Systemic Acid-base Homeostasis Research Articles

A pH imbalance is linked to autophagic dysregulation of inner ear hair cells in Atp6v1ba-deficient zebrafish

V-ATPase is an ATP hydrolysis-driven proton pump involved in the acidification of intracellular organelles and systemic acid-base homeostasis through H+ secretion in the renal collecting ducts. V-ATPase dysfunction is associated with hereditary distal renal tubular acidosis (dRTA). ATP6V1B1 encodes the B1 subunit of V-ATPase that is integral to ATP hydrolysis and subsequent H+ transport. Patients with pathogenic ATP6V1B1 mutations often exhibit an early onset of sensorineural hearing loss. However, the mechanisms underlying this association remain unclear. We employed morpholino oligonucleotide-mediated knockdown and CRISPR/Cas9 gene editing to generate Atp6v1ba-deficient (atp6v1ba−/−) zebrafish as an ortholog model for ATP6V1B1. The atp6v1ba−/− zebrafish exhibited systemic acidosis and significantly smaller otoliths compared to wild-type siblings. Moreover, deficiency in Atp6v1ba led to degeneration of inner ear hair cells, with ultrastructural changes indicative of autophagy. Our findings indicate a critical role of ATP6V1B1 in regulating lysosomal pH and autophagy in hair cells, and the results provide insights into the pathophysiology of sensorineural hearing loss in dRTA. Furthermore, this study demonstrates that the atp6v1ba−/− zebrafish model is a valuable tool for further investigation into disease mechanisms and potential therapies for acidosis-related hearing impairment.

Acid base homeostasis and serum bicarbonate concentration in syndrome of inappropriate anti-diuretic hormone secretion (SIADH) with hyponatremia.

The Syndrome of Inappropriate ADH secretion (SIADH) presents with excess ADH release caused by a range of conditions; including pneumonia, brain tumors, certain lung cancers, and diseases of the hypothalamus. It presents with significant reduction in both sodium and chloride concentrations in the blood. However, reports examining the acid base status indicate a normal serum bicarbonate concentration and systemic acid base homeostasis. The mechanisms for the absence of abnormalities in acid base homeostasis remain speculative. This mini review is highlighting the recent advances in renal molecular physiology to provide answers for the maintenance of acid base status and serum bicarbonate in a physiological range.

Loss of the Secretin Receptor Impairs Renal Bicarbonate Excretion and Aggravates Metabolic Alkalosis in Mice during Acute Base-Loading.

During acute base excess, the renal collecting duct β -intercalated cells ( β -ICs) become activated to increase urine base excretion. This process is dependent on pendrin and cystic fibrosis transmembrane regulator (CFTR) expressed in the apical membrane of β -ICs. The signal that leads to activation of this process was unknown. Plasma secretin levels increase during acute alkalosis, and the secretin receptor (SCTR) is functionally expressed in β -ICs. We find that mice with global knockout for the SCTR lose their ability to acutely increase renal base excretion. This forces the mice to lower their ventilation to cope with this challenge. Our findings suggest that secretin is a systemic bicarbonate-regulating hormone, likely being released from the small intestine during alkalosis. The secretin receptor (SCTR) is functionally expressed in the basolateral membrane of the β -intercalated cells of the kidney cortical collecting duct and stimulates urine alkalization by activating the β -intercalated cells. Interestingly, the plasma secretin level increases during acute metabolic alkalosis, but its role in systemic acid-base homeostasis was unclear. We hypothesized that the SCTR system is essential for renal base excretion during acute metabolic alkalosis. We conducted bladder catheterization experiments, metabolic cage studies, blood gas analysis, barometric respirometry, perfusion of isolated cortical collecting ducts, immunoblotting, and immunohistochemistry in SCTR wild-type and knockout (KO) mice. We also perfused isolated rat small intestines to study secretin release. In wild-type mice, secretin acutely increased urine pH and pendrin function in isolated perfused cortical collecting ducts. These effects were absent in KO mice, which also did not sufficiently increase renal base excretion during acute base loading. In line with these findings, KO mice developed prolonged metabolic alkalosis when exposed to acute oral or intraperitoneal base loading. Furthermore, KO mice exhibited transient but marked hypoventilation after acute base loading. In rats, increased blood alkalinity of the perfused upper small intestine increased venous secretin release. Our results suggest that loss of SCTR impairs the appropriate increase of renal base excretion during acute base loading and that SCTR is necessary for acute correction of metabolic alkalosis. In addition, our findings suggest that blood alkalinity increases secretin release from the small intestine and that secretin action is critical for bicarbonate homeostasis.

