Acidosis Mediates the Upregulation of UT-A Protein in Livers from Uremic Rats
Janet D. Klein,
Patricia Rouillard,
Brian R. Roberts and
Jeff M. Sands
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia.
Correspondence to: Dr. Jeff M. Sands, Emory University School of Medicine, Renal Division, WMRB Room 338, 1639 Pierce Drive, NE, Atlanta, GA 30322. Phone: 404-727-2435; Fax: 404-727-3425; E-mail: jsands{at}emory.edu
ABSTRACT. Liver expresses a 49-kD UT-A protein whose abundanceis increased by uremia. Chronic renal failure causes acidosis;therefore, the role of acidosis in increasing UT-A abundancewas tested. Rats underwent 5/6 nephrectomy, and half were givenbicarbonate mixed in their food. Bicarbonate administrationsignificantly increased blood pH. Compared with sham-operatedrats, UT-A protein abundance was significantly increased by50% in livers from uremic, acidotic rats; bicarbonate administrationprevented the increase in UT-A protein. To determine whetheracidosis alone would increase UT-A protein in liver, rats weremade acidotic, but not uremic, by feeding them HCl. HCl-feedingsignificantly lowered blood pH, increased urea excretion, andincreased the abundance of the 49-kD liver UT-A protein by 36%compared with pair-fed nonacidotic rats. HCl-feeding significantlyincreased the abundance of the 117-kD UT-A1 protein in kidneyinner medulla but did not change aquaporin-2 protein. Next,rats were fed urea to determine whether elevated blood ureawould increase UT-A protein. However, urea feeding had no effecton UT-A in liver or kidney inner medulla. It was, therefore,concluded that acidosis, either directly or through a changein ammonium concentration, rather than other dietary components,stimulates the upregulation of UT-A protein in liver and kidneyinner medulla.
Urea is a highly polar, small molecule that has a low permeabilityacross artificial lipid bilayers (1). Urea is transported byfacilitated (or carrier-mediated) transport pathways in kidneyinner medullary collecting ducts (IMCD), erythrocytes, and liver(2–4). Four urea transporter cDNAs have been cloned fromrat kidney (UT-A1, UT-A2, UT-A3, and UT-A4) that originate froma single gene by alternative splicing (5–11). AlthoughUT-A protein expression was initially thought to be limitedto the kidney, we showed that liver expresses 49- and 36-kDUT-A proteins (3), and Northern analysis shows that liver expressesa 2.7-kb UT-A mRNA (12), which is consistent with the size ofUT-A2b (8).
In liver, the abundance of the 49-kD UT-A protein is significantlyincreased in rats made uremic by 5/6 nephrectomy (3). We proposed(3) that upregulation of this UT-A protein may allow hepatocytesto increase urea production (13,14) to reduce the accumulationof ammonium (15–17). In searching for signals that couldupregulate UT-A, we have studied metabolic acidosis becauserenal failure frequently causes metabolic acidosis (18). Acidosis,in turn, enhances protein degradation, both in rats and patientswith renal failure, and bicarbonate administration has beenshown to improve nitrogen balance in patients (19). The goalof our study was to determine whether metabolic acidosis causesan increased abundance of the 49-kD UT-A protein in liver, bothin uremic and nonuremic rats.
Tissue Preparation
Male Sprague-Dawley rats (National Cancer Institute, Frederick,MD) were anesthetized with intraperitoneal pentobarbitol (Nembutal,Abbott Laboratories, North Chicago, IL), an aortic blood samplewas obtained for blood gas and biochemical analysis, and theliver and kidneys were removed. The kidney inner medulla wasdissected into base and tip portions, as described previously(20). Tissue samples were homogenized in isolation buffer (10mM triethanolamine, 250 mM sucrose, 1.46 g/ml leupeptin, 0.1mg/ml phenylmethylsulfonyl fluoride, pH 7.6; 0.025 to 0.1 gtissue per ml isolation buffer) (3,20,21). Concentrated sodiumdodecyl sulfate (SDS) was added to achieve a final concentrationof 1%, and samples were sheared by passage through a 28-gaugeneedle and centrifuged for 15 min at 14,000 x g. Protein wasdetermined by using the BioRad DC protein assay kit (BioRad,Richmond, CA).
