ABSTRACT

A recent study demonstrated that in response to a feeding-induced metabolic acidosis, goldfish (Carassius auratus) adjust epithelial protein and/or mRNA expression in their kidney tubules for multiple transporters known to be relevant for acid–base regulation. These include Na+/H+ exchanger (NHE), V-type H+-ATPase (V-ATPase), cytoplasmic carbonic anhydrase, HCO3 transporters and Rhesus proteins. Consequently, renal acid output in the form of protons and NH4+ increases. However, little is known about the mechanistic details of renal acid–base regulation in C. auratus and teleost fishes in general. The present study applied the scanning ion-selective electrode technique (SIET) to measure proton flux in proximal, distal and connecting tubules of goldfish. We detected increased H+ efflux into the extracellular fluid from the tubule in fed animals, resulting from paracellular back-flux of H+ through the tight junction. By applying inhibitors for selected acid–base regulatory epithelial transporters, we found that cytosolic carbonic anhydrase and HCO3 transporters were important in mediating H+ flux in all three tubule segments of fed goldfish. Contrastingly, V-ATPase seemed to play a role in H+ flux only in proximal and distal tubules, and NHE in proximal and connecting tubules. We developed working models for transport of acid–base relevant equivalents (H+, HCO3, NH3/NH4+) for each tubule segment in C. auratus kidney. While the proximal tubule appears to play a major role in both H+ secretion and HCO3 reabsorption, the distal and connecting tubules seem to mainly serve for HCO3 reabsorption and NH3/NH4+ secretion.

INTRODUCTION

The maintenance of acid–base homeostasis is one of the most important physiological processes in all organisms as it ensures proper enzyme and protein function (Somero, 1986; Riggs, 1988). The kidney has the predominant function of generating and transporting HCO3 and ammonia (here referred to as the sum of NH3 and NH4+) in mammals, and thus ultimately regulates any metabolic acid–base disturbance by adjusting net acid (both titratable acid and NH4+) and base (HCO3) excretion (Hamm et al., 2015). In teleost fishes, by contrast, the predominant organ involved in regulating acid–base disturbances is the gill (Perry and Gilmour, 2006), while the kidney seems to play only a small but significant role in acid–base balance. For instance, the kidney of the freshwater rainbow trout, Oncorhynchus mykiss, is known to compensate for environmental disturbances that induce acidosis, such as exposure to low environmental pH (McDonald and Wood, 1981), hyperoxia (Wheatly et al., 1984), hypercapnia (Perry and Fryer, 1997) and exhaustive exercise (Wood, 1988), as well as for a feeding-induced alkaline tide (Bucking et al., 2010). The mechanisms of renal ammonia and acid–base regulation in teleosts, however, remain poorly studied to date. This difficulty stems, in part, from the structural heterogeneity and architectural complexity of the teleost kidney, which contains multiple tissue types and a convoluted tubule (Sakai, 1985). Consequently, most physiological studies have only investigated acid–base regulation by the kidney as a whole (e.g. Wood et al., 1999; Lawrence et al., 2015; Wright et al., 2014). However, recently, we (Fehsenfeld and Wood, 2018) micro-dissected the kidney of the goldfish, Carassius auratus, enabling us to profile mRNA and protein expression of renal epithelial transporters involved in acid–base homeostasis along the renal tubule and its different segments.

In general, renal ionoregulation in freshwater teleosts involves epithelial transporters that are important for the re-absorption of ions to minimize solute loss into the dilute external medium (Wheatly et al., 1984; Curtis and Wood, 1992; Perry et al., 2003b). Transport of primary ions and electrolytes is coupled to the transport of acid–base equivalents, hence linking ionoregulation and acid–base balance in the teleost kidney (Perry et al., 2003b; Wheatly et al., 1984; Dantzler, 2016). Many of the same transporters, including V-type H+-ATPase (V-ATPase), Na+/H+ exchanger (NHE), Na+/HCO3 co-transporter and HCO3/Cl-exchanger, can also be found in the teleost gill epithelium (Perry et al., 2003b). Similarly, these transporters are involved in acid–base regulation in the mammalian kidney (Weiner and Verlander, 2013).

In fasting C. auratus, the kidney produces alkaline urine, resulting in net renal base excretion. Exposure of animals to a low pH environment, however, results in net renal acid excretion (Lawrence et al., 2015). In contrast to the rainbow trout, the goldfish is agastric, like the freshwater killifish Fundulus heteroclitus (Wood et al., 2010), having an alkaline digestive tract, and therefore exhibits a systemic acidic tide after feeding. We (Fehsenfeld and Wood, 2018) were able to show that a systemic acid-load (metabolic acidosis) caused by feeding resulted in increased acid excretion by the goldfish kidney, as indicated by a decrease in urine pH and an increase in urine inorganic phosphate and ammonia concentrations. This process was in part accomplished by differentially adjusting expression levels of both mRNA and protein of a variety of transporters in the proximal, distal and connecting tubule of the goldfish nephron (Fehsenfeld and Wood, 2018).

The present study aimed to further investigate the mechanism of renal acid–base regulation in response to feeding in C. auratus. Micro-dissection of intact kidney tubules in combination with the scanning ion-selective electrode technique (SIET) (Smith et al., 1994; Piñeros et al., 1998) allowed direct measurement of H+ flux at the basolateral surface of distinct segments of the renal tubule. Measurements from fasted and fed goldfish were used to demonstrate the effects of feeding on the acid–base transport physiology of the proximal, distal and connecting tubule. Furthermore, with the application of specific inhibitors, we were able to identify epithelial transporters involved in renal acid–base regulation by promoting and/or inhibiting H+ flux in the various renal tubule segments. We hypothesized that V-ATPase, NHE, HCO3 transporters (Na+/HCO3 co-transporter and/or HCO3/Cl-exchanger) and carbonic anhydrase (the enzyme converting CO2 to H+ and HCO3 and vice versa) contribute to the H+ flux through the renal epithelium. Furthermore, we hypothesized that (i) H+ flux would differ among the segments of the renal tubule and (ii) would be differentially affected by the inhibitors, as a result of the differential expression of transporters (at the mRNA and protein level) in each tubule segment, as shown in our previous study (Fehsenfeld and Wood, 2018).

