alpha -Melanocyte-simulating hormone and interleukin-10 do not protect the kidney against mercuric chloride-induced injury

Takehiko Miyaji, Xuzhen Hu, and Robert A. Star

Renal Diagnostics and Therapeutics Unit, National Institutes of Health, Bethesda, Maryland 20892-1268


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The anti-inflammatory cytokines alpha -melanocyte-stimulating hormone (MSH) and interleukin (IL)-10 inhibit acute renal failure (ARF) after ischemia or cisplatin administration; however, these agents have not been tested in a pure nephrotoxic model of ARF. Therefore, we examined the effects of alpha -MSH and IL-10 in HgCl2-induced ARF. Mice were injected subcutaneously with HgCl2 and then given vehicle, alpha -MSH, or IL-10 by intravenous injection. Animals were killed to study serum creatinine, histology, and myeloperoxidase activity. Treatment with either alpha -MSH or IL-10 did not alter the increase in serum creatinine, tubular damage, or leukocyte accumulation at 48 h after HgCl2 injection. Because alpha -MSH and IL-10 are active in other injury models that involve leukocytes, we studied the time course of tubular damage and leukocyte accumulation to investigate whether leukocytes caused the tubular damage or accumulated in response to the tubular damage. Tubular damage was present in the outer stripe 12 h after HgCl2 injection. In contrast, the number of leukocytes and renal myleoperoxidase activity were normal at 12 h but were significantly increased at 24 and 48 h after injection. We conclude that neither alpha -MSH nor IL-10 altered the course of HgCl2-induced renal injury. Because the tubular damage preceded leukocyte infiltration, the delayed leukocyte accumulation may play a role in the removal of necrotic tissue and/or tissue repair in HgCl2-induced ARF.

mercury; acute renal failure; leukocytes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HUMAN ACUTE RENAL FAILURE (ARF) is caused by both ischemic and nephrotoxic insults acting alone or in combination (33, 37). Because it is often difficult to identify the cause of ARF, it would be useful to have therapeutic agents that are effective in both ischemic and nephrotoxic forms of ARF. We have recently shown that the anti-inflammatory cytokines alpha -melanocyte-stimulating hormone (MSH) and interleukin (IL)-10 inhibit renal injury in both ischemia and cisplatin models of ARF (3, 5, 6, 20, 21). They inhibit renal injury even when started 1 (IL-10 in cisplatin-induced ARF) or 6 h (alpha -MSH in ischemic ARF) after the injury process is initiated (3, 5, 6). Both agents have multiple effects; they inhibit the production of cytokines [tumor necrosis factor (TNF)-alpha ], chemokines (IL-8, monocyte chemoattractant protein-1), adhesion molecules [intracellular adhesion molecule (ICAM)-1], and inducible nitric oxide synthase (iNOS) (3, 5, 6). alpha -MSH also increases blood pressure after hemorrhagic shock in rats and in healthy hemodialysis patients (1, 34).

There is evidence for vasoconstriction after HgCl2-induced nephrotoxic ARF, because renal blood flow is reduced and because verapamil decreases HgCl2-induced renal injury (15, 38). However, the decrement in glomerular filtration rate is much larger than the change in renal blood flow and remains when renal blood flow is normalized by volume expansion (38). Furthermore, verapamil has some antioxidant properties (13). Therefore, recent work has focused on the role of oxidant-induced renal injury in HgCl2-induced renal injury. For example, HgCl2 increases the production of many endogenous oxidants, such as superoxide and hydrogen peroxide, and causes depletion of protective glutathione (14, 27, 28). Indeed, some (12, 28), but not all (27), studies showed that HgCl2-induced injury can be ameliorated by superoxide dismutase or the antioxidants N-acetylcysteine and melatonin. In contrast, the role of inflammation in the HgCl2 model is controversial. Early studies by Solez et al. (32) found decreased medullary blood flow and accumulation of neutrophils in the vasa recta in rats treated with HgCl2, suggesting a role for leukocyte-endothelial interactions. In more recent studies, Yanagisawa et al. (40) found that HgCl2 increases renal mRNA accumulation of TNF-alpha and iNOS. They suggest that TNF-alpha and iNOS cause injury rather than being associated with injury, because modest protection was afforded by pretreatment with agents that inhibit TNF-alpha or iNOS, such as the angiotensin II receptor antagonist TCV-116, TNF-alpha antibody, or the iNOS inhibitor aminoguanidine (40). The degree of protection with TNF-alpha antibody or iNOS inhibition was rather modest, especially compared with more complete projection in renal ischemia-reperfusion injury (7, 23, 29). Furthermore, antibodies to the adhesion molecule ICAM-1 or lymphocyte function-associated antigens (LFA) did not protect against HgCl2-induced injury (9); in contrast, antibodies to ICAM-1 protected against ischemia- and cisplatin-induced renal injury (8, 18, 19, 31).

