ARTICLES J Am Soc Nephrol 13: 581–587, 2002

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.

Abstract. Liver expresses a 49-kD UT-A protein whose abun- pH, increased urea excretion, and increased the abundance of dance is increased by uremia. Chronic renal failure causes the 49-kD liver UT-A protein by 36% compared with pair-fed acidosis; therefore, the role of acidosis in increasing UT-A nonacidotic rats. HCl-feeding significantly increased the abun- abundance was tested. Rats underwent 5/6 nephrectomy, and dance of the 117-kD UT-A1 protein in kidney inner medulla half were given bicarbonate mixed in their food. Bicarbonate but did not change aquaporin-2 protein. Next, rats were fed administration significantly increased blood pH. Compared urea to determine whether elevated blood urea would increase with sham-operated rats, UT-A protein abundance was signif- UT-A protein. However, urea feeding had no effect on UT-A icantly increased by 50% in livers from uremic, acidotic rats; in liver or kidney inner medulla. It was, therefore, concluded bicarbonate administration prevented the increase in UT-A that acidosis, either directly or through a change in ammonium protein. To determine whether acidosis alone would increase concentration, rather than other dietary components, stimulates UT-A protein in liver, rats were made acidotic, but not uremic, the upregulation of UT-A protein in liver and kidney inner by feeding them HCl. HCl-feeding significantly lowered blood medulla.

Urea is a highly polar, small molecule that has a low perme- determine whether metabolic acidosis causes an increased ability across artificial lipid bilayers (1). Urea is transported by abundance of the 49-kD UT-A protein in liver, both in uremic facilitated (or carrier-mediated) transport pathways in kidney and nonuremic rats. inner medullary collecting ducts (IMCD), erythrocytes, and liver (2–4). Four urea transporter cDNAs have been cloned Materials and Methods from rat kidney (UT-A1, UT-A2, UT-A3, and UT-A4) that Tissue Preparation originate from a single gene by alternative splicing (5–11). Male Sprague-Dawley rats (National Cancer Institute, Frederick, Although UT-A protein expression was initially thought to be MD) were anesthetized with intraperitoneal pentobarbitol (Nembutal, limited to the kidney, we showed that liver expresses 49- and Abbott Laboratories, North Chicago, IL), an aortic blood sample was 36-kD UT-A proteins (3), and Northern analysis shows that obtained for blood gas and biochemical analysis, and the liver and liver expresses a 2.7-kb UT-A mRNA (12), which is consistent kidneys were removed. The kidney inner medulla was dissected into with the size of UT-A2b (8). base and tip portions, as described previously (20). Tissue samples In liver, the abundance of the 49-kD UT-A protein is sig- were homogenized in isolation buffer (10 mM triethanolamine, 250 nificantly increased in rats made uremic by 5/6 nephrectomy mM sucrose, 1.46 g/ml leupeptin, 0.1 mg/ml phenylmethylsulfonyl (3). We proposed (3) that upregulation of this UT-A protein fluoride, pH 7.6; 0.025 to 0.1 g tissue per ml isolation buffer) may allow hepatocytes to increase urea production (13,14) to (3,20,21). Concentrated sodium dodecyl sulfate (SDS) was added to reduce the accumulation of ammonium (15–17). In searching achieve a final concentration of 1%, and samples were sheared by passage through a 28-gauge needle and centrifuged for 15 min at for signals that could upregulate UT-A, we have studied met- 14,000 ϫ g. Protein was determined by using the BioRad DC protein abolic acidosis because renal failure frequently causes meta- assay kit (BioRad, Richmond, CA). bolic acidosis (18). Acidosis, in turn, enhances protein degra- dation, both in rats and patients with renal failure, and bicarbonate administration has been shown to improve nitro- Animal Models gen balance in patients (19). The goal of our study was to Plasma and urine chemistries were measured by the Emory Uni- versity Veterinary Services Laboratory. Arterial blood gas was mea- sured by using a blood gas analyzer (Opti 1; AVL Scientific, Roswell, Received August 14, 2001. Accepted November 3, 2001. GA). Urine osmolality was measured by using a vapor pressure Correspondence to: Dr. Jeff M. Sands, Emory University School of Medicine, osmometer (model 5500; Wescor, Logan, UT). Renal Division, WMRB Room 338, 1639 Pierce Drive, NE, Atlanta, GA Chronic Renal Failure. To induce uremia, rats underwent 5/6 30322. Phone: 404-727-2435; Fax: 404-727-3425; E-mail: [email protected] nephrectomy, were fed 40% protein, and drank 0.225 mg/dl NaCl; 1046-6673/1303-0581 control rats underwent sham-operation and were pair-fed the same Journal of the American Society of Nephrology food and drink (22,23). To test for an effect of acidosis, a second Copyright © 2002 by the American Society of Nephrology group of rats underwent 5/6 nephrectomy but were given bicarbonate 582 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 581–587, 2002