List of symbols and abbreviations
     
  • AE

    anion exchanger (Cl/HCO3)

  •  
  • xi

    buffering capacity (SIET)

  •  
  • CA

    carbonic anhydrase

  •  
  • CB

    background ion concentration (μmol l–1) (SIET)

  •  
  • ΔC

    concentration gradient (mol cm−3) (SIET)

  •  
  • ΔV

    voltage gradient obtained from the software ASET2 (μV) (SIET)

  •  
  • Δx

    distance between the two measuring points of an electrode (cm) (SIET)

  •  
  • D

    diffusion coefficient (cm2 s−1) (SIET)

  •  
  • DIDS

    4,4'-diisothiocyanatostilbene-2,2'-disulfonate

  •  
  • ECF

    extracellular fluid

  •  
  • EIPA

    5-(N-ethyl-N-isopropyl)amiloride

  •  
  • V-ATPase

    V-type H+-ATPase

  •  
  • J

    flux (mol cm−2 s−1)

  •  
  • Ka

    acid dissociation constant (SIET)

  •  
  • NBC

    Na+/HCO3 co-transporter

  •  
  • NHE

    Na+/H+ exchanger

  •  
  • PNA+/−

    peanut lectin agglutinin cell

  •  
  • S

    slope of the SIET-electrode

  •  
  • SIET

    scanning ion-selective electrode technique

  •  
  • TAP

    2,4,6-triaminopyrimidine

MATERIALS AND METHODS

Animal care

Goldfish (∼5 cm, 2.5–3.0 g) were obtained from commercial suppliers and were held in 20 l aquaria at room temperature at a maximum density of 1 fish per 2 l for a minimum of 7 days before the experiments began. The recirculating and filtered tap water composition was (in mequiv l−1): 0.6 Na+, 0.8 Cl, 1.8 Ca2+, 0.3 Mg2+, 0.05 K+; titration alkalinity 2.1; hardness ∼140 mg l−1 as CaCO3 equivalents. Water parameters (pH 7.4, ammonia, nitrate and nitrite) were closely monitored and water changes were performed 2–3 times a week as necessary. Fish were fed to satiation 2–3 times a week with commercial flaked food (Nutrafin Max, Hagen, Montreal, QC, Canada). For experiments, fish were either fasted for 96 h (‘fasted’) or fasted for 93 h, then fed to satiation and used 3 h after feeding (‘fed’). Each animal was killed by cephalic concussion. All procedures were conducted under the approval of the University of British Columbia Animal Care Committee (licence no. A14-0251) and the guidelines of the Canadian Council on Animal Care.

Preparation of renal tubules for measurements

Immediately following the experimental procedures described above, kidneys were removed and transferred into control goldfish Ringer solution containing (in mmol l−1): 100.0 NaCl, 2.5 KCl, 1.5 CaCl2·2H2O, 1.0 MgCl2·6H2O, 5.0 NaHCO3, 0.7 NaH2PO4·H2O, 10 glucose, pH 7.3 (modified after Hoar and Hickman, 1975). First, larger clusters of tubules were pulled from the Wolffian duct as it runs through each half of the kidney. Subsequently, minuten pins glued to needles and screwed into syringes for better handling were used to carefully isolate single tubules including the glomerulus, proximal tubule, distal tubule and connecting tubule out of these clusters, as described in Fehsenfeld and Wood (2018). One intact tubule was transferred into the lid of a 5 cm Nunc cell culture dish and pinched down onto the dish at both ends using the needles. Lids, rather than the bottom part of the dishes, were used because tubules would stick to them due to apparent adhesive forces holding them in place that did not seem to be present in the bottom dish. The preparation was then transferred into the scanning ion-selective electrode technique (SIET) set-up and measurements were started immediately in control goldfish Ringer solution.

Measurement of renal H+ flux by SIET

SIET hardware was obtained from Applicable Electronics (New Haven, CT, USA) and Automated Scanning Electrode Technique version 2 (ASET2) software from Science Wares (Falmouth, MA, USA). Hardware, software and methodology for acquiring SIET data and calculating ion flux have been described in detail in previous publications (Donini and O'Donnell, 2005; Pacey and O'Donnell, 2014; D'Silva et al., 2017). Briefly, ion-selective microelectrodes were pulled to a ∼5 µm tip diameter using a programmable P-97 micropipette puller (Sutter Instruments, Novato, CA, USA). Silanized microelectrodes were back-filled with 100 mmol l−1 NaCl/100 mmol l−1 sodium citrate (pH 6.0). Electrodes were then tip-filled with ∼200 µm column length of H+ Ionophore I Cocktail B (Millipore Sigma, Burlington, MA, USA). Slopes of microelectrodes calibrated in pH 6.5 and pH 7.5 solutions were 59.5±0.4 mV per 10-fold change in H+ concentration. Voltage gradients obtained using SIET readings were converted into concentration gradients (ΔC) and subsequently into flux (J) using Fick's law by applying the following equations from Donini and O'Donnell (2005):
formula
(1)
formula
(2)
where ΔC is the concentration gradient in mol cm−3; CB is the background ion concentration (calculated as the average of the concentration at each point) in μmol l–1; ΔV is the voltage gradient obtained from the software ASET2 in μV; S is the slope of the electrode; J is the flux in mol cm−2 s−1; D is the diffusion coefficient in cm2 s−1; and Δx is the distance between the two points in cm. Buffering capacity (xi) for H2O, HCO3 and H2PO4, their diffusion coefficients (DB) (H2O, DB=9.31E−05/HCO3, DB=1.20E−05/H2PO4, DB=8.30E−06) and acid dissociation constants (Ka) were taken into account to adjust H+ flux values (JH+total) for the buffering capacity of the measuring media as follows (Messerli et al., 2006):
formula
(3)
formula
(4)

