Dietary salt regulates expression of Tamm-Horsfall glycoproteinin rats

WEI-ZHONG YING and PAUL W. SANDERS

Nephrology Research and Training Center, Comprehensive Cancer Center, and Cell Adhesion and Matrix Research Center, Division ofNephrology, Department of Medicine and Department of Physiology & Biophysics, University of Alabama at Birmingham, andDepartment of Veterans Affairs Medical Center, Birmingham, Alabama, USA

Dietary salt regulates expression of Tamm-Horsfall glycoproteinin rats.

Background. Tamm-Horsfall glycoprotein (THP) is a uniqueprotein that is produced exclusively by cells of the thick ascendinglimb of Henle’s loop (TALH). This study examined whetherdietary salt altered renal THP production.

Methods. Male Sprague-Dawley rats were examined on days 1,4, and 15 following placement in metabolic cages on diet thatcontained 0.3%, 1.0% or 8.0% NaCl. THP expression was quan-tified using Northern hybridization and Western blotting analysis.

Results. An increase in dietary salt produced sustained increasesin relative steady-state mRNA and protein levels of THP in thekidney. Addition of furosemide, but not chlorothiazide, to animalson the 8.0% NaCl diet further augmented steady-state mRNAlevels of THP.

Conclusions. An increase in dietary salt and the loop diuretic,furosemide, increased expression of THP in the rat. The datasupport the involvement of this unique protein in the function ofthe TALH during changes in dietary salt. These findings alsosuggest that restriction of dietary salt may be beneficial in castnephropathy in multiple myeloma and recurrent nephrolithiasis,two diseases in which THP can play an important pathogeneticrole.

Tamm-Horsfall glycoprotein (THP) was described ini-tially as a urinary protein that inhibited viral hemaggluti-nation [1]. Subsequently, Muchmore and Decker identifieda urinary protein that possessed immunosuppressive prop-erties in vitro and hence coined the term uromodulin [2].Interest in uromodulin resulted in cloning of this protein,which was identified as THP [3, 4]; however, when furtherstudies did not suggest a major physiological function inimmune modulation, enthusiasm for the study of THPdiminished. The function of THP remained undefined, but

the exclusive production by cells of the thick ascending limbof the loop of Henle (TALH) along with a striking absenceof THP mRNA in the macula densa [5], a specializedportion of the TALH involved in the detection of theluminal concentration of sodium chloride, suggested aninvolvement in salt and water transport in the TALH.However, the absence of aquaporins in the TALH [6–8]has been proposed as the mechanism of water imperme-ability of that segment. Along with studies showing thatwater intake did not alter THP production [9], involvementof THP in water transport in the TALH appeared unlikely.Although not proven, THP may modulate salt transport inthe TALH.

We hypothesized that changes in dietary salt and theaddition of a loop diuretic altered the production of THP.Understanding the involvement of these parameters inTHP expression is also potentially useful in the therapy ofdiseases where THP plays an important role, such as castnephropathy (“myeloma kidney”) [10–15] and nephrolithi-asis [16]. Using Northern hybridization and Western blot-ting, the current study determined whether dietary saltaffected relative steady-state mRNA and renal proteincontent of THP in Sprague-Dawley rats.

METHODS

Animal preparation

Seventy-six male Sprague-Dawley rats (Charles RiverLaboratories, Wilmington, MA, USA), 28 days of age, werehoused under standard conditions and given 0.3% (low-salt) diet (AIN-76A with 0.3% NaCl; Dyets, Inc., Bethle-hem, PA, USA) and water ad libitum for four days beforeinitiating the experiment. Animals were used at this agebecause previous experiments showed preservation ofblood pressure and inulin clearance for two weeks on an8.0% NaCl diet [17]. Maintaining the animals on a 0.3%NaCl diet allowed the establishment of a baseline standard-ized diet before initiation of study. Rats were then placed inmetabolic cages and allowed free access to a water and ratdiet, which contained 0.3% (termed the Lo-salt group),

Key words: uromodulin, thick ascending limb of the loop of Henle,furosemide, chlorothiazide, gene expression.