Table 1. Blood chemistries in uremic ratsa

Parameter Sham Operation Uremia Uremia ϩ Bicarbonate

Albumin (g/dl) 3.3 Ϯ 0.1cd 2.4 Ϯ 0.2b 2.4 Ϯ 0.1b Total protein (g/dl) 4.9 Ϯ 0.1 4.6 Ϯ 0.3 4.6 Ϯ 0.2 Creatinine (mg/dl) 0.5 Ϯ 0.1cd 1.3 Ϯ 0.1b 1.3 Ϯ 0.1b Sodium (mEq/L) 137 Ϯ 2 131 Ϯ 3 135 Ϯ 3 Chloride (mEq/L) 100 Ϯ 1 105 Ϯ 2d 92 Ϯ 3c BUN (mg/dl) 26 Ϯ 3cd 191 Ϯ 7b 159 Ϯ 13b Arterial blood pH 7.41 Ϯ 0.02c 7.02 Ϯ 0.05bd 7.43 Ϯ 0.02c Ϯ Ϯ Ϯ Arterial blood pCO2 37.9 1.9 32.2 2.7 35.1 2.5 Ϯ c Ϯ bd Ϯ c Serum HCO3 (mEq/L) 24.9 0.7 7.7 1.1 25.3 0.9 a Data are mean Ϯ SE; n ϭ 6 rats/group; BUN, blood urea nitrogen. b P Ͻ 0.01 versus sham operation. c P Ͻ 0.01 versus CRF. d P Ͻ 0.01 versus CRF ϩ Bicarbonate.