To determine control H+ flux in fasted and fed animals, renal tubules were scanned in control goldfish Ringer solution in 100 µm increments starting at the glomerulus and ending at the connecting tubule. To test for potential effects of time on tissue viability, the goldfish Ringer solution was renewed after the first scan, the tubules incubated for 10 min (to mimic addition of inhibitors as described below), and then rescanned at the same positions. For investigation of the effects of inhibitors on H+ flux, the tubules of fed goldfish were incubated with the respective inhibitor for 10 min after the control scan, and then rescanned at the same locations as for the control Ringer solution. Inhibitors and their concentrations (and targets) were: 10 mmol l−1 2,4,6-triaminopyrimidine (TAP; paracellular pathway), 100 µmol l−1 5-(N-ethyl-N-isopropyl)amiloride (EIPA; NHE), 100 µmol l−1 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; HCO3/Cl-exchanger and/or Na+/HCO3 co-transporter), 5 µmol l−1 bafilomycin (V-ATPase) and 1 µmol l−1 acetazolamide (carbonic anhydrase). There was no effect of the inhibitors at the indicated concentrations on the slope/responsiveness of the H+ microelectrodes.

Statistical analysis

Data are expressed as means±s.e.m. (N), where N represents the number of animals. All statistical analyses were performed with PAST3 (Hammer et al., 2001) or Minitab® Statistical Software (State College, PA, USA). Data were checked for normal distribution with the Shapiro–Wilks test and homogeneity of variances with the F-test. If data did not meet these criteria for parametric testing, they were log transformed.

To investigate the effect of feeding (Fig. 1) and/or time (Fig. 2) on tubular H+ flux, the mean H+ flux for each segment (as indicated by horizontal lines in Figs 1 and 2 for proximal, distal and connecting tubule) was calculated for fasted and fed animals, respectively. Data were then analyzed by two-way ANOVA using a general linear model as implemented in Minitab 18 (Fig. 1: factor 1=fasted versus fed, factor 2=tubule segment; Fig. 2: repeated measures two-way ANOVA, factor 1=1st run versus 2nd run, factor 2=tubule segment). Subsequently, pairwise comparisons were conducted using Tukey's post hoc test.

Fig. 1.

Proton flux along the nephron of fastedand fed goldfish (Carassius auratus). (A) Scanning ion micro-electrode measurements of H+ flux in renal proximal, distal and connecting tubules in fasted and fed goldfish. G, glomerulus. Lowercase letters indicate significant differences among mean flux rates for each tubule segment in fed animals, while asterisks denote significant differences in the mean flux in the respective tubule segment between fed and fasted animals (two-way ANOVA and Tukey's post hoc analysis, P<0.05). Data are represented as means±s.e.m. (N=5). (B) Representative visualization of calculated H+ flux in the nephron of a fed goldfish as depicted by ASET2 software (arrow length indicates magnitude, arrowheads indicate direction of the flux). H+ flux was directed from the tubule lumen towards the external medium. Because of the length of the tissue, the figure was generated by combining two screenshots; these have been overlaid as much as possible and the right image was then cropped to match the transition from the left (indicated by black line between the screenshots).

Fig. 1.

Proton flux along the nephron of fastedand fed goldfish (Carassius auratus). (A) Scanning ion micro-electrode measurements of H+ flux in renal proximal, distal and connecting tubules in fasted and fed goldfish. G, glomerulus. Lowercase letters indicate significant differences among mean flux rates for each tubule segment in fed animals, while asterisks denote significant differences in the mean flux in the respective tubule segment between fed and fasted animals (two-way ANOVA and Tukey's post hoc analysis, P<0.05). Data are represented as means±s.e.m. (N=5). (B) Representative visualization of calculated H+ flux in the nephron of a fed goldfish as depicted by ASET2 software (arrow length indicates magnitude, arrowheads indicate direction of the flux). H+ flux was directed from the tubule lumen towards the external medium. Because of the length of the tissue, the figure was generated by combining two screenshots; these have been overlaid as much as possible and the right image was then cropped to match the transition from the left (indicated by black line between the screenshots).

Fig. 2.

Effect of time on H+ flux along the renal tubules of fed goldfish. Data were obtained by re-scanning the same tubule to exclude changes in tissue viability for inhibitor experiments. After a first scan in 100 µm increments, Ringer solution was exchanged and left for 10 min before a second scan of the same tubule was performed. Letters indicate significant differences among mean flux rates for each tubule segment in fed animals (lowercase, first run; uppercase, second run); no difference was found between the first and second run (repeated measures two-way ANOVA with Tukey's post hoc analysis, N=3). Data are represented as means±s.e.m.

Fig. 2.

Effect of time on H+ flux along the renal tubules of fed goldfish. Data were obtained by re-scanning the same tubule to exclude changes in tissue viability for inhibitor experiments. After a first scan in 100 µm increments, Ringer solution was exchanged and left for 10 min before a second scan of the same tubule was performed. Letters indicate significant differences among mean flux rates for each tubule segment in fed animals (lowercase, first run; uppercase, second run); no difference was found between the first and second run (repeated measures two-way ANOVA with Tukey's post hoc analysis, N=3). Data are represented as means±s.e.m.

