Tamm-Horsfall glycoprotein (THP) was described initially as a urinary protein that inhibited viral hemagglutination1. Subsequently, Muchmore and Decker identified a urinary protein that possessed immunosuppressive properties in vitro and hence coined the term uromodulin2. Interest in uromodulin resulted in cloning of this protein, which was identified as THP3,4; however, when further studies did not suggest a major physiological function in immune modulation, enthusiasm for the study of THP diminished. The function of THP remained undefined, but the exclusive production by cells of the thick ascending limb of the loop of Henle (TALH) along with a striking absence of THP mRNA in the macula densa5, a specialized portion of the TALH involved in the detection of the luminal concentration of sodium chloride, suggested an involvement in salt and water transport in the TALH. However, the absence of aquaporins in the TALH6,7,8 has been proposed as the mechanism of water impermeability of that segment. Along with studies showing that water intake did not alter THP production9, involvement of THP in water transport in the TALH appeared unlikely. Although not proven, THP may modulate salt transport in the TALH.
We hypothesized that changes in dietary salt and the addition of a loop diuretic altered the production of THP. Understanding the involvement of these parameters in THP expression is also potentially useful in the therapy of diseases where THP plays an important role, such as cast nephropathy ("myeloma kidney")10,11,12,13,14,15 and nephrolithiasis16. Using Northern hybridization and Western blotting, the current study determined whether dietary salt affected relative steady-state mRNA and renal protein content of THP in Sprague-Dawley rats.
METHODS
Animal preparation
Seventy-six male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, USA), 28 days of age, were housed under standard conditions and given 0.3% (low-salt) diet (AIN-76A with 0.3% NaCl; Dyets, Inc., Bethlehem, PA, USA) and water ad libitum for four days before initiating the experiment. Animals were used at this age because previous experiments showed preservation of blood pressure and inulin clearance for two weeks on an 8.0% NaCl diet17. Maintaining the animals on a 0.3% NaCl diet allowed the establishment of a baseline standardized diet before initiation of study. Rats were then placed in metabolic cages and allowed free access to a water and rat diet, which contained 0.3% (termed the Lo-salt group), 1.0% (AIN-76A with 0.3% NaCl; Dyets, Inc.; the Mid-salt group) or 8.0% (AIN-76A containing 8.0% NaCl, Dyets, Inc.; the Hi-salt group) sodium chloride. Urine was collected under oil to prevent desiccation. Food consumption, urine flow and body weights were recorded daily. In other experiments, rats on the high-salt diet received intraperitoneal injections of either furosemide (Elkins-Sinn, Inc., Cherry Hill, NJ, USA), 5 mg/kg, (denoted Hi-F), or chlorothiazide (Merck & Co., West Point, PA, USA), 4 mg/kg (Hi-C), twice daily. Animals on the low-salt and high-salt diets were killed on days 1, 4 and 15. Because of the potential variable food and water intake, experiments were not concluded before 24 hours. Both kidneys were immediately harvested under aseptic conditions and either snap-frozen in liquid nitrogen to isolate total RNA (see below) or placed in a chilled solution that contained 250 mM sucrose, 10 mM triethanolamine, 5 
g/ml leupeptin, 5 g/ml aprotinin and 0.1 mg/ml phenylmethylsulfonyl fluoride (pH 7.6; all from Sigma Chemical Co., St. Louis, MO, USA) in order to obtain protein for Western blotting (see below). Blood was collected for determination of serum sodium concentration. Plasma and urine samples were analyzed for sodium and potassium using flame photometry (model IL-943; Instrumentation Laboratories, Inc., Lexington, MA, USA).
Rat THP purification and description of monoclonal anti-THP antibody
Rat THP was prepared by precipitation from urine using 0.64 M NaCl, according to the original protocol of Tamm and Horsfall1. Purity of the isolated THP was established by Coomassie blue staining following electrophoresis (SDS-PAGE) using a 10% polyacrylamide gel. A mouse monoclonal anti-human THP antibody (#5B4E2) was raised in our laboratory and purified from the medium using ammonium sulfate precipitation, as described previously10. This monoclonal antibody reacted with deglycosylated human THP and thus was directed against a peptide portion of the molecule. Immunoblotting was performed to determine whether this monoclonal antibody also recognized rat THP. Following SDS-PAGE using 10% polyacrylamide gel under reducing conditions, rat THP was transferred onto a nitrocellulose membrane. After washing and blocking unbound sites on the nitrocellulose membrane in standard fashion, the membrane was incubated with our mouse anti-human THP monoclonal antibody, 50 g/ml in phosphate-buffered saline containing 5% nonfat milk (Sigma Chemical Co.), followed by goat anti-mouse IgG conjugated with horseradish peroxidase (Bio-Rad, Melville, NY, USA), 1:2000 dilution in phosphate-buffered saline. After additional washes, the membrane was developed using ECL Western blotting system (Amersham International plc., Buckinghamshire, UK) and XAR-5 film (Kodak, Rochester, NY, USA). Under these conditions, a single band at approximately 100 kD was identified, demonstrating immunoreactivity with rat THP (data not shown).