The Less Well-Known Little Brothers: The SLC9B/NHA Sodium Proton Exchanger Subfamily-Structure, Function, Regulation and Potential Drug-Target Approaches.

The SLC9 gene family encodes Na+/H+ exchangers (NHEs), a group of membrane transport proteins critically involved in the regulation of cytoplasmic and organellar pH, cell volume, as well as systemic acid-base and volume homeostasis. NHEs of the SLC9A subfamily (NHE 1–9) are well-known for their roles in human physiology and disease. Much less is known about the two members of the SLC9B subfamily, NHA1 and NHA2, which share higher similarity to prokaryotic NHEs than the SLC9A paralogs. NHA2 (also known as SLC9B2) is ubiquitously expressed and has recently been shown to participate in renal blood pressure and electrolyte regulation, insulin secretion and systemic glucose homeostasis. In addition, NHA2 has been proposed to contribute to the pathogenesis of polycystic kidney disease, the most common inherited kidney disease in humans. NHA1 (also known as SLC9B1) is mainly expressed in testis and is important for sperm motility and thus male fertility, but has not been associated with human disease thus far. In this review, we present a summary of the structure, function and regulation of expression of the SLC9B subfamily members, focusing primarily on the better-studied SLC9B paralog, NHA2. Furthermore, we will review the potential of the SLC9B subfamily as drug targets.

NBCn1 Increases NH4 + Reabsorption Across Thick Ascending Limbs, the Capacity for Urinary NH4 + Excretion, and Early Recovery from Metabolic Acidosis.

The electroneutral Na+/HCO3 - cotransporter NBCn1 (Slc4a7) is expressed in basolateral membranes of renal medullary thick ascending limbs (mTALs). However, direct evidence that NBCn1 contributes to acid-base handling in mTALs, urinary net acid excretion, and systemic acid-base homeostasis has been lacking. Metabolic acidosis was induced in wild-type and NBCn1 knockout mice. Fluorescence-based intracellular pH recordings were performed and NH4 + transport measured in isolated perfused mTALs. Quantitative RT-PCR and immunoblotting were used to evaluate NBCn1 expression. Tissue [NH4 +] was measured in renal biopsies, NH4 + excretion and titratable acid quantified in spot urine, and arterial blood gasses evaluated in normoventilated mice. Basolateral Na+/HCO3 - cotransport activity was similar in isolated perfused mTALs from wild-type and NBCn1 knockout mice under control conditions. During metabolic acidosis, basolateral Na+/HCO3 - cotransport activity increased four-fold in mTALs from wild-type mice, but remained unchanged in mTALs from NBCn1 knockout mice. Correspondingly, NBCn1 protein expression in wild-type mice increased ten-fold in the inner stripe of renal outer medulla during metabolic acidosis. During systemic acid loading, knockout of NBCn1 inhibited the net NH4 + reabsorption across mTALs by approximately 60%, abolished the renal corticomedullary NH4 + gradient, reduced the capacity for urinary NH4 + excretion by approximately 50%, and delayed recovery of arterial blood pH and standard [HCO3 -] from their initial decline. During metabolic acidosis, NBCn1 is required for the upregulated basolateral HCO3 - uptake and transepithelial NH4 + reabsorption in mTALs, renal medullary NH4 + accumulation, urinary NH4 + excretion, and early recovery of arterial blood pH and standard [HCO3 -]. These findings support that NBCn1 facilitates urinary net acid excretion by neutralizing intracellular H+ released during NH4 + reabsorption across mTALs.

Adenylyl cyclase 6 in acid-base balance – adding complexity

Systemic acid-base balance is tightly controlled within a narrow range of pH. Disturbances in systemic acid-base homeostasis are associated with diverse detrimental effects. The kidney is a key regulator of acid-base balance, capable of excreting HCO3- or H+, and chronic kidney disease invariably leads to acidosis. However, the regulatory pathways underlying the fine-tuned acid-base sensing and regulatory mechanisms are still incompletely understood. In the article published recently in Clinical Science (vol 132 (16) 1779-1796), Poulson and colleagues investigated the role of adenylyl cyclase 6 (AC6) in acid-base homeostasis. They uncovered a complex role of AC6, specifically affecting acid-base balance during HCO3- load, which causes pronounced alkalosis in AC6-deficient mice. However, the phenotype of AC6-deficient mice appears much more complex, involving systemic effects associated with increased energy expenditure. These observations remind us that there is much to be learned about the intricate signaling pathways involved in renal control of acid-base balance and the complex ramifications of acid-base regulation.