For the inhibitor experiments (Figs 36), Student's paired t-tests were applied for each inhibitor in each section. Data were considered significantly different at P≤0.05. All graphs were generated with Inkscape 0.92.0 r15299 (https://www.inkscape.org).

Fig. 3.

Effects of inhibition of the paracellular pathway on renal tubule H+ back-flux in preparations from fed goldfish. Data are represented as means±s.e.m. Asterisks denote significant differences between H+ flux with control saline and the application of 10 mmol l−1 2,4,6-triaminopyrimidine (TAP; Student's paired t-test with P<0.05 and N=4).

Fig. 3.

Effects of inhibition of the paracellular pathway on renal tubule H+ back-flux in preparations from fed goldfish. Data are represented as means±s.e.m. Asterisks denote significant differences between H+ flux with control saline and the application of 10 mmol l−1 2,4,6-triaminopyrimidine (TAP; Student's paired t-test with P<0.05 and N=4).

Fig. 4.

Effects of inhibitors of acid–base relevant epithelial transporters on H+ back-flux in the proximal tubule in preparations from fed goldfish. H+ flux was directed from the lumen into the extracellular fluid and decreased with the application of all inhibitors: 100 µmol l−1 EIPA for Na+/H+ exchanger (NHE), 100 µmol l−1 DIDS for HCO3 transporters, 5 µmol l−1 bafilomycin for V-type H+-ATPase (V-ATPase) and 1 µmol l−1 acetazolamide for carbonic anhydrase. Asterisks denote significant differences between H+ back-flux with control saline and following application of inhibitor for the respective transporters (Student's paired t-test with P<0.05 and N=6–8). Data are represented as means±s.e.m.

Fig. 4.

Effects of inhibitors of acid–base relevant epithelial transporters on H+ back-flux in the proximal tubule in preparations from fed goldfish. H+ flux was directed from the lumen into the extracellular fluid and decreased with the application of all inhibitors: 100 µmol l−1 EIPA for Na+/H+ exchanger (NHE), 100 µmol l−1 DIDS for HCO3 transporters, 5 µmol l−1 bafilomycin for V-type H+-ATPase (V-ATPase) and 1 µmol l−1 acetazolamide for carbonic anhydrase. Asterisks denote significant differences between H+ back-flux with control saline and following application of inhibitor for the respective transporters (Student's paired t-test with P<0.05 and N=6–8). Data are represented as means±s.e.m.

Fig. 5.

Effects of inhibitors of acid–base relevant epithelial transporters on H+ back-flux in the distal tubule in preparations from fed goldfish. H+ flux was directed from the lumen into the extracellular fluid and decreased with the application of most inhibitors: 100 µmol l−1 EIPA for NHE; 100 µmol l−1 DIDS for HCO3 transporters; 5 µmol l−1 bafilomycin for V-ATPase and 1 µmol l−1 acetazolamide for carbonic anhydrase. Asterisks denote significant differences between H+ back-flux with control saline and following application of inhibitor for the respective transporters (Student's paired t-test with P<0.05 and N=6–8). Data are represented as means±s.e.m.

Fig. 5.

Effects of inhibitors of acid–base relevant epithelial transporters on H+ back-flux in the distal tubule in preparations from fed goldfish. H+ flux was directed from the lumen into the extracellular fluid and decreased with the application of most inhibitors: 100 µmol l−1 EIPA for NHE; 100 µmol l−1 DIDS for HCO3 transporters; 5 µmol l−1 bafilomycin for V-ATPase and 1 µmol l−1 acetazolamide for carbonic anhydrase. Asterisks denote significant differences between H+ back-flux with control saline and following application of inhibitor for the respective transporters (Student's paired t-test with P<0.05 and N=6–8). Data are represented as means±s.e.m.

Fig. 6.

Effects of inhibitors of acid–base relevant epithelial transporters on H+ back-flux in the connecting tubule in preparations from fed goldfish. H+ flux was directed from the lumen into the extracellular fluid and decreased with the application of most inhibitors: 100 µmol l−1 EIPA for NHE, 100 µmol l−1 DIDS for HCO3 transporters, 5 µmol l−1 bafilomycin for V-ATPase and 1 µmol l−1 acetazolamide for carbonic anhydrase. Asterisks denote significant differences between H+ back-flux with control saline and following application of inhibitor for the respective transporters (Student's paired t-test with P<0.05 and N=6–8). Data are represented as means±s.e.m.

Fig. 6.

Effects of inhibitors of acid–base relevant epithelial transporters on H+ back-flux in the connecting tubule in preparations from fed goldfish. H+ flux was directed from the lumen into the extracellular fluid and decreased with the application of most inhibitors: 100 µmol l−1 EIPA for NHE, 100 µmol l−1 DIDS for HCO3 transporters, 5 µmol l−1 bafilomycin for V-ATPase and 1 µmol l−1 acetazolamide for carbonic anhydrase. Asterisks denote significant differences between H+ back-flux with control saline and following application of inhibitor for the respective transporters (Student's paired t-test with P<0.05 and N=6–8). Data are represented as means±s.e.m.

RESULTS

In fasted goldfish, H+ efflux was observed from the tubule lumen and/or the epithelial cells over the basolateral epithelium to the external solution (Fig. 1B). Mean H+ flux rates did not differ significantly between the different segments of fasted animals (Fig. 1A). Two-way ANOVA revealed a significant effect of both tubule segment and feeding status on H+ flux, but not their interaction. Tukey's post hoc analysis identified H+ flux in the distal tubule of fed animals to be significantly lower (−57%) than that in the proximal and connecting tubule of fed animals. Mean H+ flux increased significantly in fed animals compared with fasted animals in all three tubule segments (Tukey's post hoc analysis), and the difference was most pronounced in the proximal tubule (3.5-fold versus 2.4- and 2.2-fold increases in the distal and connecting tubule, respectively) (Fig. 1).

