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Am J Physiol Renal Physiol 294: F433-F439, 2008. First published December 5, 2007; doi:10.1152/ajprenal.00436.2007
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Renal localization and regulation of 15-hydroxyprostaglandin dehydrogenase

Bing Yao, Jie Xu, Raymond C. Harris, and Ming-Zhi Zhang

Division of Nephrology, Vanderbilt University School of Medicine, Nashville, Tennessee

Submitted 17 September 2007 ; accepted in final form 3 December 2007


   ABSTRACT
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Tissue prostaglandin levels are determined by both biosynthesis and catabolism. The current studies report the expression and localization of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin catabolism in the kidneys. We also investigated potential interactions between 15-PGDH and cyclooxygenase (COX), a key enzyme in prostaglandin biosynthesis. Both 15-PGDH mRNA and protein levels were significantly higher in kidney cortex than in papilla, which is opposite to the expression pattern of COX-2. In situ hybridization indicated that 15-PGDH mRNA was mainly localized to the tubular epithelial cells in kidney cortex and outer medulla but not in the glomerulus or papilla. Dual immunofluorescent staining indicated that 15-PGDH was expressed in the proximal tubule, cortical, and outer medullary thick ascending limb and collecting duct but not in the macula densa or papilla. 15-PGDH levels were significantly lower in a macula densa cell line (MMDD1) than in a proximal tubule cell line. Although a high-salt diet decreased COX-2 expression in macula densa, it increased macula densa 15-PGDH expression in both mouse and rat kidneys. In MMDD1 cells, a COX-2 inhibitor increased 15-PGDH, whereas a COX-1 inhibitor had no effect. Furthermore, intense 15-PGDH immunofluorescent staining was found in both macula densa and glomerulus in COX-2 knockout mice. The intrarenal distribution of 15-PGDH and its interactions with COX-2 suggest that differential regulation of COX-2 and 15-PGDH may play an important role in determining levels of prostaglandins involved in regulation of salt, volume, and blood pressure homeostasis.

kidney; prostaglandin catabolism; cyclooxygenase; macula densa

IN ADULT MAMMALIAN KIDNEY, prostaglandins regulate renal hemodynamics and salt and water homeostasis (11). Prostaglandin production depends on the release of arachidonic acid from membrane phospholipids by specific phospholipases and subsequent conversion to prostaglandin H2 (PGH2) by cyclooxygenase (COX). Further metabolism by specific synthases produces individual prostanoid species. COX exists in two isoforms: constitutive COX-1 and inducible COX-2 (11, 12). In kidney cortex, COX-2 is localized to macula densa and adjacent cortical thick ascending limbs (cTAL), and COX-2-derived prostaglandins have two proposed functions: dilation of afferent arterioles and control of renin biosynthesis and release (11). Renal cortical COX-2 expression/activity is regulated by different stimuli, including alterations of dietary salt intake and inhibition or stimulation of the renin-angiotensin system (11).

The steady-state kidney levels of active prostaglandins depend on the relative rates of biosynthesis and inactivation. It has been proposed that two major steps are involved in prostaglandin inactivation: uptake from plasma membrane by prostaglandin transporter and oxidation by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) (20, 21, 27). The prostaglandin transporter has been reported to be expressed in many renal cell types (4). 15-PGDH oxidizes the 15(S)-hydroxyl group of prostaglandins to yield inactive 15-keto metabolites (8, 16). Previous reports have shown that 15-PGDH activity is higher in kidney cortex (particularly in the proximal tubule) and outer medulla than in inner medulla/papilla (14, 19, 27). Kidney 15-PGDH activity has been reported to be increased in obstructive kidneys (10) but inhibited by estradiol (5). Kidney 15-PGDH activity was significantly lower in young spontaneously hypertensive rats than in Wistar Kyoto rats (18). In 15-PGDH knockout mice, the ductus arteriosus failed to close after birth, possibly due to increased local PGE2 levels (7).

In the current studies, we examined the expression and localization of 15-PGDH in the kidney by in situ hybridization, immunostaining, dual immunofluorescent staining, and Northern and Western analyses. We also examined the role of COX-2 activity in regulation of 15-PGDH expression in the macula densa.