Because of the only modest success of narrowly based anti-inflammatory agents inhibiting renal damage after HgCl2 administration, we reasoned that the broad spectrum of action of alpha -MSH or IL-10 (21, 26) might allow these agents to be effective in the HgCl2 model of acute renal injury. As noted above, alpha -MSH and IL-10 inhibit induction of both TNF-alpha and iNOS mRNA after both ischemia and cisplatin administration (3, 5). Furthermore, unlike ICAM-1 antibodies (18), delay of IL-10 treatment for 1 h after cisplatin still yields excellent protection against cisplatin-induced injury (5). Therefore, we tested IL-10 and alpha -MSH in a HgCl2 model of acute renal injury.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male BALB/C mice, aged 6-7 wk, were purchased from the National Institutes of Health (NIH). All animals had free access to water and chow (NIH-07 Rodent Chow, Zeigler Bros., Gardners, PA) before surgery. Animal care followed NIH criteria for the care and use of laboratory animals in research.

Chemicals. IL-10 was purchased from CN Biosciences (Boston, MA). alpha -MSH was purchased from Phoenix Pharmaceuticals (Mountain View, CA). All other chemicals were purchased from Sigma (St. Louis, MO).

HgCl2 model of nephrotoxic ARF. The mice were anesthetized with isoflurane (Baxter Healthcare, Deerfield, IL) inhalation. The animals were given 6 mg/kg body wt of HgCl2 (1 mg/ml) in normal saline by subcutaneous injection, and then either vehicle, 1 µg of IL-10, or 50 µg of alpha -MSH dissolved in 0.3 ml normal saline were given by intravenous injection. The first dose of IL-10 or alpha -MSH was given immediately after the HgCl2 injection. Additional doses of alpha -MSH were given every 8 h. The animals were killed at 0.17, 0.5, 1, 2, 3, or 5 days after HgCl2 injection. At the time of death, blood was collected for measurement of serum creatinine by an Astra 8 autoanalyzer (Beckman Instruments, Fullerton, CA) and both kidneys were harvested for histological and other studies.

Histological examination. Parafolmaldehyde (4%)-fixed and paraffin-embedded kidney specimens were stained with periodic acid-Schiff reagent or naphthol AS-D chloroacetate esterase (kit no. 91-C, Sigma). Histological changes in the cortex and the outer stripe of the outer medulla (OSOM) were evaluated by semiquantitative measurements of tissue damage and leukocyte infiltration. Tubular damage, defined as tubular epithelial swelling, loss of brush border, vacuolar degeneration, necrotic tubules, and desquamation, was estimated by counting the percentage of damaged tubules in 10 high-power fields/section. Because the esterase stain identifies infiltrating neutrophils and macrophages (41), we use the term leukocytes. Leukocyte accumulation was evaluated by counting the number of esterase-positive cells in 10 high-power fields in the cortex and 10 high-power fields in the OSOM.

