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Am J Physiol Renal Physiol 293: F1238-F1247, 2007. First published July 25, 2007; doi:10.1152/ajprenal.00151.2007
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Effect of reduced renal mass on renal ammonia transporter family, Rh C glycoprotein and Rh B glycoprotein, expression

Hye-Young Kim,1,2 Chris Baylis,1,3 Jill W. Verlander,1 Ki-Hwan Han,4 Sirirat Reungjui,1 Mary E. Handlogten,1 and I. David Weiner1,5

1Division of Nephrology, Hypertension and Transplantation, University of Florida College of Medicine, Gainesville, Florida; 2Chungbuk National University College of Medicine, Cheongju, Korea; 3Department of Physiology, University of Florida College of Medicine, Gainesville, Florida; 4Department of Anatomy, Ewha Womans University, Seoul, Korea; and 5Nephrology and Hypertension Section, North Florida/South Georgia Veterans Health System, Gainesville, Florida

Submitted 30 March 2007 ; accepted in final form 24 July 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Kidneys can maintain acid-base homeostasis, despite reduced renal mass, through adaptive changes in net acid excretion, of which ammonia excretion is the predominant component. The present study examines whether these adaptations are associated with changes in the ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). We used normal Sprague-Dawley rats and a 5/6 ablation-infarction model of reduced renal mass; control rats underwent sham operation. After 1 wk, glomerular filtration rate, assessed as creatinine clearance, was decreased, serum bicarbonate was slightly increased, and Na+ and K+ were unchanged. Total urinary ammonia excretion was unchanged, but urinary ammonia adjusted for creatinine clearance, an index of per nephron ammonia metabolism, increased significantly. Although reduced renal mass did not alter total Rhcg protein expression, both light microscopy and immunohistochemistry with quantitative morphometric analysis demonstrated hypertrophy of both intercalated cells and principal cells in the cortical and outer medullary collecting duct that was associated with increased apical and basolateral Rhcg polarization. Rhbg expression, analyzed using immunoblot analysis, immunohistochemistry, and measurement of cell-specific expression, was unchanged. We conclude that altered subcellular localization of Rhcg contributes to adaptive changes in single-nephron ammonia metabolism and maintenance of acid-base homeostasis in response to reduced renal mass.

intercalated cell; principal cell; cortical collecting duct; outer medullary collecting duct

REDUCED RENAL MASS IS ASSOCIATED with maintenance of fluid and electrolyte homeostasis until very substantial changes in glomerular filtration rate develop. For many components of fluid and electrolyte homeostasis, such as H2O, Na+, and phosphate, homeostasis involves decreased tubular reabsorption that increases fractional excretion until there are appropriate rates of renal excretion. However, acid-base homeostasis requires fundamentally different mechanisms. Renal ammonia excretion, the predominant component of renal net acid excretion (10, 18, 42), does not depend on glomerular filtration of ammonia (37, 39). Instead, ammonia metabolism involves coordinated intrarenal ammonia production and epithelial segment transport (10, 18, 28). Thus maintenance of normal ammonia excretion in the presence of decreased nephron number associated with reduced renal mass requires adaptive changes in individual renal epithelial segment ammonia metabolism.

Recent evidence suggests that the nonerythroid ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), are important for renal ammonia metabolism. Rhbg and Rhcg transport ammonia (2, 36, 60) and are expressed in the distal convoluted tubule, connecting segment, initial collecting tubule, cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD) (11, 20, 40, 44, 51), the sites responsible for secreting the majority of urinary ammonia (18, 42). In vitro studies using cultured mouse collecting duct cells, mIMCD-3, that express Rhbg and Rhcg show that plasma membrane ammonia transport involves carrier-mediated mechanisms with functional characteristics similar to those identified for Rhbg and Rhcg (21, 22). Finally, in metabolic acidosis, a condition in which there is increased ammonia metabolism, there is enhanced Rhcg expression (44, 45).