Animal Models
Plasma and urine chemistries were measured by the Emory UniversityVeterinary Services Laboratory. Arterial blood gas was measuredby using a blood gas analyzer (Opti 1; AVL Scientific, Roswell,GA). Urine osmolality was measured by using a vapor pressureosmometer (model 5500; Wescor, Logan, UT).
Chronic Renal Failure.
To induce uremia, rats underwent 5/6 nephrectomy, were fed 40%protein, and drank 0.225 mg/dl NaCl; control rats underwentsham-operation and were pair-fed the same food and drink (22,23).To test for an effect of acidosis, a second group of rats underwent5/6 nephrectomy but were given bicarbonate for 8 d by adding1.7 g NaHCO3/100 g chow to their food and 0.125 mg/dl NaHCO3to their drinking fluid; these rats were pair-fed to the 5/6nephrectomy rats that were not given bicarbonate (23).
Acidosis.
To induce acidosis in nonuremic rats, normal rats were fed a50:50 mixture of normal chow and chow with 0.8 M HCl (wt/vol)for 4 to 11 d and given water ad libitum; control rats werepair-fed a 50:50 mixture of normal chow and chow with water(24).
To control for the increase in water intake of the HCl-fed rats,a second group of HCl-fed rats were water-restricted by limitingtheir water intake to that of the pair-fed control rats. Thewater-restricted, HCl-fed rats were pair-fed to the HCl-fedrats receiving water ad libitum.
Urea Feeding.
To increase blood urea nitrogen (BUN), rats were fed standardchow supplemented with 30% urea for 7 d; control rats were pair-fedwith standard chow (25).
Western Blot Analyses
Proteins (10.46 g/lane) were separated on 10% SDS-polyacrylamidegels and then transferred to a polyvinylidene difluoride membrane.Membranes were probed with our affinity-purified polyclonalantibody (3.8 mg/ml used at 1:5000 for Western blot) to UT-A(3,20). This antibody was prepared against the C-terminal portionof UT-A1 and will also detect UT-A2 and UT-A4 (7,20). The immunoreactiveproteins were visualized by enhanced chemiluminescence (ECL;Amersham, Arlington Heights, IL) (3,20,21). Autoradiograms werescanned using the Bio-Rad Gel Doc 1000 digital imaging densitometer(Bio-Rad Laboratories, Hercules, CA). Scanned bands were quantifiedby using the system’s Multi-Analyst version 1.0.1 software.Results are expressed as arbitrary units/0.46 g protein loaded.
Statistical Analyses
All data are presented as mean ± SEM, and n = numberof rats. The t test was used to test for statistical significancebetween two groups. An ANOVA was used to test for statisticalsignificance between three groups, followed by Tukey’sprotected t test (26) to determine which groups were significantlydifferent. The criterion for statistical significance was P< 0.05.
Uremic Rats
The rats that underwent a 5/6 nephrectomy had significantlyincreased serum creatinine and BUN and decreased pH and serumbicarbonate and albumin compared with sham-operated rats (Table 1).Feeding bicarbonate to uremic rats significantly increasedpH and serum bicarbonate and decreased serum chloride (Table 1)compared with uremic rats that were not given bicarbonate.Compared with sham-operated rats, the 49-kD UT-A protein abundancewas significantly increased by 50% in liver from uremic, acidoticrats, which is consistent with our previous study (3); administeringbicarbonate to uremic rats reversed the increase in the 49-kDUT-A protein (Figure 1).
Figure 1. Urea transporter protein abundance in livers from rats made uremic by 5/6 nephrectomy. (Left panel) representative Western blot showing UT-A protein bands in liver from a sham-operated rat (Sham), a uremic rat (Uremic), and a uremic rat given bicarbonate (Uremic + Bicarb). Each lane represents a sample from a separate rat. (Right panel) densitometric summary of the 49-kD band on the gels. There was a significant increase in the density of the 49-kD band in liver from uremic rats compared with control rats. There was no change in the abundance of the 36-kD band (data not shown). Data are mean ± SE; n = 6 rats/group; *P < 0.05.