Received for publication March 4, 1998and in revised form May 11, 1998Accepted for publication May 11, 1998

© 1998 by the International Society of Nephrology

Kidney International, Vol. 54 (1998), pp. 1150–1156

1150

1.0% (AIN-76A with 0.3% NaCl; Dyets, Inc.; the Mid-saltgroup) or 8.0% (AIN-76A containing 8.0% NaCl, Dyets,Inc.; the Hi-salt group) sodium chloride. Urine was col-lected under oil to prevent desiccation. Food consumption,urine flow and body weights were recorded daily. In otherexperiments, rats on the high-salt diet received intraperito-neal injections of either furosemide (Elkins-Sinn, Inc.,Cherry Hill, NJ, USA), 5 mg/kg, (denoted Hi-F), or chlo-rothiazide (Merck & Co., West Point, PA, USA), 4 mg/kg(Hi-C), twice daily. Animals on the low-salt and high-saltdiets were killed on days 1, 4 and 15. Because of thepotential variable food and water intake, experiments werenot concluded before 24 hours. Both kidneys were imme-diately 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 mg/ml leupeptin, 5 mg/mlaprotinin and 0.1 mg/ml phenylmethylsulfonyl fluoride (pH7.6; all from Sigma Chemical Co., St. Louis, MO, USA) inorder to obtain protein for Western blotting (see below).Blood was collected for determination of serum sodiumconcentration. Plasma and urine samples were analyzed forsodium and potassium using flame photometry (modelIL-943; Instrumentation Laboratories, Inc., Lexington,MA, USA).

Rat THP purification and description of monoclonalanti-THP antibody

Rat THP was prepared by precipitation from urine using0.64 M NaCl, according to the original protocol of Tammand Horsfall [1]. Purity of the isolated THP was establishedby Coomassie blue staining following electrophoresis (SDS-PAGE) using a 10% polyacrylamide gel. A mouse mono-clonal anti-human THP antibody (#5B4E2) was raised inour laboratory and purified from the medium using ammo-nium sulfate precipitation, as described previously [10].This monoclonal antibody reacted with deglycosylated hu-

man THP and thus was directed against a peptide portionof the molecule. Immunoblotting was performed to deter-mine whether this monoclonal antibody also recognized ratTHP. Following SDS-PAGE using 10% polyacrylamide gelunder reducing conditions, rat THP was transferred onto anitrocellulose membrane. After washing and blocking un-bound sites on the nitrocellulose membrane in standardfashion, the membrane was incubated with our mouseanti-human THP monoclonal antibody, 50 mg/ml in phos-phate-buffered saline containing 5% nonfat milk (SigmaChemical Co.), followed by goat anti-mouse IgG conju-gated with horseradish peroxidase (Bio-Rad, Melville, NY,USA), 1:2000 dilution in phosphate-buffered saline. Afteradditional washes, the membrane was developed usingECL Western blotting system (Amersham Internationalplc., Buckinghamshire, UK) and XAR-5 film (Kodak,Rochester, NY, USA). Under these conditions, a singleband at approximately 100 kD was identified, demonstrat-ing immunoreactivity with rat THP (data not shown).

Western blot analysis

Protein was harvested from kidneys using the protocol ofEcelbarger and associates; this protocol recovered mem-brane-bound proteins and was used to examine THPexpression in that study [18]. Following placement of thekidney in chilled membrane-isolation solution containing250 mM sucrose, 10 mM triethanolamine, 5 mg/ml leupeptin,5 mg/ml aprotinin and 0.1 mg/ml phenylmethylsulfonylfluoride (pH 7.6; all from Sigma Chemical Co.), the me-dulla was dissected and then homogenized using a tissuehomogenizer (Omni-Mixer 17105; Omni Co., Waterbury,CT, USA) in the same isolation solution. The homogenizedtissue was initially centrifuged at 1,000 3 g for 10 minutes,and the supernatant was collected. To increase yield and toeffect more thorough homogenization, the pellet was againhomogenized and centrifuged at 1,000 3 g for 10 minutes.