The present study examines whether reduced renal mass alters single nephron ammonia metabolism and whether this is associated with altered expression of the ammonia transporter family members, Rhbg and Rhcg. We used normal Sprague-Dawley rats and a 5/6 ablation-infarction model of reduced renal mass; control rats underwent sham surgery without ablation-infarction. After 1 wk, we examined the physiological response to decreased nephron number in terms of acid-base and electrolyte homeostasis. We then examined the effect of reduced renal mass on Rhbg and Rhcg expression and localization.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Animals

Normal Sprague-Dawley rats weighing 350–400 g were divided into sham and reduced renal mass groups. The 5/6 ablation-infarction and sham surgery were performed under general anesthesia using 1.5–5% isoflurane, as described previously (12). Briefly, 5/6 ablation-infarction involved removal of the right kidney and ligation of two of the three branches of the left renal artery. In sham-operated animals, both kidneys were accessed and gently manipulated, but nephrectomy and renal artery ligation were not performed. After surgery, animals were allowed free access to water and rodent chow. All studies were approved by the University of Florida College of Medicine Institutional Animal Care and Use Committee.

On postoperative day 6, animals were placed in metabolic cages, and urine was collected under mineral oil for 24 h. The urine volume was recorded, and an aliquot was frozen for later analysis. Blood was taken from the abdominal aorta for serum collection. After centrifugation, serum was stored at –80°C until analyzed. Kidneys were removed, and cortex, outer medulla, and inner medulla were dissected. The tissues were homogenized with Tissue Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL). The sample was then centrifuged at 14,000 rpm for 15 min at 4°C and stored at –80°C until analyzed.

Antibodies

Affinity-purified antibodies to rodent Rhbg and Rhcg have been characterized previously (20, 23, 44, 45, 51, 55). Antibodies to the B1/B2 subunit of H+-ATPase were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Immunoblot Analysis

Immunoblot analysis was performed using renal tissue lysate protein (25 µg/lane) and visualized using enhanced chemiluminescence (SuperSignal West Pico Substrate, Pierce Biotechnology) and a Kodak Image Station 440CF digital imaging system, as described previously (20–22, 44, 51, 55). Band density was quantified using Kodak 1D, version 3.5.4 software (Kodak Scientific Imaging Systems, New Haven, CT).

Blood and Urine Analysis

Serum and urine electrolytes, urea nitrogen, and creatinine were determined using the VetACE clinical chemistry system (Alfa Wassermann, West Caldwell, NJ). Ammonia (Ammonia Reagent Set, Pointe Scientific, Canton, MI) and aldosterone (Aldosterone EIA Kit, Cayman Chemical, Ann Arbor, MI) concentrations were measured using commercially available assays.

Tissue Processing for Immunohistochemistry

On the day of fixation, animals were anesthetized with isoflurane inhalation, and the kidneys were preserved by in vivo perfusion fixation through the abdominal aorta. Briefly, the distal aorta was cannulated, and blood pressure was measured (Gilson Physiography, Middleton, WI). The kidneys were then perfused briefly with PBS (pH 7.4) to rinse away all blood and, subsequently, with periodate-lysine-2% paraformaldehyde for 7 min. The kidneys were removed, cut transversely into 2- to 4-mm-thick slices, and immersed overnight at 4°C in the same fixative. Samples of kidney from each animal were embedded in polyester wax (polyethylene glycol 400 distearate, Polysciences, Washington, PA), and 3-µm-thick sections were cut and mounted on gelatin-coated glass slides.

General Histological Examination

Routine hematoxylin and eosin staining of tissue sections was performed in the clinical laboratory of the Gainesville Veterans Affairs Medical Center.

Immunohistochemistry

Immunolocalization of Rhbg and Rhcg was accomplished using immunoperoxidase procedures and a commercially available kit (Mach2, Biocare Medical, Concord, CA) using techniques described in detail previously (33, 44, 51, 55, 59). Anti-Rhbg and anti-Rhcg antibodies were used at 1:2,000 and 1:30,000 dilution, respectively. Tissue was examined on a Nikon E600 microscope equipped with differential interference contrast optics and photographed using a DXM1200F digital camera and ACT-1 software (Nikon USA).

Double-Labeling Procedure

Double-labeling was performed using sequential immunoperoxidase procedures and commercially available kits (Mach2, Biocare Medical, Concord, CA, and Vector-SG, Vector Laboratories, Burlingame, CA) using techniques described previously (20, 23, 44, 51, 55).