HCl-Fed Rats
To determine whether acidosis alone can increase UT-A proteinabundance, normal rats were fed HCl. Arterial blood pH and bicarbonatewere reduced after 4, 6, 7, and 11 d of HCl feeding (Table 2).The 49-kD UT-A protein in liver was increased at each time point(Figure 2).
Figure 2. Western blot showing UT-A protein bands in liver from rats fed HCl (HCl) or pair-fed control (C) rats. Rats were fed HCl for the number of days indicated. The abundance of the 49-kD band is increased in the HCl-fed rat at each time point. Each lane represents a sample from a separate rat; two HCl-fed and control rats were studied at each time point.
Additional rats were fed HCl for 7 d, and compared with pair-fedcontrol rats, the rats fed HCl had significantly decreased arterialblood pH, serum bicarbonate, and urine pH and significantlyincreased arterial blood pCO2 and urine urea excretion (Table 3).The abundance of the 49-kD liver UT-A protein was significantlyincreased by 36% (Figure 3).
Figure 3. Urea transporter protein abundance in livers from rats fed HCl for 7 d. (Left panel) representative Western blot showing UT-A protein bands in liver from control (C) and HCl-fed (HCl) rats. Each lane represents a sample from a separate rat. (Right panel) densitometric summary of the 49-kD band on the gels. There was a significant increase in the density of the 49-kD band in HCl-fed rats compared with pair-fed control rats. There was no change in the abundance of the 36-kD band (data not shown). Data are mean ± SE; n = 8 rats/group; *P < 0.05.
The HCl-fed rats also had a significant increase in urine volume(Table 3). Therefore, we measured UT-A1 and aquaporin-2 (AQP2)protein abundances in the renal inner medulla (IM). In the IMtip, the abundance of the 117-kD UT-A1 protein was significantlyincreased by 129% and the abundance of the 97-kD UT-A1 proteinwas unchanged (Figure 4). In the IM base, the 117-kD UT-A1 proteinwas nearly undetectable in control rats but clearly presentin HCl-fed rats; the abundance of the 97-kD UT-A1 protein wasunchanged (Figure 5). AQP2 protein abundance was unchanged inthe IM tip of control versus HCl-fed rats (data not shown).
Figure 4. Urea transporter protein abundance in kidney inner medullary (IM) tip from rats fed HCl for 7 d. (Left panel) representative Western blot showing UT-A1 protein bands in IM tip from control (C) and HCl-fed (HCl) rats. Each lane represents a sample from a separate rat. (Right panel) densitometric summary of the 117- and 97-kD UT-A1 bands on the gels. There was a significant increase in the density of the 117-kD band in HCl-fed rats compared with pair-fed control rats. There was no change in the abundance of the 97-kD band. Data are mean ± SE; n = 4 rats/group; *P < 0.05.
Figure 5. Urea transporter protein abundance in kidney inner medullary (IM) base from rats fed HCl for 7 d. (Left panel) representative Western blot showing UT-A1 protein bands in IM base from control (C) and HCl-fed (HCl) rats. Each lane represents a sample from a separate rat. (Right panel) densitometric summary of the 117- and 97-kD UT-A1 bands on the gels. There was a significant increase in the density of the 117-kD band in HCl-fed rats compared with pair-fed control rats. There was no change in the abundance of the 97-kD band. Data are mean ± SE; n = 4 rats/group; *P < 0.05.
To determine whether the increase in UT-A1 protein in the kidneyof HCl-fed rats was due to the increase in urine volume, HCl-fedrats were water-restricted by limiting their water intake tothe amount drunk by the control rats (Table 4). In the IM tip,the abundance of the 117-kD UT-A1 protein was significantlyincreased and the abundance of the 97-kD UT-A1 protein was unchanged,which is similar to the results obtained in the HCl-fed ratsdrinking water ad libitum (Figure 6).