Renal tissue myeloperoxidase activity. Myeloperoxidase (MPO) activity, an indicator of leukocyte accumulation in tissues, was measured as described by Laight et al. (22). We used kidney tissues subjected to 40 min of ischemia and reperfused for 4 or 12 h as a positive control for the MPO assay (3). Kidney tissue was homogenized in a solution containing 0.5% (wt/vol) hexadecyltrimethylammonium bromide dissolved in 50 mM potassium phosphate buffer (pH 6.0) and centrifuged for 30 min at 20,000 g at 4°C. Samples were incubated at 60°C for 2 h in a water bath and then centrifuged at 4,000 g for 12 min. The supernatant (40 µl) was incubated with 160 µl of a reaction mixture containing 1.6 mM tetramethylbenzidine and 3 mM H2O2 diluted in 80 mM phosphate buffer (pH 5.4) in a 96-well microplate. The rate of change in absorbance at 630 nm was measured spectrophotometrically by using a SPECTRAmax 190 (Molecular Devices, Sunnyvale, CA). MPO activity was expressed as absorbance per minute per milligram of wet tissue.

Statistical analysis. All data are expressed as means ± SE. Differences between data sets were examined for statistical significance by using ANOVA with post hoc analysis (Bonferroni-Dunn or Dunnett tests; StatView 5.0, Berkeley, CA). A P value <0.05 was accepted as statistically significant.


    RESULTS
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HgCl2-induced ARF. In preliminary studies, we investigated the dose-response of HgCl2 in mice by using serum creatinine at 48 h as the end point. We found that 6 and 8 mg/kg body wt but not 2 and 4 mg/kg body wt of HgCl2 induced a significant increase in serum creatinine in male BALB/C mice (Fig. 1A). Therefore, we used 6 mg/kg body wt of HgCl2 in all subsequent experiments. Injection of 6 mg/kg body wt of HgCl2 induced a significant but transient increase in serum creatinine (Fig. 1B). The serum creatinine level significantly increased at 24 h (1.87 ± 0.35 mg/dl vs. day 0, P < 0.01), peaked at 48 h (2.20 ± 0.53 mg/dl vs. day 0, P < 0.01), and then gradually returned to the basal values by day 5. At 12 h after HgCl2 injection, histological examination of periodic acid-Schiff stained sections revealed that tubular epithelial swelling, loss of brush border, and vacuolar degeneration were limited to the OSOM (see Fig. 3E). At 24-48 h after the injection, extensive tubular damage and necrosis had spread to the cortex (see Fig. 3A).


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Fig. 1.   A: dose-response to HgCl2 administration in BALB/C mice. Male mice were injected subcutaneously with 0-8 mg/kg body wt (BW) of HgCl2, then killed at 48 h. , individual data points; black-triangle, mean values ± 1 SE. *P < 0.01 vs. vehicle. B: time course of HgCl2-induced acute renal failure (ARF) in BALB/C mice. Male mice were injected subcutaneously with 6 mg/kg body wt of HgCl2. Blood samples were collected for measurement of serum creatinine on days 0.5, 1, 2, 3, and 5. Values are means ± SE; n = 4 animals/time point. *P < 0.01 vs. day 0.

Effect of alpha -MSH and IL-10 in HgCl2-induced ARF. To evaluate the effects of alpha -MSH and IL-10 in HgCl2-induced renal injury, we investigated serum creatinine levels and histological changes at 48 h after injection. Treatment with alpha -MSH or IL-10 did not decrease the level of serum creatinine compared with vehicle-treated mice in HgCl2-induced ARF at 48 h (alpha -MSH, 2.35 ± 0.07, vs. vehicle, 2.15 ± 0.23 mg/dl; IL-10, 2.10 ± 0.35, vs. vehicle, 2.25 ± 0.32 mg/dl) or 72 h (Fig. 2). Similarly, tubular damage in alpha -MSH- or IL-10-treated mice at 48 h was not significantly different from that in vehicle-treated animals in either cortex (alpha -MSH, 28.20 ± 5.57, vs. vehicle, 28.75 ± 1.31%; IL-10, 27.40 ± 2.41, vs. vehicle, 25.77 ± 3.16%) or the OSOM (alpha -MSH, 38.63 ± 1.71, vs. vehicle, 38.00 ± 1.84%; IL-10, 37.75 ± 1.27, vs. vehicle, 33.38 ± 2.64%; Figs. 3, A-C, and 4).