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Cell culture. LLCPKc14 is an established proximal tubule epithelial cell line derived from pig kidney (1). MMDD1 is a renal epithelial cell line with properties of macula densa cells (kindly provided by J. Schnermann, National Institutes of Health, Bethesda, MD) (30). LLCPKc14 and MMDD1 cells were cultured in DMEM nutrient mixture/Ham's F-12 (DMEM/F-12; Invitrogen) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) and incubated at 37°C in a humidified atmosphere of 95% air-5% CO2.

Animals. Male Sprague-Dawley rats were purchased from Harlan (150–200 g; Indianapolis, IN). Heterozygous breeding pairs with genetic deletion of the COX-2 gene maintained on a mixed B6/129 background were originally obtained from Jackson Laboratories (stock no. 002476; Bar Harbor, ME). Male rats and B6/129 mice (3 mo old) were maintained on normal rat chow, rat chow deficient in sodium (LS; 0.02–0.03% Na+), or rat chow with high salt (HS; 8% NaCl) for 2 wk (ICN Biochemicals, Costa Mesa, CA). Low salt-treated rats were given a single intraperitoneal injection of furosemide (1 mg/kg) before being placed on the low-salt diet. At the end of the experiments, one kidney was fixed in buffered formalin (10%) and then embedded in paraffin for light microscopy. The other kidney was separated into cortex and papilla for protein and RNA isolation. All animal study protocols were reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee. All experiments were conducted according to National Institutes of Health guidelines.

Northern analysis. 15-PGDH mRNA expression in kidney cortex and papilla was determined by Northern analysis. Total RNA from male adult B6/129 mouse kidney cortex and papilla was isolated using TriReagent (Molecular Research Center), and concentrations were quantitated by absorbance at 260 nm with a Bio-Rad spectrophotometer. Total RNA (15 µg) was electrophoresed on 1% formaldehyde-agarose gels, transferred to nylon membranes (Schleicher & Schuell), and probed with a 1.3-kb 32P-labeled cDNA Kpn/Xho1 fragment of the 3'-untranslated region of rat 15-PGDH. Northern analysis was repeated three times from independent RNA isolations.

In situ hybridization. Digoxigenin (DIG)-labeled sense and antisense probes of rat 15-PGDH were synthesized with the DIG RNA labeling kit (Roche) from linearized plasmids containing a rat 15-PGDH fragment (600 bp in the coding region of 15-PGDH mRNA). After fixation in freshly prepared buffered formalin (10%), hemisected kidneys were cut into 5-µm sections and subjected to in situ hybridization. The DIG nucleic acid detection kit (Roche) was used to visualize hybridized probes according to the manufacturer's directions. Eosin was used for counter staining. The slides were examined via a microscope equipped with differential interference contrast optics.

Immunohistochemical/immunofluorescent staining. The animals were anesthetized with Nembutal (70 mg/kg ip), given heparin (1,000 U/kg ip) to minimize coagulation, and perfused with FPAS (3.7% formaldehyde, 10 mM sodium m-periodate, 40 mM phosphate buffer, and 1% acetic acid) through the aortic trunk, cannulated by means of the left ventricle. FPAS is an acidified aldehyde, which provides excellent preservation of tissue structure, antigenicity, and mRNA and is suitable for immunofluorescent staining with low background (34). The fixed kidney was dehydrated through a graded series of ethanols, embedded in paraffin, sectioned (4 µm), and mounted on glass slides. Immunostaining was carried out as described in previous reports (33). For immunofluorescent staining, deparaffinized sections were blocked with 10% normal donkey serum for 1 h and then incubated with primary antibodies overnight at 4°C. After being washed with PBS, the sections were incubated with Rhodamine red-X-conjugated donkey anti-rabbit IgG (red; Jackson ImmunoResearch Laboratories), washed with PBS, and then incubated with fluorescein Dolichos biflorus agglutinin (green; FITC-labeled DBA; a marker for collecting duct epithelial cells), fluorescein Lotus tetragonolobus agglutinin (green; FITC-labeled LTA; a marker for proximal tubule epithelial cells) (Vector Laboratories, Burlingame, CA), or FITC-conjugated donkey anti-sheep IgG [green; detecting Tamm-Horsfall protein (THP), a marker for thick ascending limb] (Santa Cruz Biotechnology). Sections were viewed and imaged with a Nikon TE300 fluorescence microscope and spot-cam digital camera (Diagnostic Instruments). The primary antibodies that were used for immunofluorescence studies included rabbit anti-rat 15-PGDH antibody (1:150, no. 160615; Cayman Chemicals), rabbit polyclonal anti-murine COX-2 antibody (1:2,000, no. 160106; Cayman Chemicals), and sheep anti-human THP (1:5,000, no. 8595-0054; Biogenesis).

