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Increased Endothelin Activity Mediates Augmented Distal Nephron Acidification Induced by Dietary Protein

Apurv Khanna^*, Jan Simoni, Callenda Hacker^*, Marie-Josée Duran and Donald E. Wesson^*,

Departments of *Internal Medicine, Surgery, and Physiology, Texas Tech University Health Sciences Center, Texas Tech University School of Medicine, Lubbock, Texas

Correspondence to Dr. Donald E. Wesson, Texas Tech University Health Sciences Center, Renal Section, 3601 Fourth Street, Lubbock, TX 79430. Phone: 806-743-3107; Fax: 806-743-3177; E-mail: donald.wesson{at}ttuhsc.edu

	Abstract

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ABSTRACT. The hypothesis that increased dietary protein augments distal nephron acidification and does so through an endothelin (ET-1)-dependent mechanism was tested. Munich-Wistar rats that ate minimum electrolyte diets of 50% (HiPro) and 20% (CON) casein-provided protein, the latter comparable to standard diet, were compared. HiPro versus CON had higher distal nephron net HCO₃ reabsorption by in vivo microperfusion (37.8 ± 3.2 versus 16.6 ± 1.5 pmol/mm per min; P < 0.001) as a result of higher H⁺ secretion (41.3 ± 4.0 versus 23.0 ± 2.1 pmol/mm per min; P < 0.002) and lower HCO₃ secretion (–3.5 ± 0.4 versus –6.4 ± 0.8 pmol/mm per min; P < 0.001). Perfusion with H⁺ inhibitors support that increased H⁺ secretion was mediated by augmented Na⁺/H⁺ exchange and H⁺-ATPase activity without augmented H⁺,K⁺-ATPase activity. HiPro versus CON had higher levels of urine ET-1 excretion, renal cortical ET-1 addition to microdialysate in vivo, and renal cortical ET-1 mRNA, consistent with increased renal ET-1 production. Oral bosentan, an ET A/B receptor antagonist, decreased distal nephron net HCO₃ reabsorption (22.4 ± 1.9 versus 37.8 ± 3.2 pmol/mm per min; P < 0.001) as a result of lower H⁺ secretion (28.4 ± 2.4 versus 41.3 ± 4.0 pmol/mm per min; P < 0.016) and higher HCO₃ secretion (–6.0 ± 0.7 versus –3.5 ± 0.4 pmol/mm per min; P < 0.006). The H⁺ inhibitors had no additional effect in HiPro ingesting bosentan, supporting that ET mediated the increased distal nephron Na⁺/H⁺ exchange and H⁺-ATPase activity in HiPro. Increased dietary protein augments distal nephron acidification that is mediated through an ET-sensitive increase in Na⁺/H⁺ exchange and H⁺-ATPase activity.

	Introduction

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The routine acid challenges to systemic acid-base status faced by humans are modest compared with the large acid loads administered to animals in most experimental protocols. Augmented distal rather than proximal nephron acidification is the predominant renal regulatory response in experimental animals to modest dietary acid loads induced by acid-producing mineral salts (1,2). Augmented distal nephron acidification induced by dietary acid is mediated by multiple mechanisms, including (1) increased net HCO₃ reabsorption (3), consistent with increased H⁺ secretion; (2) reduced HCO₃ delivery to the terminal distal nephron (4) that facilitates NH₄⁺ secretion (5) and permits secreted H⁺ to effect acid excretion rather than HCO₃ reclamation; and (3) decreased distal nephron HCO₃ secretion (1) mediated by endogenous endothelins (ET) (2).

In contrast with the acid-producing mineral salts that are most commonly used to induce an acid challenge in experimental protocols, increased intake of dietary protein that contains acid-producing amino acids constitutes the acid challenge that humans more routinely face. Intake of acid-producing amino acids increases systemic acid production and urine net acid excretion (6), but its effect on distal nephron acidification or its hormonal and/or transport mediators are not known. Recognizing that ET mediate increased distal nephron acidification induced by modest dietary acid loads as a result of intake of acid-producing mineral salts (1,2), the present studies tested the hypothesis that increased intake of acid-producing amino acids as dietary protein increases distal nephron acidification and that this increased acidification is mediated by enhanced ET activity.