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Fig. 2.   Effect of alpha -melanocyte-stimulating hormone (MSH) and interleukin (IL)-10 on serum creatinine obtained at 48 and 72 h after HgCl2 injection. Mice were injected subcutaneously with 6 mg/kg body wt of HgCl2 and then given alpha -MSH, IL-10, or vehicle by intravenous injection. The first dose of 1 µg IL-10 or 50 µg alpha -MSH was given immediately after HgCl2 injection. Additional doses of alpha -MSH were given every 8 h. Animals were killed at either 48 or 72 h. NS, not significant. Values are means ± SE; n = 4 animals/group.



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Fig. 3.   Typical renal histology after HgCl2 administration. Kidney sections were stained with either periodic acid-Schiff (A-C, E, and F) or naphthol AS-D chloroacetate esterase (D). Comparisons are of typical histology in animals treated with vehicle (A and D), alpha -MSH (B), or IL-10 (C) at 48 h. Typical histology in vehicle-treated animals on days 0.5 (E) and 5 (F) are shown. Arrows, esterase-positive cells. Original magnification, ×250 (A-C), ×400 (D-F).

Time course of tubular damage and leukocyte accumulation. We studied the time course of tubular damage and leukocyte accumulation in the kidney to investigate whether leukocytes caused the tubular damage or accumulated in response to the tubular damage after HgCl2 administration. Esterase-positive cells were found only occasionally in both control kidneys and at 12 h after HgCl2 injection (0.21 ± 0.15 cells/mm2). The number of esterase-positive cells in the OSOM were significantly increased at 24 h (10.10 ± 2.73 cells/mm2 vs. day 0, P < 0.05) and then rapidly increased and reached a maximum at 48 h after HgCl2 injection (31.10 ± 6.26 cells/mm2 vs. day 0, P < 0.01; Fig. 5A). In the cortex, esterase-positive cells were only sporadically present even at 48 h (3.15 ± 1.31 cells/mm2). Most of the esterase-positive cells were exclusively located around necrotic tubules, not normal or degenerated tubules, which were located mainly in the OSOM (Fig. 3D).

In contrast to the delayed entry of leukocytes, tubular damage was present in the OSOM as early as 12 h after HgCl2 injection (16.19 ± 2.75% vs. day 0, P < 0.05; Figs. 3E and 5). The majority of necrotic tubules disappeared by day 5, and some denuded basement membranes were covered with flattened regenerating epithelial cells. Hyperproliferative cells occasionally could be seen in some proximal tubules on day 5 (Fig. 3F). However, we could still observe some casts in damaged tubular lumens.

MPO activity in HgCl2-induced acute renal injury. The time course of leukocyte accumulation in the kidney was also determined by renal tissue MPO assay. The basal level of MPO activity was 2.67 ± 0.22 absorbance · min-1 · 100 mg tissue-1. MPO activity remained unchanged by 12 h after exposure to HgCl2. MPO activity significantly increased at 24 h after HgCl2 injection (5.28 ± 0.56 absorbance · min-1 · 100 mg tissue-1 vs. day 0, P < 0.01) and then peaked at 48 h (8.77 ± 0.13 absorbance · min-1 · 100 mg tissue-1 vs. day 0, P < 0.01; Fig. 5B). These results were consistent with the number of esterase-positive cells detected by histological analysis. However, increased kidney tissue MPO activity in HgCl2-induced acute renal injury was much less than that in ischemia- or reperfusion-injured kidney tissues (24.98 ± 2.86 absorbance · min-1 · 100 mg tissue-1 at 4 h and 46.29 ± 16.39 absorbance · min-1 · 100 mg tissue-1 at 24 h after reperfusion; Fig. 5B).


    DISCUSSION
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In contrast to their effects in ischemic and cisplatin models of renal injury, neither alpha -MSH nor IL-10 inhibited injury in the HgCl2 model. Because the role of leukocytes is unknown, we investigated the role of leukocytes in the HgCl2 model.