The role of renal proximal tubule transport in the regulation of blood pressure.

The electrogenic sodium/bicarbonate cotransporter 1 (NBCe1) on the basolateral side of the renal proximal tubule plays a pivotal role in systemic acid-base homeostasis. Mutations in the gene encoding NBCe1 cause severe proximal renal tubular acidosis accompanied by other extrarenal symptoms. The proximal tubule reabsorbs most of the sodium filtered in the glomerulus, contributing to the regulation of plasma volume and blood pressure. NBCe1 and other sodium transporters in the proximal tubule are regulated by hormones, such as angiotensin II and insulin. Angiotensin II is probably the most important stimulator of sodium reabsorption. Proximal tubule AT1A receptor is crucial for the systemic pressor effect of angiotensin II. In rodents and rabbits, the effect on proximal tubule NBCe1 is biphasic; at low concentration, angiotensin II stimulates NBCe1 via PKC/cAMP/ERK, whereas at high concentration, it inhibits NBCe1 via NO/cGMP/cGKII. In contrast, in human proximal tubule, angiotensin II has a dose-dependent monophasic stimulatory effect via NO/cGMP/ERK. Insulin stimulates the proximal tubule sodium transport, which is IRS2-dependent. We found that in insulin resistance and overt diabetic nephropathy, stimulatory effect of insulin on proximal tubule transport was preserved. Our results suggest that the preserved stimulation of the proximal tubule enhances sodium reabsorption, contributing to the pathogenesis of hypertension with metabolic syndrome. We describe recent findings regarding the role of proximal tubule transport in the regulation of blood pressure, focusing on the effects of angiotensin II and insulin.

Carbonic Anhydrase: A New Therapeutic Target for Managing Diabetes

Background: Carbonic Anhydrase (CA) is a zinc metallo-enzyme that is critical to regulation of systemic acid-base homeostasis by facilitating urinary acidification. Inhibition of carbonic anhydrase results in metabolic acidosis which leads to decrease in pH. Aim and objectives: The study aims to highlight the potential utility of erythrocyte carbonic anhydrase as therapeutic target for managing diabetes, by investigating changes of erythrocyte carbonic anhydrase activity in STZ induced diabetic rats. Methods: Carbonic anhydrase activity was determined by the absorbance of p-nitrophenol at 345nm released from p-nitrophenyl acetate. HbA1c was determined by ion exchange method (Spectrum diagnostics). Biochemical parameters were determined by Accutrend GCT meters with cobias® test strips. Results: The result revealed that inhibition of erythrocyte carbonic anhydrase results in significant increase in both blood lactate concentration and HbA1c level with significant reduction in blood glucose concentration. Metformin was found to reduce carbonic anhydrase activity and HbA1c level significantly and increased blood lactate concentration. The extract of Cadaba farinosa was found to reduce blood glucose concentration. Conclusions: Inhibition of carbonic anhydrase can be associated with reduced circulating blood glucose level. Metformin may therefore reduce circulating blood glucose by inhibiting carbonic anhydrase. Increased level of HbA1c may probably be due to inhibition of erythrocyte carbonic anhydrase. Therefore Carbonic anhydrase can potentially serve as a therapeutic target for managing diabetes in combination as serving as valuable marker for lactic acidosis.

Can the manipulation of urinary pH by beverages assist with the prevention of stone recurrence?