H+ flux during two consecutive runs with control saline in tubules of fed animals did not decrease significantly over time (Fig. 2; repeated measures two-way ANOVA). There was, however, a significant effect of tubule segment according to findings in fed tubules as mentioned above, with the distal tubule having significantly lower H+ flux compared with both the proximal and connecting tubule (Tukey's post hoc analysis). There was no significant interaction of time and tubule segment (P=0.7).

Consequently, tubules from fed animals were used in testing the effects of inhibitors. The application of 2,4,6-triaminopyrimidine (TAP; 10 mmol l−1) (Fig. 3) to inhibit the paracellular pathway resulted in a dramatic ∼90% decrease of H+ flux in all three segments. Targeting epithelial transporters and enzymes with specific inhibitors differentially affected H+ flux along the tubule. Fig. 4 shows results for the proximal tubule, Fig. 5 shows results for the distal tubule and Fig. 6 shows those for the connecting tubule. Inhibiting NHE with EIPA decreased H+ flux by ∼55% in the proximal and connecting tubule, but did not affect H+ flux in the distal tubule. Applying DIDS to inhibit HCO3 transporters resulted in a decrease of H+ flux by 70% in the proximal tubule, and approximately 50% in the distal and connecting tubule. When V-ATPase was blocked by bafilomycin, H+ flux decreased by 67% in the proximal tubule and 45% in the distal tubule, while no effect was observed in the connecting tubule. Targeting carbonic anhydrase with acetazolamide decreased H+ flux by roughly 60% in all three segments.

DISCUSSION

General observations

Goldfish, C. auratus, produce an alkaline urine (pH 7.8–8.5) under fasted conditions (Lawrence et al., 2015; Fehsenfeld and Wood, 2018). This indicates renal reabsorption of H+ from the tubule lumen into the extracellular fluid/plasma and, indeed, this is what we measured in the present study by applying SIET to the proximal, distal and connecting tubule (Fig. 1). In response to the acidic tide caused by feeding, however, urine pH was observed to drop by up to 0.6 units (Fehsenfeld and Wood, 2018); under these conditions the kidney reversed this flux to net H+ secretion. Surprisingly, the results here appear to show the opposite effect, suggesting a pronounced increase in H+ reabsorption in response to feeding that was highest in the proximal tubule (3.5-fold versus ∼2.3-fold in distal and connecting tubule with reference to fasted conditions; Fig. 1). When the paracellular pathway was blocked with TAP, however, the observed H+ reabsorption was reduced to 10% of the control values for fed animals in all segments (Fig. 3). The teleost kidney has been reported to express a multitude of tight junction proteins: cytosolic scaffolding proteins cingulin and ZO-1 (Kolosov et al., 2014), as well as transmembrane proteins like occludin (Chasiotis and Kelly, 2008; Kolosov et al., 2017) and over 35 claudins (Kolosov et al., 2013), and the tricellular proteins ILDR2 and tricellulin (Kolosov and Kelly, 2013, 2018; Kolosov et al., 2017). Despite the fact that many tight junction proteins have been detected in the kidney, their individual role and distribution in the different regions of the fish nephron are unknown. Chasiotis and Kelly, however, showed that occludin was expressed in the distal and not the proximal tubule of goldfish (Chasiotis and Kelly, 2008) and the African clawed frog Xenopus laevis (Chasiotis and Kelly, 2009). Accordingly, rather than indicating a direct reabsorption of protons, the observed H+ flux probably represents H+ back-flux via the paracellular pathway down the concentration gradient established by active H+ secretion into the small closed tubular lumen.

We are confident that the tissue was intact and that this H+ back-flux was not due to tissue deterioration, because we took particular care to avoid touching the tubules themselves during the microdissection (i.e. they were not pulled on or squeezed). Rather, needles were mainly used to carefully tease apart the connective tissue. Additionally, we carefully inspected every tubule visually during and after the microdissection for any potential rips. Measurements were highly reproducible, and tubules of fasted animals generally had very low H+ flux.

As it was not possible to directly measure apical H+ flux, the observed H+ back-flux through the tight junction can be interpreted as a proxy for apical H+ secretion. Consequently, a proton gradient directed from the lumen to the plasma, resulting in the observed H+ back-flux, can be explained by the following: (1) active apical ion transport resulting in higher [H+] in the tubular lumen compared with extracellular [H+] and/or (2) ‘trapped’ urine of fed animals having increased [H+] in the lumen (pH 7.2) compared with the extracellular fluid (pH 7.85 of fasted animals; Fehsenfeld and Wood, 2018). Differences in the magnitude of H+ back-flux in the different segments of the tubule isolated from fed animals (Fig. 1A) can be attributed to a combination of regional alterations in (i) paracellular permeability, i.e. the tight junctions, and (ii) active ion transporter (epithelial channels and pumps) expression. In the intact kidney of fed freshwater fishes, such back-flux of H+ through the paracellular cleft would be minimized by continuous production and flow of copious amounts of dilute urine that would probably increase in the face of a systemic acidosis (Lawrence et al., 2015).