	Materials and Methods

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Animals and Diet Protocol
Male and female Munich-Wistar rats (Harlan Sprague-Dawley, Houston, TX) that weighed 200 to 220 g ate standard rat chow (Prolab RMH 2500 with 23% protein) for 1 wk (week 0), then ate a custom minimum electrolyte diet with protein as purified high nitrogen casein (ICN Nutritional Biochemicals, Cleveland, OH) for 3 wk (weeks 1, 2, and 3). High-protein rats (HiPro) ate custom diet with 50% protein, and controls (CON) ate 20%. In preliminary studies, similar-weight rats ate 24.6 ± 0.9 and 27.1 ± 1.2 g/d, respectively (n = 4, P = 0.15), and so all rats received 24 g/d diet to ensure similar diet intake. Some animals received bosentan (Hoffman-LaRoche, Basel, Switzerland), a nonpeptide ET A/B receptor antagonist (7), mixed with study diet at 100 mg/kg body wt per d and so was completely ingested. This oral dose blocks action of pressor doses of intravenous big ET-1 for >24 h (7). All drank distilled H₂O except for a separate CON group that was given 4.0% dextrose drinking solution to approximate the increased urine output associated with the HiPro diet. Animals drank ad libitum.

Urine Net Acid and ET-1 Excretion
We measured daily excretion of urine net acid (NAE) (8) and ET-1 (9) in a 24-h sample collected on days 7 (week 0), 14 (week 1), 21 (week 2), and 28 (week 3) of the protocol from eight each of HiPro and CON in metabolic cages. We examined the effect of ET receptor blockade with bosentan on urine NAE in paired and separate groups of eight each (four with and four without drug) of HiPro and CON. NAE was the mean for each animal group.

Arterial Blood Parameters
We measured pH, PCO₂, calculated [HCO₃] (IRMA Blood Analysis System, Diametrics Medical, St. Paul, MN), and total CO₂ (TCO₂) by ultrafluorometry (see below) in 1.0 ml of blood from a chronic carotid arterial catheter in eight each of awake, gently restrained, and calm HiPro and CON at weeks 1 and 3 to assess the effects of HiPro on plasma acid-base parameters. We also measured arterial BP through this chronic arterial catheter as done previously (9).

Microdialysis Technique for Measurement of Renal Cortical Fluid ET-1
Renal cortical fluid ET-1 addition was measured using microdialysis of the renal cortex as done in our laboratory (10) at weeks 0, 1, 2, and 3. Three consecutive 20-min collection periods were done in four each of HiPro, CON, and CON + 4.0% dextrose animals for microdialysate ET-1 measurements.

Micropuncture Protocol
Animals were prepared for micropuncture of accessible distal tubules (9) at weeks 1 and 3. In situ early distal flow rate for HiPro and CON was 9.4 ± 0.7 (n = 6) and 6.4 ± 0.4 nl/min (n = 8), respectively. Separate superficial distal nephrons of HiPro and CON were each perfused 9 and 6 nl/min with a Hampel pump to approximate respective in situ flow rates. We measured distal tubule transepithelial potential difference to calculate blood-to-lumen HCO₃ permeability (9). After weighing the micropunctured (left) kidney, perfused nephron length was determined by measuring the length of a latex cast injected after micropuncture, recovered after acid digestion of the kidney (9). We measured [HCO₃] in stellate vessel plasma to determine peritubular blood-to-lumen HCO₃ gradient for calculating transepithelial H⁺/HCO₃ passive permeability (9). Diet but not H₂O was withheld the evening before micropuncture to yield higher baseline HCO₃ reabsorption (11), as done previously (9).