The formation of various types of stones in the urinary tract is strongly influenced by urinary pH. An acidic urinary pH promotes the crystallization of uric acid and cystine, respectively. Moreover, changes in systemic acid-base homeostasis alter urinary excretion of citrate, an important inhibitor of calcium oxalate stone formation. The effect of beverages on urinary pH and citrate excretion is mainly determined by the presence of bicarbonate and citrate. The bicarbonate content of mineral water can replace alkalization therapy with potassium citrate and contribute to urine inhibitory power by increasing urinary pH and citrate excretion. Citrus juices are rich sources of citrate. Oral citrate is absorbed in the intestine and nearly completely metabolized to bicarbonate, providing an alkali load, which in turn increases urinary pH and citrate excretion. However, data from observational and interventional studies on the effect of different types of citrus juices on the risk of urinary stone formation are conflicting. In conclusion, favourable changes in urinary pH and citrate excretion can be attained by various beverages. However, the long-term efficacy of certain beverages for the recurrence prevention of different types of stones has yet to be determined.

Characterization of Atp6ap2 ((Pro)‐Renin Receptor) in Receptor Mediated Endocytosis by the Renal Proximal Tubule

Atp6ap2 may be an accessory subunit of the V‐type H+ ATPase regulating organellar, cellular, and systemic acid‐base homeostasis. Here we examine whether the knock‐down of Atp6ap2 affects renal V‐ATPase function in the proximal tubule. There, V‐ATPases are important for receptor‐mediated endocytosis of low molecular weight proteins from urine via the megalin/cubilin receptors. In order to elucidate the role of Atp6ap2 in this function we used an inducible shRNA Atp6ap2 rat model and a doxycyline‐inducible kidney specific Atp6ap2 KO mouse model. Knock‐down of Atp6ap2 reduced Atp6ap2 expression by approximately 90 %. Atp6ap2 knock‐down animals showed higher proteinuria (albumin and Vitamin D binding protein).To further examine proximal tubular endocytosis, we injected animals with markers for fluid phase endocytosis (FITC‐ dextran, 10 kDa) and a for receptor mediated endocytosis (human transferrin). Immunofluorescence of kidney slices from both animal groups did not show a difference in FITC‐dextran intensity or localization. Furthermore, evaluation of receptor mediated endocytosis of human transferrin, showed in immunofluorescence stainings no differences between the animal groups but the knock‐down animals excreted human transferrin in the urine while control animal did not so. Hence, our data suggests a possible role for ATP6ap2 for proximal tubule function in the kidney; however, the defect in receptor‐mediated endocytosis is mild in rats and mice.Funded by the Swiss National Science Foundation.

PH-responsive, gluconeogenic renal epithelial LLC-PK1-FBPase+cells: a versatile in vitro model to study renal proximal tubule metabolism and function.

Ammoniagenesis and gluconeogenesis are prominent metabolic features of the renal proximal convoluted tubule that contribute to maintenance of systemic acid-base homeostasis. Molecular analysis of the mechanisms that mediate the coordinate regulation of the two pathways required development of a cell line that recapitulates these features in vitro. By adapting porcine renal epithelial LLC-PK1 cells to essentially glucose-free medium, a gluconeogenic subline, termed LLC-PK1-FBPase(+) cells, was isolated. LLC-PK1-FBPase(+) cells grow in the absence of hexoses and pentoses and exhibit enhanced oxidative metabolism and increased levels of phosphate-dependent glutaminase. The cells also express significant levels of the key gluconeogenic enzymes, fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK). Thus the altered phenotype of LLC-PK1-FBPase(+) cells is pleiotropic. Most importantly, when transferred to medium that mimics a pronounced metabolic acidosis (9 mM HCO3 (-), pH 6.9), the LLC-PK1-FBPase(+) cells exhibit a gradual increase in NH4 (+) ion production, accompanied by increases in glutaminase and cytosolic PEPCK mRNA levels and proteins. Therefore, the LLC-PK1-FBPase(+) cells retained in culture many of the metabolic pathways and pH-responsive adaptations characteristic of renal proximal tubules. The molecular mechanisms that mediate enhanced expression of the glutaminase and PEPCK in LLC-PK1-FBPase(+) cells have been extensively reviewed. The present review describes novel properties of this unique cell line and summarizes the molecular mechanisms that have been defined more recently using LLC-PK1-FBPase(+) cells to model the renal proximal tubule. It also identifies future studies that could be performed using these cells.