Effects of ion transporter inhibitors targeting epithelial transporters on proton flux

While the recent study by Fehsenfeld and Wood (2018) established a general molecular basis for renal acid–base regulation in different tubule segments of the goldfish kidney, the SIET measurements of the present study allowed us to establish real-time H+ flux data. Furthermore, by application of specific inhibitors, our results allow us to compare the observed H+ flux to the presence of epithelial transport proteins and further characterize their role in renal acid–base balance in the different tubule segments. Note that we elected to test the potential inhibitors only on preparations from fed animals, because H+ flux rates were low in fasted animals, so we cannot eliminate the possibility that different response patterns would have been seen in the latter. Here, we suggest models for general mechanisms of transport of acid–base equivalents (i.e. H+, HCO3 and NH4+) in the proximal (Fig. 7), distal (Fig. 8) and connecting tubule (Fig. 9). No inhibitors are currently available for Rhesus proteins, the major transporters for NH3/NH4+. They are, however, likely to play a role in acid–base regulation, so we have incorporated them into our proposed models where appropriate. The models take into account inhibitor results from the present study, protein and mRNA expression data from Fehsenfeld and Wood (2018), as well as aspects from the mammalian models for renal acid–base (Hamm et al., 2013) and ammonia transport (Weiner and Verlander, 2013, 2017). While depicted here as one cell, it is possible that certain processes are instead associated with multiple, rather specific cell types as in the mammalian system.

Fig. 7.

Proposed model for renal flux of H+ and otheracid–base equivalents in the proximal tubule of the goldfish. Solid lines represent primary and secondary active ion transport, dashed lines are indicative of channels that allow bi-directional flow, and wavy lines (see Figs 8 and 9) represent vesicular transport. The negative electric potential of the cell towards both the plasma/extracellular fluid (ECF) and the urine/lumen is indicated by the oval containing a minus sign. Major contributors to acid–base regulation are indicated by bold lines and text. We propose the proximal tubule to mainly contribute to HCO3 reabsorption into the extracellular fluid, as well as H+ secretion into the luminal space, comparable to what has been observed in the mammalian kidney. H+ is suggested to mainly be generated by cytoplasmic carbonic anhydrase (CAII-like) and secreted by apical Na+/H+ exchanger (NHE3). Apical Rhesus proteins (Rhcg1b/Rhcg2) potentially titrate urine H+ and trap it in the form of NH4+ in the lumen. Apical Na+ entry via NHE3 is energized by the basolateral Na+/K+-ATPase. H+ is also partly reabsorbed into the plasma via basolateral V-ATPase (in contrast to apical V-ATPase in mammals) and might help to titrate plasma NH3 reabsorbed via Rhesus protein Rhbg to ‘trap’ NH4+. The major source of HCO3 in this tubule region is probably ammoniagenesis (AMM) as observed in mammals. HCO3 is reabsorbed into the extracellular fluid via Na+/HCO3 co-transport (probably electrogenic 1:3 by NBC1). The processes depicted here indicate a proximal tubule cell in goldfish to resemble an intermediate between mammalian proximal tubule cells and B-intercalated cells of the collecting duct. ap, apical; bl, basolateral.

Fig. 7.

Proposed model for renal flux of H+ and otheracid–base equivalents in the proximal tubule of the goldfish. Solid lines represent primary and secondary active ion transport, dashed lines are indicative of channels that allow bi-directional flow, and wavy lines (see Figs 8 and 9) represent vesicular transport. The negative electric potential of the cell towards both the plasma/extracellular fluid (ECF) and the urine/lumen is indicated by the oval containing a minus sign. Major contributors to acid–base regulation are indicated by bold lines and text. We propose the proximal tubule to mainly contribute to HCO3 reabsorption into the extracellular fluid, as well as H+ secretion into the luminal space, comparable to what has been observed in the mammalian kidney. H+ is suggested to mainly be generated by cytoplasmic carbonic anhydrase (CAII-like) and secreted by apical Na+/H+ exchanger (NHE3). Apical Rhesus proteins (Rhcg1b/Rhcg2) potentially titrate urine H+ and trap it in the form of NH4+ in the lumen. Apical Na+ entry via NHE3 is energized by the basolateral Na+/K+-ATPase. H+ is also partly reabsorbed into the plasma via basolateral V-ATPase (in contrast to apical V-ATPase in mammals) and might help to titrate plasma NH3 reabsorbed via Rhesus protein Rhbg to ‘trap’ NH4+. The major source of HCO3 in this tubule region is probably ammoniagenesis (AMM) as observed in mammals. HCO3 is reabsorbed into the extracellular fluid via Na+/HCO3 co-transport (probably electrogenic 1:3 by NBC1). The processes depicted here indicate a proximal tubule cell in goldfish to resemble an intermediate between mammalian proximal tubule cells and B-intercalated cells of the collecting duct. ap, apical; bl, basolateral.

Fig. 8.

Proposed model for renal flux of H+ and other acid–base equivalents in the distal tubule of the goldfish. Please see Fig. 7 for formatting details and sources. The distal tubule may contribute substantially to NH3/NH4+ secretion into the luminal space, as well as to HCO3 reabsorption into the extracellular fluid. In contrast to mammals, it may play only a minor role in direct H+ secretion into the lumen. As in the proximal tubule, cytoplasmic carbonic anhydrase (CAII-like) may play the major role in the generation of H+. H+ and HCO3 are reabsorbed basolaterally by V-ATPase and Na+/HCO3 co-transport (probably electrogenic 1:3 by NBC1). NBC1-mediated reabsorption of HCO3 into the plasma would be driven by the negative membrane potential generated by basolateral Na+/K+-ATPase. NH3/NH4+ secretion via apical Rhcg1a and/or Rhcg1b, and basolateral Rhcg1a may be supported by exocytosis of acidified vesicles as proposed for branchial epithelia in green crabs (Carcinus maenas; Fehsenfeld and Weihrauch, 2016) and rats experiencing a metabolic acidosis (Seshadri et al., 2006).

Fig. 8.

