Abstract
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.
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
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).
Table 1. Perfusate composition (mM)
Identification of the H+ Transport Mediators of HiPro-Induced Changes in Distal Nephron Acidification
We compared the net decrease in distal nephron H+ secretion in response to specific H+ transport inhibitors in HiPro versus CON to determine the contribution of Na+/H+ exchange (EIPA, 10−5 M), H+-ATPase (bafilomycin, 10−7 M), and H+,K+-ATPase (Sch 28080, 10−5 M) as done previously in our laboratory (14). Greater inhibitor-induced decrease in H+ secretion in HiPro versus CON determined increased activity of the H+-transport inhibited by that compound (14).
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 1× 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
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.
Table 2. Plasma acid-base data in conscious animals after three weeks of dietary protein (HiPro)a
Effect of HiPro on Renal Acidification
Figure 1 shows higher urine NAE in HiPro than CON at weeks 1, 2, and 3 (7067 ± 937 versus 4460 ± 639 μM/d; P < 0.04 at week 3). Higher NAE in HiPro was due to higher ammonium (NH4+) excretion (5117 ± 613 versus 2455 ± 353 μM/d; P < 0.003) and lower HCO3 excretion (56 ± 21 versus 257 ± 68 μM/d; P < 0.002), but titratable acid excretion was not different between HiPro and CON (2006 ± 338 versus 2261 ± 345 μM/d respectively; NS). Distal nephron net HCO3 reabsorption was higher at week 1 in HiPro than CON whether perfused at 6 nl/min (26.1 ± 2.2 versus 12.2 ± 1.4 pmol/mm per min; P < 0.001) or 9 nl/min (39.0 ± 3.6 versus 18.2 ± 1.6 pmol/mm per min; P < 0.001) as shown in Figure 2. Higher distal nephron net HCO3 reabsorption in HiPro than CON at week 1 was due to higher H+ secretion (29.1 ± 2.7 versus 21.6 ± 2.0 pmol/mm per min, P < 0.02 for 6 nl/min; 43.5 ± 3.9 versus 26.2 ± 2.5 pmol/mm per min, P < 0.002 for 9 nl/min) and less so to lower HCO3 secretion (−3.0 ± 0.5 versus −6.1 ± 0.8 pmol/mm per min, P < 0.001 for 6 nl/min; −4.5 ± 0.6 versus −8.0 ± 1.0 pmol/mm per min, P < 0.001 for 9 nl/min). Similarly, Figure 2 shows that distal nephron net HCO3 reabsorption was higher at week 3 in HiPro than CON whether perfused at 6 nl/min (25.8 ± 2.2 versus 14.4 ± 2.0 pmol/mm per min; P < 0.004) or 9 nl/min (37.8 ± 3.2 versus 16.6 ± 1.5 pmol/mm per min; P < 0.001) as shown in Figure 2. Higher distal nephron net HCO3 reabsorption in HiPro than CON at week 3 was due to higher H+ secretion (28.7 ± 2.8 versus 19.9 ± 1.9 pmol/mm per min, P < 0.03 for 6 nl/min; 41.3 ± 4.0 versus 23.0 ± 2.1 pmol/mm per min, P < 0.002 for 9 nl/min) and less so to lower HCO3 secretion (−2.8 ± 0.3 versus −5.5 ± 0.5 pmol/mm per min, P < 0.001 for 6 nl/min; −3.5 ± 0.4 versus −6.4 ± 0.8 pmol/mm per min, P < 0.001 for 9 nl/min).
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.
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.