Quantitative Analysis of Immunohistochemistry

High-resolution, 36-megapixel, digital micrographs were taken of defined tubular segments using a Nikon E600 microscope equipped with a DXM1200F digital camera and ACT-1 software (Nikon USA). Differential interference contrast optics and other image enhancement techniques were not used to avoid changes in pixel intensity other than due to Rhbg and Rhcg immunoreactivity. Freely available software (NIH ImageJ, version 1.34s) quantified pixel intensity across a line drawn from the tubule lumen through the center of an individual cell. These data were then analyzed using custom-written software. Pixel intensity at each point of the line was displayed graphically. The apical and basolateral edges were determined by the user as the point at which intensity diverged from baseline. Background pixel intensity was calculated as mean pixel intensity outside the cell regions and was subtracted from the pixel intensity at each point to yield net intensity. Total cellular expression was determined by integrating net pixel intensity through the entire cell. Cell height was determined as the distance in pixels between the apical and basolateral edges of the cells. Immunoreactivity expression in the apical and basolateral 25% of the cell was determined by integrating pixel intensity in these respective regions of the cell. In preliminary experiments, there was <5% variability in these measurements during repetitive analysis of a single cell.

The individual performing the microscopy, photography, and quantitative analysis was blinded to the treatment status of the animal. A minimum of five tubules in the CCD, OMCD, and IMCD were selected randomly in regions removed from the infarct area to minimize artifactual responses possibly related to altered renal perfusion in the kidney tissue immediately adjacent to the infarct zone and were digitally photographed. We then averaged data from all cells examined of a given type, i.e., CCD principal cell, in an individual kidney to yield a single data point for statistical analysis.

Statistics

Results are presented as means ± SD. Statistical analyses were performed using Student's unpaired t-test, and P < 0.05 was taken as statistically significant. N refers to the numbers of animal studied.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Physiological Parameters

Physiological and serum data are summarized in Table 1. Serum urea nitrogen and creatinine in reduced renal mass rats were significantly higher than in controls. There were no significant differences in plasma sodium or potassium concentrations. Plasma bicarbonate level was slightly elevated in reduced renal mass rats compared with control rats. Plasma aldosterone levels were increased significantly in reduced renal mass rats.


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  		Table 1. Serum chemistries and electrolytes in response to reduced renal mass

 
Table 2 summarizes studies based on 24-h urine collections. Creatinine clearance was decreased ~60% in response to reduced renal mass compared with control. Consistent with the lack of substantial change in acid-base balance, 24-h urinary ammonia excretion rates were not significantly different in reduced renal mass rats compared with controls. However, urinary ammonia excretion adjusted for creatinine clearance was increased significantly, indicating adaptive changes in individual nephron ammonia metabolism. Urinary sodium, potassium, and urea excretion were unchanged in response to reduced renal mass, but fractional excretion of sodium, potassium, and urea were increased significantly.


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  		Table 2. 24-h Urine parameters in response to reduced renal mass

 
General Histological Response

We used hematoxylin and eosin staining of kidney sections to characterize the renal histological response to reduced renal mass (Fig. 1). After 1 wk of reduced renal mass, the histological appearance of the portion of the noninfarcted region of kidney appeared relatively normal, without evident edema or interstitial fibrosis. However, generalized hypertrophy was evident in both the CCD and the OMCD.


Figure 1
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  		Fig. 1. Histological response to reduced renal mass (RRM). General histological examination of control and RRM kidneys performed using hematoxylin and eosin staining. A and B: low-power micrographs of renal cortex from control, sham-operated, and RRM kidneys, respectively. No edema or interstitial fibrosis is evident in the uninfarcted portions of the RRM kidney. C: high-power micrograph of cortical collecting duct (CCD) from control kidney demonstrating a relatively flat epithelium (asterisks denote tubule lumen in C–F). D: high-power micrograph of CCD in RRM kidney with substantial generalized hypertrophy. E: high-power micrograph of outer medullary collecting duct (OMCD) from control kidney demonstrating a relatively flat epithelium. F: high-power micrograph of OMCD from RRM kidney demonstrating substantial generalized hypertrophy.