The perfusion solutions used are in Table 1. Solution 1 contained 5 mM HCO₃ and 40 mM Cl^– to approximate their concentrations at the early distal nephron in situ (12). Solution 2 contained Cl^– but no HCO₃ to measure Cl^–-dependent luminal HCO₃ accumulation and to calculate an "apparent" blood-to-lumen H⁺/HCO₃ permeability (9). Solution 3 was HCO₃^–- and Cl^–-free and contained 0.5 mM acetazolamide to inhibit transtubule H⁺/HCO₃ transport and was used to determine "passive" blood-to-lumen H⁺/HCO₃ permeability (9,13). We used this "passive" permeability determined using solution 3 to calculate passive blood-to-lumen HCO₃ secretion when perfusing with the HCO₃-containing solution 1 (9). We used the "apparent" blood-to-lumen H⁺/HCO₃ permeability determined from perfusing with solution 2 to calculate "total" HCO₃ secretion when perfusing with HCO₃-containing solution 1 (9,13). We subtracted calculated "passive" HCO₃ secretion from calculated "total" HCO₃ secretion to obtain "net" HCO₃ secretion when perfusing with solution 1 (9,13). The HCO₃ secretion reported herein is the "net" HCO₃ secretion that excludes the passive HCO₃ secretion calculated as described above. Distal nephron H⁺ secretion was calculated by subtracting the calculated "net" HCO₃ secretion (a negative value) from the measured net HCO₃ reabsorption (HCO₃ perfused into the distal nephron minus HCO₃ collected) (13). All perfusing solutions contained raffinose to minimize fluid transport and gluconate substituted for Cl^– when necessary (9). Each surface distal nephron was perfused with each perfusing solution in the following order: 1, 2, 3. Previous studies that conducted random perfusions of these solutions showed that the order of perfusing solutions did not affect calculations of the components of distal nephron HCO₃ reabsorption (9).

Qualitative Comparison of ET-1 mRNA Expression
Total RNA Extraction and Reverse Transcription-PCR.
After treatment, kidneys were removed from anesthetized rats. Pieces of tissues were immediately frozen in liquid nitrogen and stored at –80°C until use.

Total RNA Isolation.
Total RNA was isolated using 1 ml of TRI-Reagent (Molecular Research Center, Cincinnati, OH) for 50 mg of tissue, a commercial variant of the guanidium thiocyanate-phenol-chloroform reagent, using the manufacturer’s suggested protocol (15). The resulting RNA was dissolved in DNase/Rnase-free water and stored at –20°C until use. Only RNA preparations whose A₂₆₀/A₂₈₀ ratio exceeded 1.6 were analyzed further. The RNA quality was assessed by running the samples in a 1% formaldehyde agarose gel following standard protocol.

Reverse Transcription.
We then performed reverse transcription with 2 µg of RNA, preheated 5 min at 65°C, in a final volume of 20 µl tat contained 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 0.2 mmol of dNTP (Roche, Indianapolis, IN), 10 mM dithiothreitol, 1 mg of oligo(dT)_12–18 primers (Roche), 40 units of RNasin (Promega, Madison, WI), and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega). After 1 h at 37°C, the enzyme was inactivated by boiling (10 min at 95°C).

PCR.
Specific oligonucleotide primers (5'-CTCTGCTGTTTGTGGCTTTC-3' and 5'-GTCTGTGGTCTTTGTGGGA-3' for sense and antisense primers, respectively) were designed to hybridize the rat ET-1 (rET-1) mRNA using Vector NTI 7 (InforMax, Frederick, MD). rET-1 cDNA amplification was carried out as follows: 1 µl of the reverse-transcribed mixture was added to the PCR mixture that contained 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl₂, 200 µM of dNTP, 400 nmol of each primer, and 2.5 units of TaqDNA polymerase (Roche) up to a final volume of 50 µl. After 2 min at 94°C, samples were submitted to 30 cycles under the following conditions: 45 s at 94°C, 45 s at 48°C (specific annealing temperature for the rET-1 primers), and 45 s at 72°C. After the final cycle, an additional elongation period of 7 min was performed at 72°C.