Transport Mediators of HiPro-Induced Enhanced Distal Nephron Acidification
Figure 3 shows that at week 1, the net decrease in distal nephron H+ secretion was greater in HiPro than CON with EIPA (−19.3 ± 1.5 versus −10.9 ± 0.7 pmol/mm per min, P < 0.006 at 6 nl/min; −18.9 ± 1.6 versus −11.9 ± 0.9 pmol/mm per min, P < 0.006 at 9 nl/min) and bafilomycin (−16.4 ± 1.3 versus −7.6 ± 0.6 pmol/mm per min, P < 0.001 at 6 nl/min; −15.1 ± 1.3 versus −5.9 ± 0.7 pmol/mm per min, P < 0.006 at 9 nl/min), consistent with enhanced Na+/H+ exchange and H+-ATPase activity, respectively. Net decrease in H+ secretion induced by Sch 28080 was not different in HiPro and CON (−3.0 ± 0.7 versus −2.5 ± 0.6 pmol/mm per min, P = 0.60 at 6 nl/min; −3.6 ± 0.7 versus −2.4 ± 0.5 pmol/mm per min, P = 0.18 for 9 nl/min), consistent with no increased H+, K+-ATPase activity in HiPro. Similarly, net decrease in distal nephron H+ secretion was greater in HiPro than CON at week 3 with EIPA (−15.5 ± 1.0 versus −11.3 ± 0.8 pmol/mm per min, P < 0.006 at 6 nl/min; −15.9 ± 1.1 versus −11.1 ± 0.8 pmol/mm per min, P < 0.004 at 9 nl/min) and bafilomycin (−14.5 ± 1.1 versus −6.0 ± 0.5 pmol/mm per min, P < 0.001 at 6 nl/min; −12.4 ± 1.0 versus −5.5 ± 0.8 pmol/mm per min, P < 0.001 at 9 nl/min), consistent with enhanced Na+/H+ exchange and H+-ATPase activity, respectively, as shown in Figure 3. Net decrease in H+ secretion induced by Sch 28080 was not different in HiPro and CON (−3.4 ± 0.9 versus −2.6 ± 0.8 pmol/mm per min, P = 0.52 at 6 nl/min; −4.0 ± 0.8 versus −2.9 ± 0.6 pmol/mm per min, P = 0.29 for 9 nl/min), consistent with no increased activity of H+,K+-ATPase activity in HiPro.
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.
Effect of HiPro on Renal ET-1 Production
Figure 4 shows that HiPro compared with CON had similar urine ET-1 excretion at week 0 (42.9 ± 5.8 versus 33.2 ± 3.7 fmol/kg body wt per d; P = 0.18), but HiPro was higher at week 1 (122.4 ± 26.8 versus 39.5 ± 3.9 fmol/kg body wt per d; P < 0.009), week 2 (89.6 ± 16.1 versus 30.8 ± 3.4 fmol/kg body wt per d; P < 0.004), and week 3 (80.0 ± 15.7 versus 29.0 ± 3.9 fmol/kg body wt per d; P < 0.008). In addition, Figure 4 shows that HiPro and CON had similar ET-1 addition to microdialysate at week 0 (275.2 ± 81.0 versus 249.3 ± 30.5 fmol/g kidney wt/ per min; P = 0.77), but HiPro had greater renal cortical microdialysate addition at week 1 (612.4 ± 81.0 versus 255.2 ± 32.5 fmol/g kidney wt per min; P < 0.002), week 2 (456.8 ± 62.5 versus 216.3 ± 34.1 fmol/g kidney wt per min; P < 0.005), and week 3 (386.1 ± 49.4 versus 230.8 ± 29.2 fmol/g kidney wt per min; P < 0.02). In addition, Figure 5 shows a qualitative increase in renal cortical mRNA in HiPro than CON at week 3. HiPro had higher urine flow than CON, and high urine flow might itself increase urine ET-1 excretion (18). Consequently, we studied CON ingesting 20% protein diet and distilled H2O compared with those ingesting 4% dextrose-containing drinking water (CON-D4W) to increase daily urine volume to a level comparable to that of HiPro without providing additional dietary protein. At week 3, CON-D4W compared with CON had higher daily urine volume (40.2 ± 3 versus 14 ± 1 ml/d, respectively; n = 8; P < 0.001) and numerically higher urine ET-1 excretion (43.6 ± 9.5 fmol/kg body wt per d versus respective week 3 CON; n = 8; P = 0.18). Nevertheless, ET-1 addition to renal cortical microdialysate in animals when compared with those without the dextrose-containing drinking solution was not different (238 ± 39 fmol/g kidney wt per min versus respective week 3 CON; n = 8; P = 0.88). Although qualitative comparison of CON-D4W and CON ET-1 mRNA expression suggested a small difference (Figure 5), absolute quantitative analysis failed to show any differences (data not shown).
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.
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.
Effect of ET-1 Receptor Blockade on Arterial Blood and Urine Parameters
Table 3 shows that bosentan did not affect CON arterial plasma acid-base parameters at weeks 1 or 3. By contrast, Table 4 shows that HiPro receiving bosentan had lower plasma TCO2 at week 1 but not week 3. In addition, mean BP was not different in bosentan-ingesting compared with noningesting HiPro (109.7 ± 2.3 versus 111.5 ± 2.4 mmHg; P = 0.60) or CON (110.4 ± 2.3 versus 110.1 ± 2.2 mmHg; P = 0.93). Figure 6 shows that HiPro receiving bosentan had lower urine NAE (5704 ± 594 versus 7067 ± 937 μM/d; P < 0.05, paired t) at week 1, but NAE was comparable without and with bosentan at week 3. Lower NAE at week 1 in HiPro with bosentan was due to lower NH4+ excretion (3715 ± 416 versus 5117 ± 613 μM/d; P < 0.03, paired t) and higher HCO3 excretion (231 ± 42 versus 56 ± 21 μM/d; P < 0.001, paired t), but titratable acid excretion was not different (2006 ± 338 versus 2221 ± 304 μM/d, respectively; P = 0.64).