 
Rhcg Protein Expression

By immunoblot analysis, reduced renal mass did not alter steady-state Rhcg protein expression significantly in the cortex, outer medulla, or inner medulla (Fig. 2).


Figure 2
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  		Fig. 2. Rh B glycoprotein (Rhcg) and Rh C glycoprotein (Rhbg) expression in response to RRM. A and B: immunoblot for Rhcg. A: immunoblot of protein from renal cortex of control (sham) and RRM kidneys. B: mean densitometries from cortex, outer medulla, and inner medulla are shown, where band density is relative to mean (100%) of expression in control kidneys. There were no significant differences between the RRM and controls in Rhcg protein expression in any region. C and D: immunoblot for Rhbg. C: immunoblot of protein from renal cortex of control and RRM kidneys. D: mean densitometries from cortex, outer medulla, and inner medulla. There were no significant differences between the RRM and controls in Rhbg protein expression in any region. Data are presented as means ± SD; n = 5 per group. NS, not significant.

 
Rhcg Immunolocalization

Rhcg expression is regulated by both changes in cell-specific expression and changes in its subcellular distribution (44, 45). To determine whether similar changes might occur in response to reduced renal mass, we examined Rhcg's expression using immunohistochemistry. We observed both apical and basolateral Rhcg immunoreactivity throughout the CCD, OMCD, and IMCD in both control and reduced renal mass kidneys (Fig. 3). Both intercalated cells and principal cells in the CCD and OMCD, but not the IMCD, appeared to have a greater cell height in reduced renal mass than in control kidneys. Furthermore, apical Rhcg immunoreactivity in intercalated cells was more discrete, that is, there was a narrower, more distinct line of apical immunolabel in reduced renal mass animals compared with the generally broader, more diffuse band of staining observed in control animals.


Figure 3
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  		Fig. 3. Rhcg immunoreactivity in response to RRM. A: CCD of control kidney. CCD in control kidneys are a relatively flat epithelium, with little difference between the height of intercalated cells, with intense apical Rhcg immunoreactivity (arrows) and principal cells (arrowhead). B: CCD in RRM kidney. All CCD cells, both intercalated cells (arrows) and principal cells (arrowheads), are hypertrophic compared with control kidney CCD cells. In addition, apical Rhcg immunoreactivity appears to be more discrete than in control kidneys. C: OMCD in control kidney. Both intercalated cells (arrows) and principal cells express Rhcg immunoreactivity. In intercalated cells, apical Rhcg immunoreactivity is a broad, diffuse band. D: OMCD in RRM kidney. Both intercalated cells (arrows) and principal cells (arrowheads) are hypertrophic compared with control kidney OMCD. In addition, apical Rhcg immunoreactivity in intercalated cells (arrows) appears to be more discrete than in control OMCD. E: inner medullary collecting duct (IMCD) in control kidney. Intercalated cells with apical and basolateral Rhcg immunoreactivity are relatively flat epithelial cells. F: IMCD in RRM kidney. Intercalated cells are similar in height and Rhcg immunoreactivity as in control kidneys. All panels are representative of findings in 6 control and 6 RRM kidneys and use Rhcg antibody at 1:30,000 dilution. All panels were photographed and reproduced at identical magnifications.

 
A-type intercalated cells in the CCD, OMCD, and IMCD have more intense apical Rhcg immunoreactivity than do nonintercalated cells under basal conditions. To confirm that a similar pattern is present in the reduced renal mass kidney, we colabeled Rhcg with AE1 to identify A-type intercalated cells. In the CCD, OMCD, and the IMCD, AE1-positive intercalated cells exhibited more intense apical Rhcg immunoreactivity than observed in adjacent AE1-negative cells (Fig. 4).


Figure 4
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  		Fig. 4. Double-labeling of Rhcg with AE1 in RRM kidneys. A: CCD. In the CCD, cells with intense apical Rhcg (blue) exhibit basolateral AE1 (brown), a marker of the A-type intercalated cell (arrow). B: OMCD. In the OMCD, cells with intense apical Rhcg immunoreactivity (blue) also exhibit basolateral AE1 immunoreactivity (brown), identifying these cells as A-type intercalated cells (arrows). C: IMCD. In the IMCD, intercalated cells (arrow) with basolateral AE1 immunoreactivity (brown) exhibit intense apical Rhcg immunoreactivity (blue). In all regions, basolateral Rhcg cannot be easily seen because of colocalization with AE1 and the more intense brown dye product generated by AE1 immunolocalization.