Real-Time PCR.
Because rat kidneys express very little ET-1 mRNA, we performed a real-time PCR using the LightCycler apparatus (Roche) after the conventional PCR. The SYBR Green, which has a high affinity for double-stranded DNA (dsDNA) and exhibits enhancement of fluorescence upon binding to the dsDNA, was chosen as the fluorescent dye. Each reaction contained 2 µl of cDNA from the conventional PCR, 3 mM MgCl₂, 0.5 mM of each primer, and 1x of FastStart DNA Master SYBR Green I mix (Roche) in a 20-µl final volume. Samples were then placed in the LightCycler instrument in duplicate and underwent the following thermal cycling profile: cDNA was denatured by a preincubation of 30 s at 95°C, and the template was amplified for 35 cycles of (1) denaturation for 0 s at 95°C, (2) annealing at 48°C for 10 s, and (3) extension at 72°C for 25 s. The increase in fluorescence, dependent on the initial template concentration, was acquired after each extension phase at 83°C, a temperature above the Tm of the primer dimers and below the Tm of the specific PCR product, thus minimizing acquisition of nonspecific fluorescence intensities. After amplification, a melting curve was generated by cooling the samples to 55°C for 30 s and slowly heating the samples at 0.1°C/s to 95°C while the fluorescence was measured continuously. The LightCycler run was concluded with a 40°C incubation for 30 s. Groups were qualitatively compared, with earlier amplification indicating greater number of ET-1 mRNA copies. Product identity was confirmed by sequence analysis and electrophoresis on a 1% agarose gel stained with ethidium bromide (Expected PCR product size, 290 bp).

Analytical Methods
Immediately after experiment termination, initial and collected perfusate, as well as stellate vessel plasma samples, were analyzed for inulin (9) and for TCO₂ using flow-through ultrafluorometry (16) as described previously (17). All tubule fluid and plasma TCO₂ were measured on the experimental day by comparing fluorescence of a 7- to 8-nl sample aliquot (corrected for a distilled H₂O blank run with each sample group) to a standard curve as described previously (17). This technique actually measures TCO₂, but we refer to this measured value as HCO₃ for simplicity.

Microdialysate and urine [ET-1] was measured using a RIA kit (Peninsula Laboratories, Belmont, CA) after disposable column extraction (Sep-Pak C18, Milford, MA) preconditioned with methanol, H₂O, and acetic acid as done previously (10) at weeks 0, 1, 2, and 3.

Statistical Analyses
Data were expressed as means ± SEM. Paired perfusions of the same tubule were compared using paired t test; otherwise, ANOVA was used for multiple group comparisons. We used the Bonferroni method for multiple comparisons (P < 0.05) of the same parameter among groups.

	Results

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Effect of HiPro on Animal/Kidney/Tubule Growth and Urine Volume
HiPro and CON had similar body weight at week 0 (214 ± 4 versus 216 ± 5 g, respectively), but HiPro gained more weight (159 ± 4 versus 114 ± 3 g, respectively; P < 0.001) by week 3. Daily food intake was identical between HiPro and CON (see Materials and Methods), but HiPro daily urine volume was higher at week 3 (44.0 ± 5 versus 15 ± 2 ml, respectively; P < 0.001). Left kidney weights in HiPro and CON were similar at week 1 (1.011 ± 0.019 versus 0.988 ± 0.022 g, respectively; P = 0.44) but were higher in HiPro at week 3 (1.251 ± 0.028 versus 1.048 ± 0.025 g, respectively; P < 0.001). In addition, length of accessible distal tubule was similar in HiPro and CON at week 1 (1.041 ± 0.032 versus 0.978 ± 0.030 mm, respectively; P = 0.17) but was greater in HiPro at week 3 (1.289 ± 0.041 versus 1.023 ± 0.033 mm, respectively; P < 0.001). Bosentan did not affect animal or kidney weight, tubule length, food intake, or urine output in either group.

Effect of HiPro on Arterial Acid-Base Parameters of Conscious Animals
Table 2 shows that weeks 1 and 3 arterial pH and PCO₂ by blood gases with calculated [HCO₃] were not different, but plasma TCO₂ by ultrafluorometry was lower in HiPro than CON. Mean BP was not different in HiPro and CON (111.5 ± 2.4 versus 110.1 ± 2.2 mmHg; P = 0.67) at week 3.

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Daily urine net acid excretion (NAE) measured in weekly intervals in conscious rats that initially ate standard chow with 23% protein before being changed to the study diets with 50% (HiPro) and 20% (CON) protein provided as purified casein. *P < 0.05 versus CON.