The Murine Cl-/HCO3- Exchanger Ae3 (Slc4a3) is Not Required for Acid-Base Balance but is Involved in Magnesium Handling by the Kidney

Background: The Slc4 family of bicarbonate transporters consists of several members, many of which are highly expressed in the kidney and play an important role in acid-base homeostasis. Among them are Ae1 (Slc4a1) and Ae2 (Slc4a2). Another member, Ae3 (Slc4a3), is suggested to be expressed in the kidney, however, its localization and impact on renal function is still unknown. Ae3 has also been implicated in the central control of breathing. Aims: Here, we analyzed the expression of Ae3 transcripts in isolated nephron segments and investigated systemic and renal acid-base homeostasis and renal electrolyte handling in the absence of Ae3, using a knock out mouse model. Methods: qPCR was used to localize Ae3 transcripts in the murine nephron, metabolic studies and whole body plethysmography to assess the role of Ae3 in renal functions. Results: Two Ae3 transcripts, the brain variant bAe3 and the cardiac variant cAe3, are expressed at low levels in the murine kidney. Although differentially distributed, they localize mostly to the distal nephron and renal collecting duct system. At baseline and after an acid challenge, mice deficient for Ae3 did not show major disturbances in renal acid-base excretion. Respiratory responses in whole body plethysmography to acid loading and CO2 and O2 challenges were normal. No major differences in renal electrolyte handling were discovered except for small changes in magnesium, potassium and sodium excretion after 7 days of acid loading. We therefore challenged mice with diets with high and low magnesium diets and found no differences in renal magnesium excretion but elevated expression of the Trpm6 magnesium channel in Ae3 KO mice. In conclusion, Ae3 is expressed in murine kidney at very low levels. Conclusions: Ae3 plays no role in systemic acid-base homeostasis but may modify renal magnesium handling inducing a higher expression of Trpm6.

Proximal renal tubular acidosis mediated by mutations in NBCe1-A: unraveling the transporter's structure-functional properties

NBCe1 belongs to the SLC4 family of base transporting membrane proteins that plays a significant role in renal, extrarenal, and systemic acid-base homeostasis. Recent progress has been made in characterizing the structure-function properties of NBCe1 (encoded by the SLC4A4 gene), and those factors that regulate its function. In the kidney, the NBCe1-A variant that is expressed on the basolateral membrane of proximal tubule is the key transporter responsible for overall transepithelial bicarbonate absorption in this nephron segment. NBCe1 mutations impair transepithelial bicarbonate absorption causing the syndrome of proximal renal tubular acidosis (pRTA). Studies of naturally occurring NBCe1 mutant proteins in heterologous expression systems have been very helpful in elucidation the structure-functional properties of the transporter. NBCe1 mutations are now known to cause pRTA by various mechanisms including the alteration of the transporter function (substrate ion interaction, electrogenicity), abnormal processing to the plasma membrane, and a perturbation in its structural properties. The elucidation of how NBCe1 mutations cause pRTA in addition to the recent studies which have provided further insight into the topology of the transporter have played an important role in uncovering its critically important structural-function properties.

Acidosis-induced V-ATPase trafficking in salivary ducts is initiated by cAMP/PKA/CREB pathway via regulation of Rab11b expression

Changes in systemic acid–base homeostasis cause a series of organ-specific cellular responses, among them changes of acid–base transporter activities, and recruitment or retrieval of these transporters from intracellular pools to the plasma membrane and vice versa. The purpose of this study was to investigate the impact of protein phosphorylation in the acidosis-induced translocation of vacuolar-type H+-ATPase (V-ATPase) in salivary ducts and to identify molecular targets. Therefore, the human submandibular gland cell line HSG was exposed to acidosis and V-ATPase trafficking was investigated in the presence or absence of inhibitors and activators of sAC/PKA and Src/ERK signaling pathways. Putative target genes have been identified by RT-PCR and immunoblotting, and validated by loss-of-function experiments. Acidosis caused activation of cAMP/PKA and Src signaling and inhibition of either pathway significantly impaired acidosis-induced V-ATPase redistribution and incorporation into the plasma membrane. Activation of ERK1/2 was Src-independent, whereas activation of PKA caused phosphorylation of cAMP response element-binding (CREB) and activation to regulate Rab11b transcription. Loss-of-function of CREB down-regulated Rab11b transcript and protein and significantly impaired acidosis-induced V-ATPase translocation in HSG cells. These data demonstrate that the cAMP/PKA/CREB signaling pathway initiates acidosis-induced V-ATPase trafficking in salivary ducts via regulation of Rab11b expression and provide first evidence for a molecular mechanism underlying cAMP/PKA-dependent transporter trafficking that could account for accumulation and activity of transporters in other cellular systems as well.