 
Quantitative Immunohistochemical Analysis

We next quantified the changes in cell height, cell-specific Rhcg expression, and subcellular Rhcg distribution observed by light microscopy. Figure 5 summarizes this technique. To verify that this technique yields similar results as immunogold electron microscopy, we compared the two techniques using kidney samples from our laboratory's recently reported metabolic acidosis study (44, 45). Table 3 shows that there was excellent concordance between these two analytic techniques when examining cell-specific, apical, and basolateral Rhcg expression OMCD intercalated cells and principal cells.


Figure 5
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  		Fig. 5. Quantitative immunohistochemistry analysis. Individual micrographs are analyzed using ImageJ software (version 1.34s, NIH, http://rsb.info.nih.gov/ij). A: pixel intensity is determined along a line drawn perpendicular to the cell. Pixel intensity values are then electronically transferred to custom-written data analysis software. B: the edges of the cell (arrows) are identified as the point at which immunoreactivity increases from baseline (arrows). Cell height is calculated as the number of pixels between the apical and basolateral edges of the cell. Total cellular immunoreactivity is determined by integrating pixel intensity inside the cell borders.

 

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  		Table 3. Changes in cellular and subcellular Rh C glycoprotein: comparison of immunogold electron microscopy and digital light microscopy

 
Digital morphometric analysis was then used to quantify the effects of reduced renal mass on cell height and on cell-specific and subcellular expression protein expression.

Cell height. Cell height of both intercalated and principal cells in the CCD and OMCD was increased significantly in response to reduced renal mass (Table 4). In the IMCD, intercalated cell height was not statistically changed, and height of nonintercalated cells, i.e., the IMCD cell and principal cell, could not be quantified, because nonintercalated cells in the IMCD cell lack detectable Rhcg immunoreactivity (11, 44, 51). Thus there is hypertrophy of both intercalated cells and principal cells in the CCD and OMCD in response to reduced renal mass.


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  		Table 4. Relative changes in cell height in response to reduced renal mass

 
Cell-specific Rhcg expression. The absence of changes in Rhcg protein expression by immunoblot assay could indicate no changes in any cells or that change in one cell counterbalanced opposing changes in another. To differentiate these possibilities, we quantified cell-specific Rhcg expression. Table 5 summarizes these results. Reduced renal mass decreased total Rhcg immunoreactivity in the CCD intercalated cell and increased total Rhcg immunoreactivity in the CCD principal cell significantly. Otherwise, Rhcg immunoreactivity was not changed significantly in other cell types.


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  		Table 5. Changes in cell-specific Rhcg and Rhbg expression in response to reduced renal mass

 
Subcellular Rhcg expression. Reduced renal mass significantly increased apical Rhcg immunoreactivity in CCD A-type intercalated cell, CCD principal cell, OMCD intercalated cell, and the OMCD principal cell, but not in the IMCD intercalated cell. Basolateral Rhcg expression increased significantly in the CCD principal cell, the OMCD intercalated cell, and the OMCD principal cell. Basolateral Rhcg expression was not significantly altered in the CCD A-type intercalated cell or the IMCD intercalated cell. Table 6 summarizes these changes.


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  		Table 6. Apical and basolateral Rhcg expression in response to reduced renal mass

 
Rhbg Protein Expression

Rhbg is a second member of the renal ammonia transporter family and is expressed in the same cellular distribution as Rhcg, but with basolateral-specific expression (34, 40, 51). Reduced renal mass did not change Rhbg protein expression in the cortex, outer medulla, or inner medulla (Fig. 2).

Rhbg Immunolocalization

Basolateral Rhbg immunoreactivity was present in both control and reduced renal mass rat kidneys in the CCD, OMCD, and in a subset of cells in the IMCD. Reduced renal mass did not induce detectable differences in Rhbg immunoreactivity or localization (Fig. 6). Colabeling Rhbg with H+-ATPase confirmed that the cells with more intense Rhbg immunoreactivity were intercalated cells (Fig. 7), consistent with the pattern under normal conditions.