Lack of effect of alpha -MSH or IL-10. Because many therapeutic agents have a golden window of opportunity, we first investigated the minimal dose of HgCl2 that would produce consistent renal injury (Fig. 1). Most previous studies of HgCl2 ARF had been performed in rats, generally by using 1-7.5 mg/kg of HgCl2 (for example, see Refs. 9, 11, 12, 15, 24, 28, 40), whereas studies in mice have used 3-7 mg/kg of HgCl2 (16, 17, 36). For example, Tanaka-Kagawa et al. (36) found differences in sensitivity to Hg-induced ARF among mouse strains by using 5.4 mg/kg of HgCl2; however, they did not study BALB/C mice. In BALB/C mice, we found that neither 2 nor 4 mg/kg of HgCl2 caused sufficient renal injury. Because 6 mg/kg of HgCl2 caused consistent renal injury, this dose was selected for subsequent studies. We then used doses and dose schedules for alpha -MSH and IL-10 that this laboratory had previously shown to be effective in ischemia- and cisplatin-induced ARF (3, 5). Despite the use of optimal doses and dose schedules, we found that neither alpha -MSH nor IL-10 had a statistically significant effect on either creatinine (Fig. 2) or renal histology (Figs. 3 and 4). The lack of effect of alpha -MSH and IL-10 is similar to the lack of effect of other anti-inflammatory strategies, including antibodies to LFA and ICAM-1 (11) or CD8 cell depletion (9). In contrast, Yanagisawa et al. (40) found that HgCl2 increased renal TNF-alpha and iNOS and that pretreatment with TNF-alpha antibody or the iNOS inhibitor aminoguanidine inhibits renal damage. However, the degree of protection with TNF-alpha antibody or iNOS inhibition was rather modest, especially compared with more complete protection in the renal ischemia or reperfusion injury model (7, 23, 29). Most of the agents that substantially inhibit HgCl2-induced ARF, such as superoxide dismutase, N-acetylcysteine, and melatonin, likely inhibit oxidant-induced cell injury (12, 14, 28), although some controversy remains (27). For example, N-acetylcysteine reduced lipid peroxidation (12), and melatonin increased the renal content of malandialdehyde and glutathione after HgCl2 (28). Therefore, the inability of alpha -MSH to inhibit renal injury after HgCl2 administration is less surprising. Although Yanagisawa et al. (40) found that pretreatment with anti-TNF-alpha antibody or the iNOS inhibitor aminoguanidine was modestly protective, there are differences between our model and Yanagisawa's model that might be significant, including species differences (mice vs. rats), route of HgCl2 administration (subcutaneous vs. intraperitoneal), and perhaps most importantly, the length of pretreatment (none vs. 1-5 days). Which of these factors, if any, is important is unknown.


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Fig. 4.   Effect of alpha -MSH and IL-10 on quantitative renal histology at 48 h after HgCl2 injection. Mice were injected subcutaneously with 6 mg/kg body wt of HgCl2 and then given intravenous alpha -MSH, IL-10, or vehicle as in Fig. 2. Animals were killed at 48 h. Data are percentage of damaged tubules per millimeters squared in the cortex and outer stripe of the outer medulla (OSOM). Values are means ± SE; n = 4 animals/group.