Figure 6. Urea transporter protein abundance in kidney inner medullary (IM) tip from rats fed HCl and water-restricted for 7 d. (Left panel) representative Western blot showing UT-A1 protein bands in IM tip from control (C), HCl-fed (HCl), and water-restricted HCl-fed (HCl/WR) rats. Each lane represents a sample from a separate rat. (Right panel) densitometric summary of the 117- and 97-kD UT-A1 bands on the gels. There was a significant increase in the density of the 117-kD band in both the HCl-fed and the water-restricted, HCl-fed rats, compared with pair-fed control rats. There was no significant change in the abundance of the 97-kD band. Data are mean ± SE; n = 3 rats/group; *P < 0.05.
Urea-Fed Rats
To determine whether an increase in BUN alone can increase UT-Ain liver, rats were fed a standard protein diet to which ureawas added. The urea-fed rats had a significantly higher BUNand urine urea excretion than pair-fed control rats (Table 4).There was no significant change in the abundance of the 49-kDUT-A protein in liver, the 117- or 97-kD UT-A1 proteins in theIM tip, or in AQP2 protein abundance in the IM tip of controlversus urea-fed rats (data not shown).
Liver
We previously showed that the liver expresses two UT-A proteins:a 49-kD protein that is present in the membrane and a 36-kDprotein that is present in the cytoplasm (3). We also foundthat our model of uremia is associated with an increase in theabundance of the 49-kD, but not the 36-kD, UT-A protein in ratliver (3). The 49-kD UT-A protein is most likely UT-A2b andis expressed in hepatocyte membranes (3,8,12). The goal of thisstudy was to identify a mediator of the in vivo increase inUT-A. Because uremia results in so many pathophysiologic changes,we chose to study metabolic acidosis because it can be manipulatedand has been shown to be a mediator of increased protein andamino acid catabolism and increased gene transcription (23).Our major result was that giving bicarbonate to uremic ratsnormalized blood pH and blocked the increase in the abundanceof the 49-kD UT-A protein in their livers. This response occurreddespite no difference in dietary constituents (other than bicarbonate).Moreover, the major endproduct of dietary protein is urea; therefore,our finding that BUN values between uremic acidotic rats anduremic nonacidotic rats were not statistically different (Table 1)indicates that the acidosis, either directly or through achange in ammonium concentration, rather than the accumulationof urea (or other nitrogenous products), was the principal causeof the increase in UT-A protein expression. This was confirmedwhen we studied HCl-fed, nonuremic rats; the abundance of the49-kD UT-A protein was increased there as well. The percentincrease (50%) in the 49-kD UT-A protein in uremic rat liverwas somewhat greater than the percent increase (36%) in HCl-fed,nonuremic rat liver. This difference could be due to the largerchange in pH in the uremic rats or to assorted consequencesof acidosis and/or uremia. In short, our results strongly suggestthat acidosis, either directly or through a change in ammoniumconcentration, is the principal cause of the upregulation ofUT-A protein expression in rat liver.
We also tested whether an increase in BUN would increase UT-Aprotein in liver by feeding rats large quantities of urea (25).The BUN of the urea-fed rats was significantly higher than inthe pair-fed control rats (25) but not as high as in the 5/6nephrectomized rats. We found that UT-A protein abundance wasunchanged in liver, which suggests that an elevated BUN alonewas not responsible for the increase in UT-A protein. However,we cannot exclude the possibility that a larger increase inBUN would have an effect on UT-A protein.
What is the physiologic advantage for an increase in hepaticUT-A protein expression since urea synthesis consumes two ammoniumand two bicarbonate molecules and should not alter acid-basebalance? One proposed function for a hepatic urea transporteris to facilitate the rapid transport of urea out of hepatocytes(following ureagenesis) and into the extracellular space (4,27,28).Several studies have shown that ureagenesis is controlled primarilyby the quantity of ammonium that needs to be removed (29,30),and studies by Mitch et al. (22,23,31–33) have shown thatacidosis increases muscle protein breakdown, thereby increasingthe nitrogen-containing waste products that need to be metabolizedand excreted. Furthermore, they showed that correcting the acidosisin uremic rats reduced muscle protein degradation (23). Althoughmetabolic acidosis increases muscle glutamine synthetase andglutamine release, thereby shifting some nitrogen to renal ammoniagenesisrather than alanine formation and hepatic ureagenesis (34,35),muscle protein degradation increases nitrogen delivery to theliver and, in turn, increases ureagenesis (29,30). We speculatethat UT-A protein expression may be upregulated in liver duringconditions associated with an increase in muscle protein degradation,such as acidosis, to facilitate urea transport out of hepatocytes.Consistent with this hypothesis, we found that urine urea excretionwas significantly increased in the HCl-fed rats (Table 3). However,previous studies of HCl-fed rats did not find an increase inurine urea excretion (24,34,35). Although the protocols forHCl-feeding differ, we do not have an explanation for this difference.