Immunoblotting. Cultured cells or kidney samples were homogenized with RIPA buffer and centrifuged, and an aliquot was taken for protein measurement. When Western blot analysis was performed, each lane was loaded with the same amount of protein. The proteins were separated on SDS-PAGE under reducing conditions and transferred to Immobilon-P transfer membranes (Millipore). After blocking with 20 mM Tris·HCl, pH 7.4, 500 mM NaCl, 5% nonfat milk, and 0.1% Tween 20 for 3 h at room temperature, the blots were incubated overnight at 4°C with affinity-purified rabbit polyclonal anti-murine PGDH antibody (1 µg/ml), rabbit polyclonal anti-murine COX-2 (0.05 µg/ml), or monoclonal anti-β-actin antibody (0.25 µg/ml). The primary antibodies were detected with peroxidase-labeled goat anti-rabbit IgG or peroxidase-labeled sheep anti-mouse IgG (Santa Cruz Biotechnology) and exposed on film by using enhanced chemiluminescence (Amersham International).


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15-PGDH expression in mouse kidney. Northern analysis and immunoblotting were used to investigate 15-PGDH mRNA and protein expression in mouse kidney. As indicated in Fig. 1A, 15-PGDH mRNA expression was significantly higher in cortex than in papilla. In immunoblots with an anti-15-PGDH antibody (Fig. 1B), loading 30 µg of protein extract from papilla produced a barely detectable band compared with the easily detectable band with 10 µg of protein from cortex, indicating substantially higher 15-PGDH protein expression in cortex than in papilla.


Figure 1
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  		Fig. 1. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) expression in the kidney. Both Northern blot analysis (A) and immunoblotting (B) demonstrated higher 15-PGDH expression in mouse kidney cortex than in inner medulla/papilla. Western blot analysis (C) indicated higher 15-PGDH expression in a cultured proximal tubule-like cell line (LLCPK1) than in a macula densa cell line (MMDD1). Each blot is representative of 3 separate experiments.

 
Localization of 15-PGDH mRNA and protein in rodent kidneys. In situ hybridization was carried out to determine the localization of 15-PGDH mRNA expression in mouse and rat kidneys. In mouse kidney cortex, 15-PGDH mRNA expression was primarily detected in the tubular epithelia but not in the macula densa or glomerulus (Fig. 2A). In normal adult rat kidney, 15-PGDH mRNA was also detected in the tubular epithelia in the cortex and outer medulla but was undetectable in glomerulus or papilla (Fig. 2, B–D).


Figure 2
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  		Fig. 2. Localization of 15-PGDH mRNA in rodent kidneys by in situ hybridization. In mouse (A) and rat (B) kidney cortex, 15-PGDH mRNA was present primarily in the tubular epithelia, including proximal tubule (PT), but minimal expression was observed in glomerulus (G) and macula densa (MD). 15-PGDH mRNA was also present in the tubular epithelia of rat kidney outer medulla (C) but not inner medulla/papilla (D). No positive signal was detected with the 15-PGDH sense probe in rat kidney cortex (E). Original magnification, x250 (A–E).

 
Immunostaining indicated that in normal adult mouse kidney, 15-PGDH protein was primarily present in the tubular epithelia in the cortex and outer medulla and was minimally detectable in the glomerulus and papilla, similar to the expression pattern of mRNA 15-PGDH protein expression (Fig. 3).


Figure 3
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  		Fig. 3. Localization of 15-PGDH protein in mouse kidney. A and B: in normal adult mouse kidney cortex, 15-PGDH was present in the tubular epithelia, including the PT, but not in G. C and D: intense 15-PGDH immunoreactivity was found in the tubular epithelial cells in outer medulla. E: 15-PGDH signal was found in outer medulla but not in inner medulla. F: no 15-PGDH signal was found in inner medulla. Original magnifications are as indicated.