Role of inflammation in HgCl2-induced ARF. Electron microscopic studies by Solez et al. (32) found neutrophils in the vasa recta after HgCl2 administration to rats. More recent studies by Ghielli et al. (11) also found renal accumulation of macrophages and CD8+ and CD4+ T cells after Hg administration. Yanagisawa et al. (40) found induction of the proinflammatory cytokine TNF-alpha . The importance of these leukocytes in tissue injury and repair is not known. Previous studies have not correlated histological changes with leukocyte accumulation soon after Hg administration. The lack of effect of alpha -MSH, IL-10, LFA, and ICAM-1, and only modest effect of anti-TNF-alpha antibodies caused us to reappraise the role of leukocytes in HgCl2-induced ARF. In the present study, leukocyte accumulation was measured by two independent methods: direct counting and MPO assay. We found evidence of tubular damage at 12 h, without any increase in leukocyte accumulation. Previous studies by Zalme and colleagues (25, 42) also found early renal damage to the proximal straight tubule as early as 6 h after Hg administration. In contrast, we first detected both leukocyte and MPO accumulation at 24 h after HgCl2 administration (Figs. 5 and 6). Thus in the HgCl2 model, tubular damage preceded leukocyte accumulation. In the renal ischemia model, in contrast, leukocyte accumulation occurs much earlier, starting ~2-4 h after injury, and either precedes or occurs at the same time as evidence of tubular injury assessed by routine histological methods (3, 4, 22, 39). Therefore, the leukocyte accumulation after Hg administration is more likely to be in response to tissue damage, whereas in the ischemia-reperfusion model, there is emerging evidence that early accumulation of macrophages and T cells may precede and perhaps cause the tubular damage (2, 4, 10, 30, 41). Leukocytes, measured by either esterase staining or MPO assay, are only transiently found in the kidney (Fig. 5), which is similar to previous findings in the uranyl acetate model, where ED-1-positive cells transiently emigrate to the region of injury and then gradually disappear after the tubules have regenerated (35). Strategies that inhibit inflammation would not be expected to decrease renal damage, and might even increase tubular damage, in the HgCl2 model.


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Fig. 5.   A: time course of histological changes in HgCl2-induced ARF. Mice were injected subcutaneously with 6 mg/kg body wt of HgCl2 and then killed 0.17, 0.5, 1, 2, 3, or 5 days later. Values are means ± SE; n = 3-4 animals/group. dagger P < 0.01 vs. day 0; dagger dagger P < 0.05 vs. day 0 tubular damage; *P < 0.01 vs. day 0; **P < 0.05 vs. day 0 leukocyte accumulation. B: time course of MPO activity in HgCl2-induced ARF. Animals were treated as in Fig. 5A. Values are means ± SE. I/R, ischemia-reperfusion; abs, absorbance.*P < 0.0 1 vs. day 0; n = 3-4 animals/group.

There are other important differences between the ischemia and HgCl2 injury models. For example, there are quantitatively more leukocytes and MPO activity in the ischemia model than in the HgCl2 model (Fig. 5). There are also differences in the amount of red blood cell pooling; red blood cell pooling is absent in the HgCl2 model and very prominent in the ischemia model. The reason for the lack of venous pooling is unknown but might be a consequence of fewer leukocytes and, hence, the absence of capillary plugging in the HgCl2 model. Alternatively, either the type of leukocyte, the activation state of the leukocytes, or the leukocyte-endothelial cell interactions might be different. Recent studies have characterized the infiltrating cell as a macrophage because it binds ED-1 (10, 41); however, there is also evidence of T cells (4, 30). Data from Solez et al. (32) suggest that neutrophils are involved in the HgCl2 model, although careful characterization of the infiltrating cells has not been performed.

Conclusion. We conclude that alpha -MSH and IL-10 do not reduce renal injury after HgCl2 administration. Leukocytes accumulate in renal tissues in response to the HgCl2-induced nephrotoxic injury. Strategies that inhibit leukocyte-endothelial interactions are either only modestly effective or fail to protect against HgCl2-induced injury. alpha -MSH (and perhaps IL-10) is more effective when the injury pathway involves leukocyte-endothelial interactions (ischemia) rather than direct tubular toxicity (HgCl2).


    ACKNOWLEDGEMENTS

This study was sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases.


    FOOTNOTES

Address for reprint requests and other correspondence: R. A. Star, Rm. 3N108, Renal Diagnostics and Therapeutics Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892-1268 (E-mail Robert_Star{at}nih.gov).

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.

First published January 2, 2002;10.1152/ajprenal.00203.2001

Received 2 July 2001; accepted in final form 29 November 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 282(5):F795-F801