Table 3. Plasma acid-base parameters of control animals in response to bosentana
Table 4. Plasma acid-base parameters of HiPro animals in response to bosentana
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.
Effect of ET-1 Receptor Blockade on HiPro-Induced Changes in Distal Nephron Acidification
Distal nephron acidification was not different between CON receiving and not receiving bosentan (data not shown). At week 1, Figure 7 shows that HiPro receiving bosentan had lower distal tubule net HCO3 reabsorption when perfused at 9 nl/min (21.5 ± 2.1 versus 39.0 ± 3.6 pmol/mm per min; P < 0.001). Lower net HCO3 reabsorption at week 1 was due to lower H+ secretion (28.7 ± 2.5 versus 43.5 ± 3.9 pmol/mm per min; P < 0.007) and higher HCO3 secretion (−6.1 ± 0.8 versus −3.0 ± 0.5 pmol/mm per min; P < 0.006). Figure 7 also shows that week 3 HiPro animals that received bosentan and perfused at 9 nl/min had lower distal tubule net HCO3 reabsorption (22.4 ± 1.9 versus 37.8 ± 3.2 pmol/mm per min; P < 0.001). This lower distal tubule net HCO3 reabsorption was due to 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).
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.
Effect of ET-1 Receptor Blockade on Enhanced H+ Transporter Activity Induced by HiPro
At week 1, net decrease in distal nephron H+ secretion was not different in bosentan-ingesting compared with noningesting HiPro perfused at 9 nl/min with EIPA (−16.4 ± 1.8 versus −18.9 ± 1.6 pmol/mm per min; P = 0.32) and bafilomycin (−14.0 ± 1.5 versus −15.1 ± 1.3 pmol/mm per min; P = 0.59), consistent with no additional effect of these H+ inhibitors on Na+/H+ exchange and H+-ATPase activity, respectively, in HiPro with ET A/B receptor blockade (Figure 8). There was no difference in net H+ secretion decrease in HiPro perfused with Sch 28080 (−4.4 ± 0.6 versus −3.0 ± 0.7 pmol/mm per min; P = 0.15), consistent with no additional effect of ET A/B receptor blockade on H+,K+-ATPase activity in HiPro. Similarly, net decrease in distal nephron H+ secretion at week 3 was not different in the bosentan-ingesting compared with the noningesting HiPro animals perfused at 9 nl/min with EIPA (−13.4 ± 1.2 versus −15.9 ± 1.1 pmol/mm per min; P = 0.15) and bafilomycin (−11.0 ± 1.0 versus −12.4 ± 1.0 pmol/mm per min; P = 0.34), consistent with no additional effect of these H+ inhibitors on Na+/H+ exchange and H+-ATPase activity, respectively, in HiPro with ET A/B receptor blockade. The was also no difference in net in H+ secretion decrease in HiPro perfused with Sch 28080 (−5.0 ± 0.8 versus −3.4 ± 0.9 pmol/mm per min; P = 0.21), consistent with no additional effect of ET A/B receptor blockade on H+, K+-ATPase activity in HiPro.
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
The present studies show that increased dietary protein as purified casein augments distal nephron acidification and does so by increasing H+ secretion through increased Na+/H+ exchange and increased H+-ATPase activity and to a lesser extent by decreasing HCO3 secretion. Increased dietary protein increased renal ET-1 production, and the data support that each component of increased distal nephron acidification was mediated by increased ET activity. That the studies used in vivo microperfusion of the distal nephron supports that the observed ET effects were mediated through effects on transport rather than hemodynamics. These studies show that ET is a mediator of increased renal acidification in response to dietary protein, the common acid challenge faced by humans.
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.
- © 2004 American Society of Nephrology
References
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- Mechanisms of Metabolic Acidosis-Induced Kidney Injury in Chronic Kidney Disease
- The Role of the Renal Ammonia Transporter Rhcg in Metabolic Responses to Dietary Protein
- Endothelin-Induced Increased Nitric Oxide Mediates Augmented Distal Nephron Acidification as a Result of Dietary Protein
- The B1-subunit of the H+ ATPase is required for maximal urinary acidification
- Endothelin-Induced Increased Aldosterone Activity Mediates Augmented Distal Nephron Acidification as a Result of Dietary Protein