Kidney
UT-A1 exists as two glycoproteins, which run at 117 and 97 kD,in rat inner medulla (36). Deglycosylation of rat inner medullaryproteins results in a single 88-kD UT-A1 band (36). Both glycoproteinsare expressed in the IMCD apical membrane, and the reason forthe two forms is not known. We found that acidosis increasedthe abundance of the 117-kD, but not the 97-kD, UT-A1 proteinin the kidney inner medullary tip. We previously showed thatUT-A1 protein abundance is increased in five conditions associatedwith increased urine volume: water diuresis, furosemide diuresis,low-protein diet, hypercalcemia, and adrenalectomy (reviewedin references (37 and (38). The HCl-fed rats also had an increasedurine volume compared with the pair-fed control rats, whichis consistent with earlier studies (24). However, the increasein the 117-kD UT-A1 protein in the inner medullary tip of HCl-fedrats is not due simply to polyuria, because it was also increasedin HCl-fed rats whose water intake was limited to the amountdrunk by control rats.
There are interesting differences between HCl-feeding and otherpolyuric conditions. One is that AQP2 protein is generally reducedin polyuric conditions (39), but in HCl-fed rats, we did notfind a reduction in AQP2 protein in the inner medullary tip.Another difference is that the 117-kD UT-A1 protein was clearlyexpressed in the inner medullary base of HCl-fed rats; the innermedullary base typically expresses only the 97-kD UT-A1 protein(reviewed in reference (37). The abundance of the 117-kD, butnot the 97-kD, UT-A1 protein was also increased in both theinner medullary base and tip regions in diabetic rats that werestudied two weeks after streptozotocin injection (40). Theserats were polyuric, but their pH was not measured.
Lastly, we tested whether an increase in BUN would alter UT-A1or AQP2 proteins in kidney inner medulla by feeding rats largequantities of urea (25). Despite significant changes in urinevolume and urea excretion, both UT-A1 and AQP2 protein abundanceswere unchanged.
In conclusion, we have identified that acidosis stimulates theexpression of UT-A protein in the liver and kidney. It is temptingto conclude that the increased expression represents transcriptionalstimulation as acidosis in uremia increases the transcriptionof genes involved in the ubiquitin-proteasome pathway, at leastin muscle (23,32,33,41). Regardless, the responses of the liverand kidney, coupled with those in muscle (19), indicate thatacidosis induces a coordinated set of physiologic responsesin different organs to defend both acid-base and nitrogen homeostasis.Additional studies will be necessary to identify the mechanismsunderlying these responses, such as whether acidosis activatesstress or immediate early response genes and whether acidosisper se or a change in ammonium concentration is the signal.
Table 5. Blood and urine chemistries in urea-fed ratsa
Acknowledgments
The authors thank Dr. William E. Mitch at Emory University forhis critical reading of this manuscript. Portions of this workhave been published in abstract form and were presented at the32nd Annual Meeting of the American Society of Nephrology, November5 to 8, 1999, Miami Beach, FL, and Experimental Biology 2000,April 15 to 18, 2000, San Diego, CA, and published in abstractform (J Am Soc Nephrol 10: 17A–18A, 1999 and FASEB J 14:A347, 2000). This work was supported by National Institutesof Health grants R01-DK41707 and P01-DK50268.
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Received for publication August 14, 2001.
Accepted for publication November 3, 2001.
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November 1, 2002;
13(11):
2795 - 2806.
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Abstract
Introduction