Proposed model for renal flux of H+ and other acid–base equivalents in the distal tubule of the goldfish. Please see Fig. 7 for formatting details and sources. The distal tubule may contribute substantially to NH3/NH4+ secretion into the luminal space, as well as to HCO3 reabsorption into the extracellular fluid. In contrast to mammals, it may play only a minor role in direct H+ secretion into the lumen. As in the proximal tubule, cytoplasmic carbonic anhydrase (CAII-like) may play the major role in the generation of H+. H+ and HCO3 are reabsorbed basolaterally by V-ATPase and Na+/HCO3 co-transport (probably electrogenic 1:3 by NBC1). NBC1-mediated reabsorption of HCO3 into the plasma would be driven by the negative membrane potential generated by basolateral Na+/K+-ATPase. NH3/NH4+ secretion via apical Rhcg1a and/or Rhcg1b, and basolateral Rhcg1a may be supported by exocytosis of acidified vesicles as proposed for branchial epithelia in green crabs (Carcinus maenas; Fehsenfeld and Weihrauch, 2016) and rats experiencing a metabolic acidosis (Seshadri et al., 2006).

Fig. 9.

Proposed model for renal flux of H+ and other acid–base equivalents in the connecting tubule of the goldfish. Please see Fig. 7 for formatting details and sources. Like the distal tubule, the connecting tubule is proposed to contribute mostly to NH3/NH4+ secretion into the luminal space, as well as HCO3 reabsorption into the extracellular fluid. In contrast to the distal tubule, however, (i) basolateral exit of H+ into the extracellular fluid is probably promoted by basolateral NHE and not basolateral V-ATPase, and (ii) NH3/NH4+ secretion might be accomplished instead via basolateral Rhesus protein Rhbg and apical Rhcg1b with less involvement of membrane-bound Rhcg1a. The presence of basolateral NHE and Na+/HCO3 co-transporter (probably electrogenic 1:3 NBC1) suggests the connecting tubule is composed of cells comparable to mammalian principal cells of the connecting segment/collecting duct of the kidney (Chaillet and Lopez, 1985; Wang and Kurtz, 1990; Weiner and Hamm, 1990).

Fig. 9.

Proposed model for renal flux of H+ and other acid–base equivalents in the connecting tubule of the goldfish. Please see Fig. 7 for formatting details and sources. Like the distal tubule, the connecting tubule is proposed to contribute mostly to NH3/NH4+ secretion into the luminal space, as well as HCO3 reabsorption into the extracellular fluid. In contrast to the distal tubule, however, (i) basolateral exit of H+ into the extracellular fluid is probably promoted by basolateral NHE and not basolateral V-ATPase, and (ii) NH3/NH4+ secretion might be accomplished instead via basolateral Rhesus protein Rhbg and apical Rhcg1b with less involvement of membrane-bound Rhcg1a. The presence of basolateral NHE and Na+/HCO3 co-transporter (probably electrogenic 1:3 NBC1) suggests the connecting tubule is composed of cells comparable to mammalian principal cells of the connecting segment/collecting duct of the kidney (Chaillet and Lopez, 1985; Wang and Kurtz, 1990; Weiner and Hamm, 1990).

NHE

NHE is generally believed to promote apical H+ secretion (and Na+ absorption) in the vertebrate kidney and specifically in the proximal tubule as the major site for renal acid excretion (Gilmour and Perry, 2009; Hamm et al., 2013; Ivanis et al. 2008; Teranishi et al. 2013). Furthermore, protein abundance of NHE (isoform 3) in the cortical brush-border apical membrane of the proximal tubule was reported to increase following chronic metabolic acidosis in rats (Ambühl et al., 1996). Likewise, in goldfish renal tubules, NHE3 protein was located in the basolateral membrane of all tubule segments of fasted goldfish, but increased in abundance and relocated to the apical membrane in the proximal tubule when animals experienced a systemic acid load as a result of feeding (Fehsenfeld and Wood, 2018). This indicates a similar mechanism in the goldfish kidney to that in the mammalian kidney.

Blockade of NHE(s) with the EIPA in the present study resulted in a decrease of H+ flux in the goldfish renal tubule, verifying its role in renal acid–base balance. Surprisingly, however, this was only observed in the proximal (Fig. 4) and connecting tubule (Fig. 6) of fed goldfish, and not in the distal segment (Fig. 5). It has to be kept in mind that the inhibitor may have acted not only on basolateral but also on apical NHE3 because of its permeability through the tight junctions (Van Itallie et al., 2008; Hou et al., 2013). Consequently, the decrease of H+ flux in the proximal tubule when inhibited by EIPA is in line with NHE-mediated apical proton secretion. Alternatively or additionally, EIPA may have blocked a different isoform of NHE, for example NHE1 that appears to be expressed in the basolateral membrane of the proximal tubules in mammals (Hamm et al., 2013; Bobulescu et al., 2005). NHE may be present in the distal segment of goldfish renal tubule, but in accordance with our measurements is probably not involved in acid–base regulation in this region, as has also been suggested for the mammalian distal tubule (Hamm et al., 2013).

HCO3 transporters

An electrogenic Na+/HCO3 co-transporter (NBCe1A/NBC1) in basolateral membranes of the proximal tubule is crucial for the reabsorption of base in mammals (Hamm et al., 2013; Weiner and Verlander, 2013; Handlogten et al., 2015). NBC1 has also been identified in the trout kidney and was proposed to mediate increased basolateral reabsorption of HCO3 in response to a respiratory acidosis caused by hypercarbia (Perry et al., 2003a). Two anion exchangers have been identified in mammalian renal cells: AE1 (SLC4A1) is present in the basolateral membrane of A-type intercalated cells in the collecting duct, whereas pendrin (SLC26A4) is found in the apical membrane of the proximal tubule and B-type intercalated cells of the collecting duct (Hamm et al., 2013; Soleimani et al., 2001). Members of the SLC26 family of membrane transporters including pendrin have also been identified in zebrafish gills (Bayaa et al., 2009) and in V-ATPase-rich cells in the elasmobranch branchial epithelium (Piermarini and Evans, 2001; Tresguerres et al., 2005; Reilly et al., 2011). In response to a metabolic alkalosis (feeding-induced alkaline tide), elasmobranch gills translocated pendrin into the apical membrane with the help of cytoplasmic carbonic anhydrase (Tresguerres et al., 2007; Roa et al., 2014). NBC1 and AE1, in contrast, were expressed basolaterally not only in the proximal but also in the distal and connecting tubule in goldfish (Fehsenfeld and Wood, 2018).