 
To characterize the tubule segments that express 15-PGDH, we performed double immunofluorescent staining. In the cortex, most 15-PGDH-positive cells were localized to LTA-positive cells, indicating 15-PGDH expression in the proximal tubules (Fig. 4A). Some 15-PGDH-positive tubular epithelial cells were also DBA positive in cortex (data not shown) and outer medulla (Fig. 4B), indicating 15-PGDH expression in the collecting ducts. In the cortex, thick ascending limbs (THP positive) adjacent to macula densa had weak 15-PGDH signal, whereas thick ascending limbs far from macula densa had strong 15-PGDH signal (Fig. 4C). In the outer medulla, many 15-PGDH-positive cells were localized to medullary thick ascending limbs (THP positive). Some 15-PGDH-positive cells were THP negative in the outer medulla (arrows). Therefore, 15-PGDH was expressed in the thick ascending limbs in cortex and outer medulla.


Figure 4
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  		Fig. 4. Dual immunofluorescent staining in mouse kidney. A: in cortex, most 15-PGDH-positive cells (red) were localized to the PT [colocalization with FITC-labeled Lotus tetragonolobus agglutinin (LTA); green, a proximal tubule marker]. No 15-PGDH signal was found in G. B: in outer medulla, some tubules were positive for 15-PGDH and FITC-labeled Dolichos biflorus agglutinin (DBA; green), an indication of 15-PGDH expression in collecting duct (CD). C: in cortex and outer medulla, 15-PGDH-positive cells were colocalized with Tamm-Horsfall protein (THP), an indication of 15-PGDH expression in thick ascending limbs (TAL). Some 15-PGDH-positive cells were TPH negative in outer medulla (arrows). cTAL, cortical thick ascending limbs; mTAL, medullary thick ascending limbs.

 
15-PGDH expression in cultured proximal tubule and macula densa cell lines. 15-PGDH expression was determined in the macula densa cell line MMDD1 (30). For comparison, expression was also determined in the cultured proximal tubule cell line LLCPKcl4 (1). As indicated in Fig. 1C, 30 µg of MMDD1 protein produced a barely detectable immunoreactive band, whereas 7.5 µg of LLCPKc14 protein produced a substantial band. This result suggests that 15-PGDH expression may be higher in proximal tubule than in macula densa.

15-PGDH expression in the macula densa was regulated by dietary salt intake. In kidney cortex, prostaglandins derived from macula densa COX-2 play an important role in regulation of vascular tone and renin biosynthesis and excretion (11). In normal adult rat cortex, only isolated macula densa cells are COX-2 positive (12). Using immunofluorescent staining, we confirmed previous reports that after salt restriction, the number of COX-2-positive cells in the renal cortex increases significantly (Fig. 5A) (12). These COX-2-positive cells were localized to macula densa and adjacent cortical thick ascending limbs (arrows). Interestingly, all COX-2-positive cells in macula densa and adjacent cortical thick ascending limbs were 15-PGDH negative (Fig. 5A, arrows). In the high salt-treated rat, macula densa COX-2 was undetectable (data not shown), whereas macula densa 15-PGDH expression was now evident (Fig. 5A). Macula densa 15-PGDH was not detectable in control wild-type mice but was evident after high-salt treatment (Fig. 5B). Glomerulus 15-PGDH was minimal in rat and mouse kidneys and was not affected by alteration of dietary salt intake. Immunoblotting indicated that whole cortical 15-PGDH expression was similar in control, low salt-, and high salt-treated rat kidneys (Fig. 6A). Therefore, alterations of dietary salt intake may selectively regulate 15-PGDH expression in the macula densa and adjacent cortical thick ascending limbs, and an inverse relationship may exist between the expression of COX-2 and 15-PGDH in the macula densa.


Figure 5
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  		Fig. 5. 15-PGDH immunofluorescent staining in kidney cortex from low salt (LS)- or high salt (HS)-treated animals and cyclooxygenase-2 (COX-2) knockout mouse (COX-2–/–). A: in LS-treated rat kidney, strong COX-2 signal was found in MD/adjacent cTAL (red; arrows). In these COX-2-positive cells, no 15-PGDH signal was found (green; arrows). 15-PGDH signal was evident in MD but was negative in G after HS treatment. B: MD 15-PGDH signal was undetectable in a control wild-type mouse but was evident after HS treatment. HS treatment did not increase 15-PGDH expression in G. In a COX-2 knockout mouse, 15-PGDH was evident in MD (arrows) and G. Original magnification, x400.