for8dbyadding 1.7 g NaHCO3/100 g chow to their food and 0.125 serum bicarbonate and albumin compared with sham-operated mg/dl NaHCO3 to their drinking fluid; these rats were pair-fed to the rats (Table 1). Feeding bicarbonate to uremic rats significantly 5/6 nephrectomy rats that were not given bicarbonate (23). increased pH and serum bicarbonate and decreased serum Acidosis. To induce acidosis in nonuremic rats, normal rats were chloride (Table 1) compared with uremic rats that were not fed a 50:50 mixture of normal chow and chow with 0.8 M HCl given bicarbonate. Compared with sham-operated rats, the (wt/vol) for 4 to 11 d and given water ad libitum; control rats were 49-kD UT-A protein abundance was significantly increased by pair-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 50% in liver from uremic, acidotic rats, which is consistent second group of HCl-fed rats were water-restricted by limiting their with our previous study (3); administering bicarbonate to ure- water intake to that of the pair-fed control rats. The water-restricted, mic rats reversed the increase in the 49-kD UT-A protein HCl-fed rats were pair-fed to the HCl-fed rats receiving water ad (Figure 1). libitum. Urea Feeding. To increase blood urea nitrogen (BUN), rats were HCl-Fed Rats fed standard chow supplemented with 30% urea for 7 d; control rats To determine whether acidosis alone can increase UT-A were pair-fed with standard chow (25). protein abundance, normal rats were fed HCl. Arterial blood pH and bicarbonate were reduced after 4, 6, 7, and 11 d of HCl Western Blot Analyses feeding (Table 2). The 49-kD UT-A protein in liver was Proteins (10.46 g/lane) were separated on 10% SDS-polyacryl- increased at each time point (Figure 2). amide gels and then transferred to a polyvinylidene difluoride mem- brane. Membranes were probed with our affinity-purified polyclonal antibody (3.8 mg/ml used at 1:5000 for Western blot) to UT-A (3,20). This antibody was prepared against the C-terminal portion of UT-A1 and will also detect UT-A2 and UT-A4 (7,20). The immunoreactive proteins were visualized by enhanced chemiluminescence (ECL; Am- ersham, Arlington Heights, IL) (3,20,21). Autoradiograms were scanned using the Bio-Rad Gel Doc 1000 digital imaging densitom- eter (Bio-Rad Laboratories, Hercules, CA). Scanned bands were quan- tified by 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 Ϯ SEM, and n ϭ number of rats. The t test was used to test for statistical significance between two Figure 1. Urea transporter protein abundance in livers from rats made groups. An ANOVA was used to test for statistical significance uremic by 5/6 nephrectomy. (Left panel) representative Western blot between three groups, followed by Tukey’s protected t test (26) to showing UT-A protein bands in liver from a sham-operated rat determine which groups were significantly different. The criterion for (Sham), a uremic rat (Uremic), and a uremic rat given bicarbonate statistical significance was P Ͻ 0.05. (Uremic ϩ Bicarb). Each lane represents a sample from a separate rat. (Right panel) densitometric summary of the 49-kD band on the gels. Results There was a significant increase in the density of the 49-kD band in Uremic Rats liver from uremic rats compared with control rats. There was no The rats that underwent a 5/6 nephrectomy had significantly change in the abundance of the 36-kD band (data not shown). Data are increased serum creatinine and BUN and decreased pH and mean Ϯ SE; n ϭ 6 rats/group; *P Ͻ 0.05. J Am Soc Nephrol 13: 581–587, 2002 Acidosis Upregulates UT-A in Liver During Uremia 583

Table 2. Arterial blood gas values in HCl-fed ratsa

Pair-Fed Control Rats HCl-Fed Rats Days HCl Feeding HCO HCO pH pCO 3 pH pCO 3 2 (mEq/L) 2 (mEq/L)

Day 4 7.29 44.6 21.1 7.05 34.9 9.5 Day 6 7.34 34.0 17.8 7.06 62.2 17.1 Day 7 7.34 47.8 25.3 7.20 54.4 20.6 Day 11 7.45 37.3 25.7 7.24 41.5 17.3

a Values shown are average of 2 rats/time point.