Western blot analysis
Protein was harvested from kidneys using the protocol of Ecelbarger and associates; this protocol recovered membrane-bound proteins and was used to examine THP expression in that study18. Following placement of the kidney in chilled membrane-isolation solution containing 250 mM sucrose, 10 mM triethanolamine, 5 g/ml leupeptin, 5 g/ml aprotinin and 0.1 mg/ml phenylmethylsulfonyl fluoride (pH 7.6; all from Sigma Chemical Co.), the medulla was dissected and then homogenized using a tissue homogenizer (Omni-Mixer 17105; Omni Co., Waterbury, CT, USA) in the same isolation solution. The homogenized tissue was initially centrifuged at 1,000 
g for 10 minutes, and the supernatant was collected. To increase yield and to effect more thorough homogenization, the pellet was again homogenized and centrifuged at 1,000 g for 10 minutes. This supernatant was combined with the previous collection. Crude membrane samples were prepared using a single-speed fractionation procedure, which was characterized previously7,18. In this protocol, the supernatant was spun at 17,000 g for 20 minutes to obtain a pellet. The pellets, which represented the crude plasma membrane preparation, were resuspended in isolation solution and protein concentration was determined using a kit (Micro BCA Protein Assay Reagent Kit; Pierce, Rockford, IL, USA). Samples containing 5 g of total protein were solubilized at 60°C for 15 minutes in Laemmli sample buffer that contained 
-mercaptoethanol, loaded into 8% SDS-polyacrylamide minigels (Mini-PROTEAN II; Bio-Rad Laboratory), and size-fractionated. The proteins were electrophoretically transferred onto nitrocellulose membrane. After blocking in 5% nonfat milk (Sigma Chemical Co.), the membrane was probed with the mouse monoclonal anti-THP antibody, 50 g/ml in phosphate-buffered saline containing 5% milk. Following addition of goat anti-mouse IgG conjugated with horseradish peroxidase (Bio-Rad Laboratory), 1:3000 dilution in phosphate-buffered saline, the membrane was developed using peroxidase substrate (0.8 mg/ml 3,3'-diaminobenzidine in phosphate-buffered saline; 0.01% hydrogen peroxide; all from Sigma Chemical Co.). To quantify the amount of THP in each sample, the density of each band was determined using a densitometer (Model 620 Video Densitometer; Bio-Rad Laboratory).
Northern hybridization analysis
Total RNA was isolated by single-step method of acid guanidinium thiocyanate-phenol chloroform extraction19. Briefly, kidneys were homogenized in a denaturing solution of 4 M guanidinium isothiocyanate, 0.5% sarcosyl, 0.1 M -mercaptoethanol in 25 mM sodium citrate (pH 7.0). After phenol/chloroform extraction, the RNA was precipitated twice with isopropanol and washed with 70% ethanol. The concentration and purity of RNA in each sample was determined using optical density at 260 and 280 nm. Fifteen micrograms of total RNA from each sample was electrophoresed in 1.0% agarose gels containing 2.2 M formaldehyde and 0.2 M MOPS, pH 7.0. Before transfer, each gel was stained with acridine orange to ensure the quality of the RNA and to determine the location of the 18S and 28S ribosomal RNA. After the gel was rinsed in distilled water, the gel was soaked in 50 mM NaOH for 10 minutes, then the samples were transferred to a nylon membrane (Boehringer Mannheim Products, Mannheim, Germany) using vacuum blotting (Model 785; Bio-Rad Laboratory) in 10 SSC. Nucleic acids were cross-linked by short ultraviolet irradiation (Stratagene, La Jolla, CA, USA). Membranes were prehybridized for 15 minutes at 68°C in a standard hybridization solution (QuikHyb; Stratagene). The membranes were hybridized at 68°C for 60 minutes with a 2.2 kb cDNA probe labeled with 
-32P-dCTP by random oligonucleotide priming (Prime-a-Gene Labeling system; Promega, Inc., WI, USA). Rat THP cDNA was kindly provided by Dr. George A. Scheele20. The blots were washed twice in 2 SSC in 0.1% SDS for 15 minutes at room temperature, followed by a single high-stringency wash using 0.1 SSC in 0.1% SDS for 20 to 30 minutes at 60°C. Membranes were exposed to XAR-5 film (Kodak), which was developed after overnight exposure at -80°C. The blots were then stripped in solution containing 1 mM Tris-HCI, pH 8.0, 0.1 mM EDTA and 0.1 Denhardt's solution, at 75°C for two hours. The membranes were then hybridized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe, which had been labeled with32P using the same method as that described above. The human GAPDH probe (plasmid pBR322) was obtained through American Type Culture Collection (Rockville, MD, USA). Autoradiography was then performed as described above. Autoradiographs were scanned using a densitometer (Model 620 Video Densitometer; Bio-Rad Laboratory). The density of the GAPDH band in the same lane was used to normalize mRNA loading. For quantification, the density of the THP bands were individually divided by the density of band for GAPDH in the same lane.