View larger version (45K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Distal nephron net HCO3 reabsorption (Net JHCO3) in tubules that were microperfused in vivo at 6 or 9 nl/min in HiPro and CON at weeks 1 and 3 of the protocol. *P < 0.05 versus CON.

View larger version (32K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Net change in distal nephron proton (H+) secretion at weeks 1 and 3 in response to in vivo microperfusion at 6 or 9 nl/min with inhibitors of Na+/H+ exchange (EIPA), H+-ATPase (Bafilomycin), and H+,K+-ATPase (Sch 28080). *P < 0.05 versus CON.

View larger version (35K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Daily urine endothelin-1 excretion (UET-1V; A) and ET-1 microdialysate addition (B) at weekly intervals in HiPro and CON conscious rats. *P < 0.05 versus CON.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Qualitative comparison of ET-1 mRNA using real-time PCR and LightCycler technology. CON indicates animals ingesting the 20% experimental diet and CON-D4W indicates CON drinking 4% dextrose solution to increase urine flow to that comparable to HiPro. Earlier amplification indicates greater number of ET-1 mRNA copies.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Daily urine NAE in HiPro animals that did an did not ingest the ET A/B receptor antagonist bosentan. *P < 0.05 versus CON.

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Distal tubule net HCO3 reabsorption (Net JHCO3) and its components, HCO3 and H+ secretion, at weeks 1 and 3 by in vivo microperfusion in HiPro animals that did and did not ingest the ET A/B receptor antagonist bosentan. *P < 0.05 versus CON.

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Net change in distal nephron proton (H+) secretion in HiPro at weeks 1 and 3 in response to in vivo microperfusion at 6 or 9 nl/min with inhibitors of Na+/H+ exchange (EIPA), H+-ATPase (Bafilomycin), and H+,K+-ATPase (Sch 28080). *P < 0.05 versus CON.

	Discussion

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ET increases Na⁺/H⁺ exchange in renal epithelia in vitro (19,20), and ET A/B receptor antagonism inhibits Na⁺/H⁺ exchange in the distal nephron in vivo (14), but we are not aware of studies showing that ET increases H⁺-ATPase activity. This suggests that the increased H⁺-ATPase activity that is reduced by ET A/B receptor blockade is an indirect effect of ET, possibly acting through another agent. A possible scenario is that increased renal ET production induced by dietary protein increases adrenal secretion of aldosterone (21) that in turn increases distal nephron H⁺-ATPase activity (22). Further studies will be necessary to explore this hypothesis.

Table 2 shows lower plasma TCO₂ in HiPro compared with CON, consistent with a relative metabolic acidosis in HiPro compared with CON. Because plasma TCO₂ remained at this slightly reduced level at weeks 1 and 3, it seems that increased dietary protein as casein leads to a steady-state but not progressive acidosis. That the metabolic acidosis was not progressive is likely due to the marked increase in urine NAE (Figure 1). Dietary mineral acid causes mild net acid retention that mediates the sustained associated increase in urine NAE (23). Also, increased [H⁺] in vitro increases ET-1 release from renal microvascular endothelium (24) and renal epithelium (20), and so increased endogenous ET might contribute to the untoward effects postulated for chronic metabolic acidosis (25).

Figure 1 shows that bosentan decreased urine NAE in HiPro at week 1 but not at week 3, suggesting greater ET dependence of HiPro-induced acidification at week 1. Although the net reduction of distal nephron acidification measured per millimeter of tubule length was not different at weeks 1 and 3, the perfused distal nephron segment was longer at week 3, consistent with tubule hypertrophy induced by HiPro. Because there was residual acidification in animals that ingested bosentan, the longer tubule at week 3 might have allowed for more overall ET-independent acidification at this time point.

In summary, increased dietary protein augments distal nephron acidification through an ET-dependent mechanism. The data support that ET contribute to the overall acidification response to this common dietary challenge to systemic acid-base homeostasis faced by humans.

	Acknowledgments

 
This work was supported by funds from the Larry and Jane Woirhaye Memorial Endowment in Renal Research the Texas Tech University Health Sciences Center.

We are grateful to Jeri Tasby and Cathy Hudson for expert technical assistance. We are also grateful to Martine Clozel, MD, for generously providing bosentan for use in these studies.

	References

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