Additional rats were fed HCl for 7 d, and compared with UT-A2b and is expressed in hepatocyte membranes (3,8,12). pair-fed control rats, the rats fed HCl had significantly de- The goal of this study was to identify a mediator of the in vivo creased arterial blood pH, serum bicarbonate, and urine pH and increase in UT-A. Because uremia results in so many patho- significantly increased arterial blood pCO2 and urine urea physiologic changes, we chose to study metabolic acidosis excretion (Table 3). The abundance of the 49-kD liver UT-A because it can be manipulated and has been shown to be a protein was significantly increased by 36% (Figure 3). mediator of increased protein and amino acid catabolism and The HCl-fed rats also had a significant increase in urine increased gene transcription (23). Our major result was that volume (Table 3). Therefore, we measured UT-A1 and aqua- giving bicarbonate to uremic rats normalized blood pH and porin-2 (AQP2) protein abundances in the renal inner medulla blocked the increase in the abundance of the 49-kD UT-A (IM). In the IM tip, the abundance of the 117-kD UT-A1 protein in their livers. This response occurred despite no dif- protein was significantly increased by 129% and the abundance ference in dietary constituents (other than bicarbonate). More- of the 97-kD UT-A1 protein was unchanged (Figure 4). In the over, the major endproduct of dietary protein is urea; therefore, IM base, the 117-kD UT-A1 protein was nearly undetectable in our finding that BUN values between uremic acidotic rats and control rats but clearly present in HCl-fed rats; the abundance uremic nonacidotic rats were not statistically different (Table of the 97-kD UT-A1 protein was unchanged (Figure 5). AQP2 1) indicates that the acidosis, either directly or through a protein abundance was unchanged in the IM tip of control change in ammonium concentration, rather than the accumu- versus HCl-fed rats (data not shown). lation of urea (or other nitrogenous products), was the principal To determine whether the increase in UT-A1 protein in the cause of the increase in UT-A protein expression. This was kidney of HCl-fed rats was due to the increase in urine volume, confirmed when we studied HCl-fed, nonuremic rats; the abun- HCl-fed rats were water-restricted by limiting their water in- dance of the 49-kD UT-A protein was increased there as well. take to the amount drunk by the control rats (Table 4). In the IM tip, the abundance of the 117-kD UT-A1 protein was significantly increased and the abundance of the 97-kD UT-A1 protein was unchanged, which is similar to the results obtained in the HCl-fed rats drinking water ad libitum (Figure 6).

Urea-Fed Rats To determine whether an increase in BUN alone can in- crease UT-A in liver, rats were fed a standard protein diet to which urea was added. The urea-fed rats had a significantly higher BUN and urine urea excretion than pair-fed control rats (Table 4). There was no significant change in the abundance of the 49-kD UT-A protein in liver, the 117- or 97-kD UT-A1 proteins in the IM tip, or in AQP2 protein abundance in the IM tip of control versus urea-fed rats (data not shown).

Discussion Liver We previously showed that the liver expresses two UT-A Figure 2. Western blot showing UT-A protein bands in liver from rats proteins: a 49-kD protein that is present in the membrane and fed HCl (HCl) or pair-fed control (C) rats. Rats were fed HCl for the a 36-kD protein that is present in the cytoplasm (3). We also number of days indicated. The abundance of the 49-kD band is found that our model of uremia is associated with an increase increased in the HCl-fed rat at each time point. Each lane represents in the abundance of the 49-kD, but not the 36-kD, UT-A a sample from a separate rat; two HCl-fed and control rats were protein in rat liver (3). The 49-kD UT-A protein is most likely studied at each time point. 584 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 581–587, 2002

Table 3. Blood and urine chemistries in HCl-fed ratsa tions associated with an increase in muscle protein degrada- tion, such as acidosis, to facilitate urea transport out of hepa- Pair-Fed HCl-Fed Parameter Control Rats Rats tocytes. Consistent with this hypothesis, we found that urine urea excretion was significantly increased in the HCl-fed rats Initial weight (g) 253 Ϯ 20 244 Ϯ 22 (Table 3). However, previous studies of HCl-fed rats did not Final weight (g) 244 Ϯ 16 234 Ϯ 19 find an increase in urine urea excretion (24,34,35). Although Average food intake 24.9 Ϯ 0.6 27.6 Ϯ 0.7 the protocols for HCl-feeding differ, we do not have an expla- (wet wt, g/d) nation for this difference. HCl load (mmol/d) N/A 11.0 Ϯ 0.3 Arterial blood pH 7.35 Ϯ 0.01 7.12 Ϯ 0.02b Kidney Ϯ Ϯ b Arterial blood pCO2 44.5 1.5 51.4 2.3 UT-A1 exists as two glycoproteins, which run at 117 and 97 Ϯ Ϯ b Serum HCO3 (mEq/L) 24.2 0.9 16.9 0.6 kD, in rat inner medulla (36). Deglycosylation of rat inner Urine pH 7.28 Ϯ 0.04 5.82 Ϯ 0.05b medullary proteins results in a single 88-kD UT-A1 band (36). Urine volume (ml/d) 9.9 Ϯ 1.0 24.2 Ϯ 2.0b Both glycoproteins are expressed in the IMCD apical mem- Urine urea excretion 3.2 Ϯ 0.4 8.2 Ϯ 1.3b brane, and the reason for the two forms is not known. We (mmol/d) found that acidosis increased the abundance of the 117-kD, but a Data are mean Ϯ SE; n ϭ 8 rats/group. b Significantly different from pair-fed control rats at P Ͻ 0.01.