 

Figure 6
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  		Fig. 6. Regulation of 15-PGDH expression in vivo and in vitro. A: renal cortical 15-PGDH expression was not regulated by dietary salt intake in rats. B: renal cortical 15-PGDH expression was comparable between wild-type and COX-2 knockout mice. C: 15-PGDH expression in MMDD1 cells. MMDD1 cells were treated with the COX-2 inhibitor SC-58236 (100 nM) or the COX-1 inhibitor SC-58560 (100 nM) for 24 h, harvested, and subjected to immunoblotting. 15-PGDH expression increased after COX-2 but not COX-1 inhibition (n = 3). *P < 0.01.

 
15-PGDH was highly expressed in the macula densa and glomerulus in COX-2 knockout mouse kidney. To investigate further the interactions between kidney COX-2 and 15-PGDH in vivo, we studied COX-2 knockout mice. In contrast to absent 15-PGDH expression in macula densa and glomerulus in normal wild-type mice, an intense 15-PGDH signal was found in these sites in COX-2 knockout mice, whereas 15-PGDH immunoreactivity was similar in tubular epithelial cells in the cortex and outer medulla in COX-2 knockout mice and littermate controls (Fig. 5B). Immunoblotting showed that 15-PGDH expression in whole cortex was comparable between wild-type and COX-2 knockout mouse kidneys (Fig. 6B). Therefore, increased 15-PGDH expression in COX-2 knockout mice was restricted to macula densa and glomerulus. These results suggest that 15-PGDH expression in macula densa and glomerulus is tonically suppressed by COX-2 activity. In COX-2 knockout mice, 15-PGDH immunoreactivity was still undetectable in inner medulla/papilla (data now shown).We treated MMDD1 cells with either a selective COX-2 inhibitor, SC-58236 (100 nM), or a selective COX-1 inhibitor, SC-58560 (100 nM), for 24 h. As indicated in Fig. 6C, COX-2 inhibition led to increased 15-PGDH in MMDD1 cells. In contrast, COX-1 inhibition had no apparent effect on MMDD1 15-PGDH expression.


   DISCUSSION
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These studies examined the renal expression of 15-PGDH, a key enzyme in prostaglandin catabolism, and its potential interactions with COX, the key enzymatic step in prostaglandin biosynthesis. In the kidney cortex, COX-1 has been localized to arteries and arterioles, glomeruli, and collecting ducts but not to the proximal or distal convoluted tubules, Henle's loop, or macula densa (22). In the outer medulla, COX-1 is expressed in the collecting duct epithelia. In the inner medulla/papilla, COX-1 is highly expressed in the collecting duct epithelial cells as well as medullary interstitial cells (12, 32). In the cortex, COX-2 has been reported to be expressed in the macula densa, cortical thick ascending limbs (12), podocytes (6, 15), mesangial cells (13, 25), and vasculature (26). In the medulla, COX-2 has been localized to medullary interstitial cells and collecting ducts of inner medulla/papilla (12, 31, 32). In the present studies, 15-PGDH was primarily localized to the proximal tubules (LTA positive) and thick ascending limbs (THP positive) and collecting duct (DBA positive) in the cortex and outer medulla. 15-PGDH expression was minimal in glomerulus, macula densa, and inner medulla/papilla in normal rodent kidney. The localization of 15-PGDH mRNA and protein in the current studies agrees with previous activity studies indicating that 15-PGDH activity was high in isolated proximal tubule and in cortical and outer medullary slices but low in inner medulla/papilla (14, 19, 27). Therefore, 15-PGDH is expressed at the sites in the kidney where COX-2 expression is either not detectable or is only minimally expressed. These results suggest that 15-PGDH expression may be tonically suppressed by COX-2-derived prostanoids through autocrine or paracrine regulation. This hypothesis is strengthened by four observations: 1) in low salt-treated rat kidney, COX-2-positive epithelial cells in the macula densa and cortical thick ascending limb were 15-PGDH negative; 2) in high salt-treated rat and mouse kidneys, in which there was minimal detectable macula densa COX-2 expression, 15-PGDH immunoreactivity was evident in the macula densa; 3) in cultured macula densa MMDD1 cells, 15-PGDH expression increased after COX-2 inhibition but not COX-1 inhibition; and 4) strong 15-PGDH immunofluorescent staining was found in the macula densa and glomerulus in COX-2 knockout mice.

