5c expression and localization are conserved in rat and human kidneys. Because Gprc5c is an orphan receptor, we used a receptor internalization assay to screen for its activation and found that extracellular alkaline pH stimulates internalization of the receptor. By studying whole animal Gprc5c KO mice, we found that KO mice exhibited abnormal pH homeostasis, with a lower blood pH and high urine pH compared with WT littermates. Additionally, using an ex vivo kidney slice preparation, we found that renal NHE3 activity is reduced in Gprc5c KO mice. These data suggest that the extracellular pH-sensitive receptor Gprc5c is involved in regulating systemic acid-base balance by regulating NHE3 activity.

CONCLUSION

Regulation of pH homeostasis is tightly controlled, and abnormalities in acid-base balance have been implicated in numerous disease states including inflammation, immune response regulation, cardiac arrhythmias, bone disorders, and chronic kidney disease (8, 12, 14, 19). Renal proximal tubules are actively involved in systemic acid-base homeostasis by regulating bicarbonate reabsorption and proton excretion in the urine (1, 3, 25). Recent advances in the field have highlighted that renal proximal tubules can sense and adapt their function to a wide range of physiological challenges by involving several sensory proteins in a complex signaling network. To date, it is unknown whether or how these sensory proteins may coordinate with one another; in addition, it will be important to understand how the activity of each of these proteins may be modulated under the stresses of acidosis and alkalosis. Further studies are necessary to provide clarity and to more completely understand these complex yet essential signaling mechanisms.

GRANTS

This work was supported by National Kidney Foundation of Maryland.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

P.R. and J.L.P. prepared figures, drafted manuscript, edited and revised manuscript, and approved final version of manuscript.