The percent increase (50%) in the 49-kD UT-A protein in uremic rat liver was somewhat greater than the percent increase (36%) in HCl-fed, nonuremic rat liver. This difference could be due to the larger change in pH in the uremic rats or to assorted consequences of acidosis and/or uremia. In short, our results strongly suggest that acidosis, either directly or through a change in ammonium concentration, is the principal cause of the upregulation of UT-A protein expression in rat liver. We also tested whether an increase in BUN would increase Figure 3. Urea transporter protein abundance in livers from rats fed UT-A protein in liver by feeding rats large quantities of urea HCl for 7 d. (Left panel) representative Western blot showing UT-A (25). The BUN of the urea-fed rats was significantly higher protein bands in liver from control (C) and HCl-fed (HCl) rats. Each than in the pair-fed control rats (25) but not as high as in the 5/6 lane represents a sample from a separate rat. (Right panel) densito- metric summary of the 49-kD band on the gels. There was a signifi- nephrectomized rats. We found that UT-A protein abundance cant increase in the density of the 49-kD band in HCl-fed rats was unchanged in liver, which suggests that an elevated BUN compared with pair-fed control rats. There was no change in the alone was not responsible for the increase in UT-A protein. abundance of the 36-kD band (data not shown). Data are mean Ϯ SE; However, we cannot exclude the possibility that a larger in- n ϭ 8 rats/group; *P Ͻ 0.05. crease in BUN would have an effect on UT-A protein. What is the physiologic advantage for an increase in hepatic UT-A protein expression since urea synthesis consumes two ammonium and two bicarbonate molecules and should not alter acid-base balance? One proposed function for a hepatic urea transporter is 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 primarily by the quantity of ammonium that needs to be removed (29,30), and studies by Mitch et al. (22,23,31–33) have shown that acidosis increases muscle protein breakdown, thereby increasing the nitrogen-containing waste products that need to be metabolized and excreted. Furthermore, they showed that correcting the acidosis in uremic rats reduced Figure 4. Urea transporter protein abundance in kidney inner medul- muscle protein degradation (23). Although metabolic acidosis lary (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) increases muscle glutamine synthetase and glutamine release, and HCl-fed (HCl) rats. Each lane represents a sample from a separate thereby shifting some nitrogen to renal ammoniagenesis rather rat. (Right panel) densitometric summary of the 117- and 97-kD than alanine formation and hepatic ureagenesis (34,35), muscle UT-A1 bands on the gels. There was a significant increase in the protein degradation increases nitrogen delivery to the liver and, density of the 117-kD band in HCl-fed rats compared with pair-fed in turn, increases ureagenesis (29,30). We speculate that UT-A control rats. There was no change in the abundance of the 97-kD band. protein expression may be upregulated in liver during condi- Data are mean Ϯ SE; n ϭ 4 rats/group; *P Ͻ 0.05. J Am Soc Nephrol 13: 581–587, 2002 Acidosis Upregulates UT-A in Liver During Uremia 585