Figure 6
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  		Fig. 6. Rhbg immunoreactivity in the cortex and the medulla in response to RRM. A: CCD in control kidney. Basolateral Rhbg immunoreactivity is present in both intercalated cells (arrows) and principal cells (arrowheads). Under control conditions, the CCD is a relatively flat epithelium. B: CCD in RRM kidney. There is hypertrophy of all cells, with increased protrusion into the tubule lumen for both intercalated cells (arrows) and principal cells (arrowhead). C: OMCD in control kidney. Intense basolateral Rhbg reactivity is present in intercalated cells (arrows), and less intense basolateral immunoreactivity is present in OMCD principal cells (arrowhead). D: OMCD in RRM kidney. Intense basolateral immunoreactivity is present in intercalated cells (arrows), and less intense basolateral immunoreactivity is present in principal cells (arrowhead). There are no evident differences in Rhbg immunoreactivity between control and RRM kidneys. All micrographs are taken and reproduced at the same magnification.

 

Figure 7
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  		Fig. 7. Colocalization of Rhbg (brown) and vacuolar H+-ATPase (blue) in the cortex in response to RRM. A: CCD in control kidney. A-type intercalated cells with apical H-ATPase (black arrow) and principal cells (arrowhead) exhibited intense and moderate-intensity basolateral Rhbg immunoreactivity, respectively. B-type intercalated cells, with diffuse H-ATPase immunoreactivity, do not exhibit detectable Rhbg immunoreactivity (white arrow) (51). B: CCD in RRM kidney. A-type intercalated cells (black arrow) with apical H+-ATPase immunoreactivity (blue) exhibit intense basolateral Rhbg immunoreactivity. Principal cells (arrowhead) exhibit less intense basolateral Rhbg immunoreactivity, and B-type intercalated cells with diffuse H+-ATPase immunoreactivity (white arrow) do not exhibit detectable Rhbg immunoreactivity. Other than hypertrophy of A-type intercalated cells and principal cells, there are no detectable differences from that observed in CCD from control kidney. C: OMCD in control kidney. Intercalated cells exhibit intense basolateral Rhbg immunoreactivity (arrows). D: OMCD in RRM kidney. Similar to OMCD in control kidney, intercalated cells (arrow) exhibit intense basolateral Rhbg immunoreactivity, whereas principal cells exhibit less intense basolateral Rhbg. Other than hypertrophy of intercalated cells, there are no detectable differences between RRM and control kidney OMCD Rhbg immunoreactivity. All micrographs were obtained and reproduced at identical magnifications.

 
Cell-Specific Quantification of Rhbg Expression

The absence of changes in Rhbg expression by immunoblot analysis and by immunohistochemistry suggests there are no cell-specific changes in Rhbg expression. To confirm this, we quantified cell-specific Rhbg immunoreactivity in intercalated cells and principal cells in the CCD and OMCD. Table 5 summarizes these results. Reduced renal mass did not result in cell-specific changes in Rhbg expression.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study provide new observations into the adaptive responses to reduced renal mass that assist in maintenance of ammonia excretion and thus acid-base homeostasis. In response to reduced renal mass, both normal acid-base homeostasis and normal total urinary ammonia excretion were maintained, despite a substantial decrease in nephron number, indicating substantial increases in individual nephron ammonia metabolism. This was associated with hypertrophy of both intercalated cells and principal cells in the CCD and OMCD, and with increased polarization of Rhcg to the apical and basolateral plasma membranes. Rhbg, in contrast to Rhcg, did not undergo detectable changes in either expression or localization. These responses are likely to be important in the maintenance of ammonia excretion and acid-base homeostasis in response to reduced renal mass.

Ammonia metabolism is the primary component of renal net acid excretion, both under basal conditions and in response to exogenous acid loads, and its metabolism involves both ammoniagenesis and integrated intrarenal transport (18, 26, 28). Previous studies show, in a variety of models of reduced renal mass, that renal ammonia excretion is either unchanged (present study) or decreases less than the reduction in renal mass (4, 7, 8, 13, 35, 43), indicating adaptive changes in single-nephron ammonia metabolism. The findings in the present study are consistent with these previous results.