Slc26a11, a chloride transporter, localizes with the vacuolar H+-ATPase of A-intercalated cells of the kidney

Chloride has an important role in regulating vacuolar H(+)-ATPase activity across specialized cellular and intracellular membranes. In the kidney, vacuolar H(+)-ATPase is expressed on the apical membrane of acid-secreting A-type intercalated cells in the collecting duct where it has an essential role in acid secretion and systemic acid base homeostasis. Here, we report the identification of a chloride transporter, which co-localizes with and regulates the activity of plasma membrane H(+)-ATPase in the kidney collecting duct. Immunoblotting and immunofluorescent labeling identified Slc26a11 (∼72 kDa), expressed in a subset of cells in the collecting duct. On the basis of double-immunofluorescent labeling with AQP2 and identical co-localization with H(+)-ATPase, cells expressing Slc26a11 were deemed to be distinct from principal cells and were found to be intercalated cells. Functional studies in transiently transfected COS7 cells indicated that Slc26a11 (designated as kidney brain anion transporter (KBAT)) can transport chloride and increase the rate of acid extrusion by means of H(+)-ATPase. Thus, Slc26a11 is a partner of vacuolar H(+)-ATPase facilitating acid secretion in the collecting duct.

Expression of Rh Glycoproteins in the Mammalian Kidney

Ammonia metabolism is a fundamental process in the maintenance of life in all living organisms. Recent studies have identified ammonia transporter family proteins in yeast (Mep), plants (Amt), and mammals (Rh glycoproteins). In mammalian kidneys, where ammonia metabolism and transport are critically important for the regulation of systemic acid-base homeostasis, basolateral Rh B glycoprotein and apical/basolateral Rh C glycoprotein are expressed along the distal nephron segments. Data from experimental animal models and knockout mice suggest that the Rh glycoproteins appear to mediate important roles in urinary ammonia excretion.

Genome-wide gene expression profiling reveals renal genes regulated during metabolic acidosis

Production and excretion of acids are balanced to maintain systemic acid-base homeostasis. During metabolic acidosis (MA) excess acid accumulates and is removed from the body, a process achieved, at least in part, by increasing renal acid excretion. This acid-secretory process requires the concerted regulation of metabolic and transport pathways, which are only partially understood. Chronic MA causes also morphological remodeling of the kidney. Therefore, we characterized transcriptional changes in mammalian kidney during MA to gain insights into adaptive pathways. Total kidney RNA from control and 2- and 7-days NH(4)Cl treated mice was subjected to microarray gene profiling. We identified 4,075 transcripts significantly (P < 0.05) regulated after 2 and/or 7 days of treatment. Microarray results were confirmed by qRT-PCR. Analysis of candidate genes revealed that a large group of regulated transcripts was represented by different solute carrier transporters, genes involved in cell growth, proliferation, apoptosis, water homeostasis, and ammoniagenesis. Pathway analysis revealed that oxidative phosphorylation was the most affected pathway. Interestingly, the majority of acutely regulated genes after 2 days, returned to normal values after 7 days suggesting that adaptation had occurred. Besides these temporal changes, we detected also differential regulation of selected genes (SNAT3, PEPCK, PDG) between early and late proximal tubule. In conclusion, the mammalian kidney responds to MA by temporally and spatially altering the expression of a large number of genes. Our analysis suggests that many of these genes may participate in various processes leading to adaptation and restoration of normal systemic acid-base and electrolyte homeostasis.

Short-Term Exposure to a High-Protein Diet Differentially Affects Glomerular Filtration Rate but Not Acid-Base Balance in Older Compared to Younger Adults

There is conflicting evidence regarding the effects of high protein intake on kidney health, especially as it relates to age. We investigated the short-term effects of a high-protein diet on kidney function and systemic acid-base homeostasis in older compared to younger adults. The subjects were healthy men and women either between the ages of 25 and 40 years (n=12) or 55 and 70 years (n=10). They underwent a two-period crossover trial with each period consisting of 2 weeks of usual diet followed by a 1-week experimental diet. During the experimental diet period subjects consumed metabolic meals that provided either low protein content (0.5 g protein/kg/day) or high protein content (2.0 g protein/kg/day). Outcome measures included blood and urine markers of renal function and acid-base balance. An analysis of variance was used to assess differences between age groups with respect to experimental diet. The older group, mainly women, showed an increase in glomerular filtration rate after the high-protein compared to low-protein diet; the younger group did not. Urinary pH was significantly lower, and ammonium excretion was significantly higher after the high-protein diet in both age groups, but neither group developed a clinically detectable acidosis after the week of receiving a high-protein diet.