Figure 5. Urea transporter protein abundance in kidney inner medul- lary (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 Figure 6. Urea transporter protein abundance in kidney inner medul- separate rat. (Right panel) densitometric summary of the 117- and lary (IM) tip from rats fed HCl and water-restricted for 7 d. (Left 97-kD UT-A1 bands on the gels. There was a significant increase in panel) representative Western blot showing UT-A1 protein bands in the density of the 117-kD band in HCl-fed rats compared with pair-fed IM tip from control (C), HCl-fed (HCl), and water-restricted HCl-fed control rats. There was no change in the abundance of the 97-kD band. (HCl/WR) rats. Each lane represents a sample from a separate rat. Data are mean Ϯ SE; n ϭ 4 rats/group; *P Ͻ 0.05. (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 not the 97-kD, UT-A1 protein in the kidney inner medullary rats, compared with pair-fed control rats. There was no significant tip. We previously showed that UT-A1 protein abundance is change in the abundance of the 97-kD band. Data are mean Ϯ SE; ϭ Ͻ increased in five conditions associated with increased urine n 3 rats/group; *P 0.05. volume: water diuresis, furosemide diuresis, low-protein diet, hypercalcemia, and adrenalectomy (reviewed in references 37 and 38). The HCl-fed rats also had an increased urine volume was also increased in both the inner medullary base and tip compared with the pair-fed control rats, which is consistent regions in diabetic rats that were studied two weeks after with earlier studies (24). However, the increase in the 117-kD streptozotocin injection (40). These rats were polyuric, but UT-A1 protein in the inner medullary tip of HCl-fed rats is not their pH was not measured. due simply to polyuria, because it was also increased in HCl- Lastly, we tested whether an increase in BUN would alter fed rats whose water intake was limited to the amount drunk by UT-A1 or AQP2 proteins in kidney inner medulla by feeding control rats. rats large quantities of urea (25). Despite significant changes in There are interesting differences between HCl-feeding and urine volume and urea excretion, both UT-A1 and AQP2 other polyuric conditions. One is that AQP2 protein is gener- protein abundances were unchanged. ally reduced in polyuric conditions (39), but in HCl-fed rats, In conclusion, we have identified that acidosis stimulates the we did not find a reduction in AQP2 protein in the inner expression of UT-A protein in the liver and kidney. It is medullary tip. Another difference is that the 117-kD UT-A1 tempting to conclude that the increased expression represents protein was clearly expressed in the inner medullary base of transcriptional stimulation as acidosis in uremia increases the HCl-fed rats; the inner medullary base typically expresses only transcription of genes involved in the ubiquitin-proteasome the 97-kD UT-A1 protein (reviewed in reference 37). The pathway, at least in muscle (23,32,33,41). Regardless, the abundance of the 117-kD, but not the 97-kD, UT-A1 protein responses of the liver and kidney, coupled with those in muscle

Table 4. Water-restricted HCl-fed ratsa

Parameter Control HCl-Fed Water-Restricted, HCl-Fed

Initial weight (g) 167 Ϯ 5 168 Ϯ 6 163 Ϯ 5 Final weight (g) 169 Ϯ 6 168 Ϯ 13 156 Ϯ 4 Water intake (ml/d) 22.7 Ϯ 1.2 49.7 Ϯ 3.7b 25.3 Ϯ 3.2 Urine volume (ml/d) 7.7 Ϯ 2.0 28.8 Ϯ 0.8b 7.0 Ϯ 1.6 Arterial blood pH 7.31 Ϯ 0.03 7.20 Ϯ 0.03b 7.22 Ϯ 0.01b Ϯ Ϯ Ϯ Arterial blood pCO2 50.8 1.3 44.0 1.6 45.5 3.5 Urine pH 8.43 Ϯ 0.64 7.77 Ϯ 0.34 6.95 Ϯ 0.57

a Data are mean Ϯ SE; n ϭ 3 rats/group. b Significantly different from control rats at P Ͻ 0.05. 586 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 581–587, 2002

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