The quantitative importance of collecting duct ammonia secretion is emphasized by the observation that ~80% of urinary ammonia derives from collecting duct secretion and only ~20% from luminal ammonia delivery to the collecting duct (18, 42). Some studies have shown that reduced renal mass increases the proportion of kidney-generated ammonia that is secreted into the urine, suggesting increased collecting duct ammonia secretion (7, 8), while another suggested that reduced renal mass decreased collecting duct ammonia secretion (5). This latter study differed from the present study in their use of Munich-Wistar instead of Sprague-Dawley rats, and their technique was used to reduce renal mass. Also, Ref. 5 demonstrated decreased total urinary ammonia excretion and the development of metabolic acidosis, which contrasts to the maintained urinary ammonia excretion and the absence of metabolic acidosis in the present study.

Reduced renal mass is associated with several changes that are likely to contribute to increased single-nephron ammonia excretion. There is hypertrophy of the remaining kidney (25, 32), and this is associated with generalized tubular and collecting duct hypertrophy (32). The present study adds to these findings by demonstrating that the collecting duct hypertrophy involves both intercalated and principal cell hypertrophy. A second alteration that may contribute to increased single-nephron ammonia secretion is increased apical polarization of H+-ATPase (3). Finally, the present study identifies increased apical and basolateral polarization of Rhcg. Because collecting duct ammonia secretion involves parallel H+ and NH3 transport (14, 15, 19, 29, 30), the identification of increased apical plasma membrane polarization of both H+-ATPase and Rhcg suggests that coordinated changes in H+ and NH3 transport contribute to the increased individual nephron ammonia metabolism.

Changes in the subcellular distribution of Rhcg expression are an important regulatory mechanism. In chronic metabolic acidosis, there is increased targeting of Rhcg to the apical plasma membrane of both the OMCD intercalated cell and principal cell and increased expression in the basolateral plasma membrane of the OMCD principal cell (45). The present study extends these observations by demonstrating changes in Rhcg polarization can develop in the CCD, as well as the OMCD, and can occur in response to conditions other than metabolic acidosis. Changes in Rhcg's subcellular distribution may be a general adaptive mechanism used to increase Rhcg-mediated ammonia transport.

Basolateral Rhcg immunoreactivity is present in the rat and human collecting duct (20, 44, 45). Moreover, conditions associated with increased single-nephron ammonia metabolism, including both chronic metabolic acidosis (45) and reduced renal mass (present study), are associated with increased basolateral Rhcg expression. Thus basolateral Rhcg appears to contribute to regulated ammonia secretion.

Reduced renal mass did not alter total Rhcg protein expression in the cortex, outer medulla, or inner medulla, which contrasts with metabolic acidosis where Rhcg expression increased in the outer and inner medulla (44). However, the relative increase in single-nephron ammonia metabolism in response to reduced renal mass, approximately threefold, is less than observed in response to chronic metabolic acidosis, ~25-fold (44). More severe reductions of reduced renal mass, where an increase in single-nephron ammonia metabolism might be greater, might be associated with increased Rhcg expression.

Rhbg expression, in contrast to Rhcg, did not change detectably with reduced renal mass. Similarly, Rhbg expression does not change in metabolic acidosis (45). These observations suggest either that Rhbg is not necessary for transepithelial ammonia secretion, or that mechanisms independent of protein expression regulated Rhbg. The observation that genetic deletion of Rhbg does not detectably alter renal ammonia metabolism (6) suggests Rhbg is not necessary for ammonia secretion.

Several previous studies have examined distal renal H+ and bicarbonate transport in response to reduced renal mass. In vivo micropuncture studies showed increased net H+ secretion in the rat superficial distal tubule and in the collecting duct distal to the late distal tubule (5, 31). This increased proton secretion may be due to increased apical polarization of vacuolar H+-ATPase (3). In contrast, studies using in vitro microperfused rabbit CCD and OMCD from remnant kidneys did not detect changes in net proton secretion (17). Possible explanations for these differences could include either species-dependent differences or the use of in vitro vs. in vivo model systems.