Unfortunately, no specific inhibitor can distinguish between NBC(s), AE(s) and pendrin (Soleimani et al., 2001; Bayaa et al., 2009). The application of DIDS, as chosen here to block HCO3 transporters in general, resulted in a decrease of proton flux in all three segments but was most pronounced in the proximal segment (70% inhibition of H+ flu, versus 50% in distal and connecting tubules; Figs 4, 5 and 6). With basolateral AE1 protein abundance being decreased in all three segments upon feeding (and with the most pronounced decrease in the proximal tubule; Fehsenfeld and Wood, 2018), the effect observed with the application of the inhibitor DIDS basolaterally might mostly be due to targeting NBC1. Blocking NBC1-promoted reabsorption of HCO3 into the extracellular fluid would result in an increase of extracellular H+ and therefore diminish the H+ gradient and the H+ back-flux as observed here. This finding emphasizes the importance of renal reabsorption of HCO3 for acid–base balance during metabolic acidosis, especially in the proximal tubule.

V-ATPase

Apical V-ATPase is generally believed to promote H+ secretion in branchial acid-secreting cells such as peanut lectin agglutinin negative (PNA) mitochondria-rich cells of the trout gill epithelium and in proton pump-rich cells of zebrafish gills (Gilmour and Perry, 2009). In contrast, branchial base-secreting cells seem to possess a basolateral V-ATPase as proposed for trout PNA positive (PNA+) mitochondria-rich cells (Gilmour and Perry, 2009) and the branchial epithelium of stingrays (Piermarini and Evans, 2001). A similar distinction between cells can be made in the vertebrate kidney, with apical V-ATPase in A-type intercalated cells (PNA; co-expressed with basolateral anion exchanger AE1) of the distal tubule, connecting segment and the collecting duct of the kidney cortex functioning as acid-secreting cells, whereas V-ATPase is more ubiquitously expressed in B-type intercalated cells (PNA+; co-expressed with apical pendrin) and is often localized in the basolateral membrane (Brown and Breton, 2000; Brown et al., 2009). In goldfish tubules, V-ATPase appeared to localize exclusively to the basolateral membrane of all three investigated segments of this study (Fehsenfeld and Wood, 2018). A basolateral V-ATPase could indeed be important for the alkalinization of the urine under control conditions (Lawrence et al., 2015).

Bafilomycin decreased H+ flux in the proximal (Fig. 4) and distal tubule (Fig. 5), confirming a role for V-ATPase in acid–base regulation of these tubule regions. With a basolateral V-ATPase presumably pumping H+ into the plasma, inhibiting this pathway would accumulate intracellular protons and result in a shift of the equilibrium for the intracellular carbonate system. Consequently, the generation of intracellular CO2 would increase while diminishing both intracellular H+ and HCO3. The observed decrease in net H+ flux upon blocking V-ATPase could then be explained by the general decrease of intracellular protons available for basolateral, and possibly apical, transport, while also less HCO3 could be reabsorbed basolaterally via Na+/HCO3 co-transporter.

Carbonic anhydrase

Carbonic anhydrase is crucial for acid–base balance in virtually all systems (Gilmour and Perry, 2009). In addition to the predominant cytosolic isoform of the enzyme (CAII-like/CAc) (Esbaugh et al., 2005; Gilmour and Perry, 2009), the kidney of freshwater teleosts expresses at least one membrane-bound carbonic anhydrase (CAIV) (Georgalis et al., 2006). CAIV is located apically and available to the tubular lumen in both mammals (Hamm et al., 2013; Schwartz et al., 2000) and fishes (Perry et al., 2003b; Georgalis et al., 2006), where it is proposed to help titrate filtered HCO3 with secreted H+, thereby effecting HCO3 reabsorption from the urine. Intracellularly, carbonic anhydrase converts intracellular CO2 into HCO3 and H+, which is then returned to the extracellular fluid or secreted into the urine, respectively.

Blocking carbonic anhydrase in the present study decreased H+ flux by 60% in all three investigated segments, indicating that the conversion of CO2 contributes considerably to the secretion and/or reabsorption of acid–base equivalents, respectively. The inhibitor acetazolamide, however, is membrane permeable and does not distinguish between different isoforms of the enzyme. Consequently, the present results confirm that carbonic anhydrase is critical for acid secretion and/or base reabsorption in all three parts of the goldfish tubule but do not reveal the site of action.

Acknowledgements

We thank Ryan Belowitz for help with fish maintenance. The authors would like to thank Dr Scott P. Kelly (York University, Toronto, ON, Canada) for donation of 2,4,6-triaminopyrimidine.

Footnotes

Author contributions

Conceptualization: S.F., D.K., C.M.W., M.J.O.; Formal analysis: S.F., D.K., M.J.O.; Investigation: S.F., D.K.; Writing - original draft: S.F.; Writing - review & editing: S.F., D.K., C.M.W., M.J.O.; Visualization: S.F.; Supervision: M.J.O.; Funding acquisition: C.M.W., M.J.O.

Funding

Financial support was provided by Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to M.J.O. (NSERC PIN-2015-14139) and C.M.W. (NSERC PIN-2017-03843).

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Competing interests

The authors declare no competing or financial interests.