Statistical analysis
All data were expressed as mean 
standard error. Significant differences among data sets were determined using either unpaired t-test or one-way analysis of variance using multiple comparisons by Fisher's protected least significant difference method, where appropriate. A P value of < 5% was considered significant.
RESULTS
Mean body wts, food intake, serum [Na+], urinary flow rate (UV
), and urinary sodium excretion (UNaV) and potassium excretion (UKV) rates of the groups of rats in this study were shown in Table 1. One animal in the Lo-salt group died of unexplained causes. Mean body wts and serum sodium concentrations did not differ among the groups of rats studied on the same days. As expected, compared to the Lo-salt and Mid-salt groups, rats on the 8.0% NaCl diet exhibited higher mean UV, UNaV, and UKV at each time of study (days 1, 4 and 15).
Mean relative steady-state mRNA levels of THP increased (P < 0.05) by the first day and remained elevated throughout the study, indicating a direct effect of dietary salt on production of THP Figures 1 and 2. The effect of furosemide was also examined. Four days following addition of furosemide to animals on the high-salt diet (Hi-F), the mean amount of mRNA of THP was greater (P < 0.05) than levels seen in animals on either the high-salt diet or the low-salt diet. However, supplementation of rats on the high-salt diet with chlorothiazide (Hi-C) produced no further increases Figure 2.
Figure 1.
Northern analysis using total RNA from kidneys of rats on an 8.0% or 0.3% NaCl diet. Samples were processed simultaneously and run on the same gel. Animals were examined after one day (N = 4 animals in each group) and after 15 days (N = 4 in Hi-salt group and N = 3 in Lo-salt group) in metabolic cages while on these two diets. Steady-state levels of mRNA of THP were greater in the Hi-salt group compared to the corresponding Lo-salt group examined at the same time point, by the first day of study, and remained elevated through day 15. The locations of 18S and 28S ribosomal RNA in the gel were represented by the arrows.
Full figure and legend (55K)Figure 2.
Summary of mean relative steady-states levels of mRNA of THP of the Hi-salt (
), Lo-salt (
), Hi-F 
, and Hi-C (
) groups in this study. Rats ingesting the 8.0% NaCl diet had sustained increases in steady-state mRNA, compared to rats on 0.3% NaCl diet for the same duration. Addition of furosemide, but not chlorothiazide, further increased steady-state mRNA, compared to the other groups of animals in the study for four days. *P < 0.05 than the corresponding Hi-salt group on that day of study;‡P < 0.05 than the other three groups on that day of study.
Using Western blotting with a monoclonal antibody (#5B4E2) that recognized rat THP (Methods section), an increase in THP protein expression in the medulla was seen after one day on 8.0% NaCl diet and persisted throughout the study Figures 3 and 4. Differences in protein expression between animals on 0.3% NaCl diet (Lo-salt) and those on 1.0% NaCl diet (Mid-salt) were also evident.
Figure 3.
Western blot of membrane preparations obtained from the medulla of rats on the three diets. All samples from the same day were processed simultaneously and run on the same gel. Animals were examined on days 1, 4 and 15. A single band at approximately 100 kD was identified by the mouse monoclonal anti-THP antibody (#5B4E2).