The mammalian kidney is a rich source of prostaglandins (9, 23). Although tissue prostaglandin levels are determined by both biosynthesis and degradation, the potential interaction of cyclooxygenase and 15-PGDH in the kidney has not been investigated to date. The role of 15-PGDH in tumorigenesis has recently received attention. Elevated COX-2 and COX-2-derived PGE2 levels are involved in tumorigenesis in a variety of tissues, and inhibition of COX-2 activity can suppress tumorigenesis (3). Recently, it has been reported that increased COX-2 activity and decreased 15-PGDH activity both contribute to increased COX-2-derived PGE2 levels in human breast cancer, colon cancer, and lung cancer, among others, and 15-PGDH has been implicated as an in vivo suppressor of tumorigenesis in these tissues (3, 17, 28, 29). In A549 human lung adenocarcinoma cells, cytokines induced COX-2 expression while simultaneously inhibiting 15-PGDH expression, and overexpression of COX-2, but not COX-1, led to decreased 15-PGDH expression in A549 cells. Conversely, overexpression of 15-PGDH attenuated interleukin-1β-induced COX-2 expression in A549 cells (24). Glucocorticoids are potent endogenous COX-2 inhibitors, and in A549 cells, dexamethasone induced 15-PGDH expression (24). Together, these data suggest that the expression of COX-2 and 15-PGDH may be reciprocally regulated.

Interactions between COX-2 and 15-PGDH are of potential importance in kidney function. In kidney cortex, COX-2-derived prostaglandins from macula densa and/or vasculature have been implicated in vasodilation and stimulation of renin biosynthesis and release. In low salt-treated animals, COX-2 expression increased in the macula densa and adjacent cortical thick ascending limbs (12), whereas 15-PGDH expression in these sites was minimal. Therefore, increased COX-2 expression and minimal 15-PGDH may both contribute to increased COX-2-derived prostaglandin levels in the macula densa and subsequent increases in renin biosynthesis and release. In high salt-treated animals, COX-2 expression in the macula densa/cortical thick ascending limbs decreased, whereas 15-PGDH expression in the macula densa increased. Therefore, decreased COX-2 expression and increased 15-PGDH may both contribute to reduced COX-2-derived prostaglandin levels in the macula densa and subsequent inhibition of renin biosynthesis and release.

Outer medullary thick ascending limb and collecting duct are important contributors to the medullary interstitial osmolarity that allows effective distal nephron salt and water reabsorption (23), and PGE2 has been reported to inhibit sodium reabsorption in these sites (2). 15-PGDH expressed in the thick ascending limb and collecting duct in the outer medulla may contribute to Na+ reabsorption through rapid degradation of prostaglandins arising from local generation, diffusion from vasculature, or filtration. Indeed, Nomura et al. (20) recently reported that both prostaglandin uptake carrier prostaglandin transporter and 15-PGDH are expressed in renal collecting ducts. Lack of 15-PGDH in inner medulla/papilla may contribute to the constitutive activity of COX-2-derived prostaglandins in medullary interstitial cells (9, 12, 32).

In summary, we have delineated for the first time the expression and localization of 15-PGDH, the key enzyme in prostaglandin degradation in the kidney. We also found that 15-PGDH was downregulated by COX-2 activity. Further studies are required to elucidate the identity of the COX-2 metabolites that downregulate 15-PGDH expression and to investigate possible physiological and/or pathophysiological roles for 15-PGDH in the kidney.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51265, DK-62794, and P60 DK-20593 (to the Vanderbilt Diabetes Research and Training Center) and by funds from the Department of Veterans Affairs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. C. Harris, Division of Nephrology, Vanderbilt Univ. School of Medicine, MCN C3121, 1161 21st St. S, Nashville, TN 37232 (e-mail: ray.harris{at}vanderbilt.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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