Postnatal expression of transport proteins involved in acid-base transport in mouse kidney.

The kidney plays a major role in maintaining and controlling systemic acid-base homeostasis by reabsorbing bicarbonate and secreting protons and acid-equivalents, respectively. During postnatal kidney development and adaptation to changing diets, plasma bicarbonate levels are increasing, the capacity for urinary acidification maturates, and the final morphology and distribution of intercalated cells is achieved. In adult kidney, at least two types of intercalated cells (IC) are found along the collecting duct characterised either by the expression of AE1 (type A IC) or pendrin (non-type A IC) where non-type A IC are found only in the convoluted distal tubule, connecting tubule and cortical collecting duct. Here we investigated in mouse kidney the relative mRNA abundance, protein expression levels and distribution of several proteins involved in renal acid-base transport, namely, the Na(+)/HCO(3)(-) cotransporter NBC1 (SLC4A4), the Na(+)/H(+)-exchanger NHE3 (SLC9A3), two subunits of the vacuolar H(+)-ATPase [ATP6V0A4 (a4), ATP6V1B1 (B1)], the Cl(-)/HCO(3)(-) exchangers AE1 (SLC4A1) and pendrin (SLC26A4). Relative mRNA abundance of all transport proteins was lowest at day 3 after birth and increased thereafter in parallel with protein levels. The numbers of type A and non-type A IC in the cortical collecting duct (CCD) increased from day 3 to days 18 and 24, whereas the number of IC in the CCD with apical staining for the vacuolar H(+)-ATPase subunits a4 and B1 decreased from day 3 to days 18 and 24, respectively. In addition, cells with characteristics of non-type A IC (pendrin expression, basolateral expression of vacuolar H(+)-ATPase subunits) were found in the inner and outer medulla 3 days after birth but were absent from the medulla of 24-day-old mice. Taken together, these results demonstrate massive changes in mRNA and protein expression levels of several acid-base transporters during postnatal kidney maturation and also show changes in intercalated cell phenotype in the medulla during these processes.

AE4 is a DIDS-sensitive Cl(-)/HCO(-)(3) exchanger in the basolateral membrane of the renal CCD and the SMG duct.

The renal cortical collecting duct (CCD) plays an important role in systemic acid-base homeostasis. The beta-intercalated cells secrete most of the HCO(-)(3), which is mediated by a luminal, DIDS-insensitive, Cl(-)/HCO(-)(3) exchange. The identity of the luminal exchanger is a matter of debate. Anion exchanger isoform 4 (AE4) cloned from the rabbit kidney was proposed to perform this function (Tsuganezawa H et al. J Biol Chem 276: 8180-8189, 2001). By contrast, it was proposed (Royaux IE et al. Proc Natl Acad Sci USA 98: 4221-4226, 2001) that pendrin accomplishes this function in the mouse CCD. In the present work, we cloned, localized, and characterized the function of the rat AE4. Northern blot and RT-PCR showed high levels of AE4 mRNA in the CCD. Expression in HEK-293 and LLC-PK(1) cells showed that AE4 is targeted to the plasma membrane. Measurement of intracellular pH (pH(i)) revealed that AE4 indeed functions as a Cl(-)/HCO(-)(3) exchanger. However, AE4 activity was inhibited by DIDS. Immunolocalization revealed species-specific expression of AE4. In the rat and mouse CCD and the mouse SMG duct AE4 was in the basolateral membrane. By contrast, in the rabbit, AE4 was in the luminal and lateral membranes. In both, the rat and rabbit CCD AE4 was in alpha-intercalated cells. Importantly, localization of AE4 was not affected by the systemic acid-base status of the rats. Therefore, we conclude that expression and possibly function of AE4 is species specific. In the rat and mouse AE4 functions as a Cl(-)/HCO(-)(3) exchanger in the basolateral membrane of alpha-intercalated cells and may participate in HCO(-)(3) absorption. In the rabbit AE4 may contribute to HCO(-)(3) secretion.