Full figure and legend (62K)Figure 4.
Quantification of protein expression in the medulla of rats on the various sodium chloride diets. The amount of THP was greatest (*P < 0.05) in preparations obtained from animals on an 8.0% NaCl diet (Hi-salt, N = 4 rats), compared to rats on a 1.0% NaCl (Mid-salt, N = 4) and 0.3% NaCl (Lo-salt, N = 4) at each of the time points studied. In addition, the amount of THP in preparations from the Mid-salt groups was greater (‡P < 0.05) than that obtained from the Lo-salt groups at each time point examined.
Full figure and legend (28K)DISCUSSION
Production of THP occurs exclusively in the medullary and cortical portions of the thick ascending limb of the loop of Henle (TALH)5,21. THP belongs to a unique class of glycophospholipid-anchored proteins22. This post-translational modification directs THP to the apical surface of cells of the TALH, but the protein remains embedded in the outer leaflet of the lipid bilayer and extends into the tubular lumen22. The unique physicochemical properties and anatomic distribution suggest a role for THP in salt transport activity in the TALH. Our current study demonstrated that a high-salt diet increased steady-state levels of mRNA, along with protein expression, of THP in the kidney. The increase in mRNA occurred by the first day on the high-salt diet and remained persistently elevated through the 15-day course of the study. Finally, twice daily administration of the loop diuretic, furosemide, but not chlorothiazide, a diuretic that inhibits salt transport in the early distal convoluted tubule, to rats on the high-salt diet produced further increases in steady-state mRNA levels of THP. Thus, although considered a constitutively produced protein, our findings showed that this process was modified under certain physiological conditions. While our present study did not demonstrate the physiologic role of THP, an increase in THP expression in the TALH, as seen in this study, along with the increase in luminal [NaCl] that occurs in rats during high-salt intake23, directly promote self-aggregation of THP24,25,26. Formation of a highly negatively charged, hydrophobic, gelatinous barrier may decrease transepithelial electrolyte flux in the TALH.
The effect of changes in dietary salt on THP production was not examined previously. Results of our experiments that used furosemide agreed with several investigators18,27, but disagreed with others9. Using Western blotting, Ecelbarger and associates clearly showed an increase in THP in kidneys of rats exposed to furosemide18. Also, Wirdnam and Milne demonstrated that furosemide stimulated release of THP directly from cortical slices27. In contrast to these studies and our current findings, however, Bachmann et al showed that THP excretion did not change one to three weeks following institution of furosemide, 12 mg/day, administered by osmotic minipumps9. Although our study administered 5 mg/kg per day by intraperitoneal injection, Ecelbarger and associates18 administered 10 mg/day by osmotic minipumps over five days. Thus, differences in the doses of furosemide used in these studies does not appear to explain the apparent discrepancy, which was not determined. Certainly, THP expression in the kidney may not correlate with protein excretion, which is technically difficult to assay in the urine28. In our present study, the combined effect of furosemide and a high-salt diet was greater than the effect of dietary salt alone.
In summary, the present study demonstrates that an increase in dietary salt facilitated production of THP in rats. The data also show that furosemide augments this effect and provides additional confirmation of previous studies18,27 that showed an effect of furosemide on THP. Our findings suggest an involvement of THP in the physiological function of the TALH. In addition, THP plays an important role in pathological conditions that includes cast nephropathy ("myeloma kidney") and nephrolithiasis. Cast nephropathy develops when nephrotoxic immunoglobulin light chains bind to THP to produce a poorly soluble complex that precipitates in the lumen of the distal nephron and blocks flow of tubule fluid10,11,12,13,14,15. Intrarenal expression of THP is particularly important in the pathogenesis of this lesion. The role of THP in nephrolithiasis is complex16,29,30. While THP from healthy volunteers serves as an inhibitor of calcium oxalate formation, THP from patients with recurrent stone formation augments calcium oxalate aggregation16. In addition, a diet high in sodium has been suggested to facilitate renal stone formation31,32. By facilitating intrarenal expression of THP, a high-salt diet, with or without furosemide, may produce detrimental effects on renal function in patients with multiple myeloma and facilitate renal stone formation in predisposed individuals.
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Acknowledgments
This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, and National Institutes of Health grant DK-46199. Portions of this manuscript have been published in abstract form (J Am Soc Nephrol 8:607A, 1997). The authors especially thank Dr. George A. Scheele for his very helpful discussions and providing the cDNA for rat THP.