Increasing evidence suggests that collecting duct principal cells contribute to acid-base homeostasis. Principal cells exhibit apical H+ secretion (53, 56), basolateral Cl–/HCO3– exchange activity (54, 56), and carbonic anhydrase immunoreactivity (9, 41, 51). They express the H+-K+-ATPase {alpha}-subunits, HK{alpha}2A and HK{alpha}2C (1, 52), and the ammonia transporter family members, Rhbg and Rhcg (44, 45, 51). Mineralocorticoids increase OMCD principal cell apical proton secretion (56), and both reduced renal mass (present study) and metabolic acidosis (45) increase OMCD principal cell apical and basolateral Rhcg expression. Thus collecting duct principal cells may contribute to transcellular acid-base and ammonia transport and, thereby, to acid-base homeostasis.

Reduced renal mass in the present study was associated with a slight, but significant, increase in plasma bicarbonate concentration. This finding is consistent with some reports (3), but differs from others that reported either mild metabolic acidosis (5, 41) or no change in acid-base homeostasis (17). These conflicting results may be due to differences in the severity or reduced renal mass, the dietary protein content, or other technical aspects. For example, reduced renal mass combined with a high-protein diet results in metabolic acidosis (36). In the present study, the increased plasma bicarbonate may partly be due to the hyperaldosteronism observed, since aldosterone stimulates renal ammonia metabolism and can lead to generation of metabolic alkalosis (47, 57).

Reductions in renal mass are well known to induce adaptive renal hypertrophy. Renal DNA synthesis increases within 6 h after uninephrectomy (49), and RNA synthesis increases within 12 h (48). Kidney size is increased within 1–2 days, and the hypertrophy is maximal within 1–2 wk (27). These adaptive responses include hypertrophy at 1 wk in both the proximal tubule (46) and the collecting duct (Ref. 50 and present study). Many factors are likely to be involved in this hypertrophic response (58); which are involved in the collecting duct response cannot be determined definitively from the current studies. Importantly, the responses observed in the present study are unlikely to be due to differences in dietary intake. Urinary sodium, potassium, and urea excretion did not statistically differ in the reduced renal mass and sham-operated groups, suggesting food intake was similar in the two groups. One likely contributor to the collecting duct hypertrophic response is the increase in plasma aldosterone. Both a previous study (50) and the present study document that reduced renal mass increases plasma aldosterone. Aldosterone levels increase, despite the elevated fractional excretion of sodium and normal serum K+, as a result of stimulation of the renin-angiotensin system (16). These changes in aldosterone appear to be critical for collecting duct hypertrophy; aldosterone clamp studies examining the entire CCD, without differentiating between principal cells and intercalated cells, document that CCD hypertrophy in response to reduced renal mass is aldosterone dependent (50). Finally, metabolic acidosis, which also induces collecting duct hypertrophy (24), is also associated with elevations in plasma aldosterone level (38).

In summary, reduced renal mass is associated with increased single-nephron ammonia metabolism, hypertrophy of both intercalated cells and principal cells in the collecting duct, and altered apical and basolateral Rhcg expression in both intercalated cells and principal cells in the collecting duct. These changes are likely to contribute to the increased single-nephron ammonia secretion and thereby contribute to the maintenance of normal acid-base homeostasis observed late in the course of reduced renal mass. Rhbg expression is not detectably changed, suggesting that it either is not involved in transepithelial ammonia secretion or is regulated by mechanisms independent of total protein expression and cellular distribution. These observations provide important new insights into the physiological mechanisms by which normal acid-base homeostasis is maintained in response to reduced renal mass.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by funds from the National Institutes of Health (DK-45788, DK-56843, and NS-47624), the Department of Veterans Affairs Merit Review Program, an International Society of Nephrology fellowship award (to H. Y. Kim), and the Post-doctoral Fellowship Program of Korea Science and Engineering Foundation (to H. Y. Kim).


   ACKNOWLEDGMENTS
 
The authors thank Gina Cowsert for secretarial assistance and Jeanice Jaroll of the University of Florida College of Medicine Electron Microscopy Core for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. D. Weiner, Division of Nephrology, Hypertension and Transplantation, Univ. of Florida College of Medicine, Gainesville, Florida (e-mail: weineid{at}ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
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 RESULTS
 DISCUSSION
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 REFERENCES
 

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