Table 1. Compiled data of the groups of animals in the study

Group Day of studyNumber of

animals Body wt g

UV̇ ml/dayUNa1V̇

mEq/minUK1V̇

mEq/minSerum [Na1]

mEq/liter per 100 g body weight

Lo-salt 1 8 110 6 6 143 6 4 7.4 6 0.6 0.13 6 0.02 0.2 6 0.04a

Nor-salt 1 4 128 6 4 ND 9.1 6 0.5 2.3 6 0.2 0.8 6 0.1Hi-salt 1 8 115 6 4 145 6 6 38.0 6 2.2a 12.2 6 1.0a 0.8 6 0.1Lo-salt 4 12 121 6 10 146 6 3 8.9 6 1.1b 0.2 6 0.03b 0.3 6 0.1a

Nor-salt 4 4 142 6 5 ND 11.2 6 0.7b 3.1 6 0.5b 1.1 6 0.2Hi-salt 4 12 135 6 7 148 6 4 57.7 6 5.6 19.8 6 2.2 1.2 6 0.2Hi-F 4 4 133 6 3 152 6 4 67.2 6 4.5 20.0 6 0.7 0.8 6 0.1Hi-C 4 4 140 6 2 154 6 5 71.1 6 3.2 22.8 6 0.7 1.1 6 0.1Lo-salt 15 7 190 6 13 137 6 4 15.6 6 3.0b 0.6 6 0.2b 1.0 6 0.2a

Nor-salt 15 4 223 6 11 ND 28.0 6 3.6b 8.3 6 1.2b 2.6 6 0.4Hi-salt 15 8 178 6 7 136 6 5 87.0 6 9.3 48.3 6 10.6 2.2 6 0.3

ND is not donea P , 0.05, compared to the other groups examined on that dayb P , 0.05, compared to the group on the 8.0% NaCl chow (Hi-salt) for the same duration

Ying and Sanders: Dietary salt controls production of THP 1151

This supernatant was combined with the previous collec-tion. Crude membrane samples were prepared using asingle-speed fractionation procedure, which was character-ized previously [7, 18]. In this protocol, the supernatant wasspun at 17,000 3 g for 20 minutes to obtain a pellet. Thepellets, which represented the crude plasma membranepreparation, were resuspended in isolation solution andprotein concentration was determined using a kit (MicroBCA Protein Assay Reagent Kit; Pierce, Rockford, IL,USA). Samples containing 5 mg of total protein weresolubilized at 60°C for 15 minutes in Laemmli samplebuffer that contained b-mercaptoethanol, loaded into 8%

SDS-polyacrylamide minigels (Mini-PROTEAN II; Bio-Rad Laboratory), and size-fractionated. The proteins wereelectrophoretically transferred onto nitrocellulose mem-brane. After blocking in 5% nonfat milk (Sigma ChemicalCo.), the membrane was probed with the mouse monoclo-nal anti-THP antibody, 50 mg/ml in phosphate-bufferedsaline containing 5% milk. Following addition of goatanti-mouse IgG conjugated with horseradish peroxidase(Bio-Rad Laboratory), 1:3000 dilution in phosphate-buff-ered saline, the membrane was developed using peroxidasesubstrate (0.8 mg/ml 3,39-diaminobenzidine in phosphate-buffered saline; 0.01% hydrogen peroxide; all from SigmaChemical Co.). To quantify the amount of THP in eachsample, the density of each band was determined using adensitometer (Model 620 Video Densitometer; Bio-RadLaboratory).

Northern hybridization analysis

Total RNA was isolated by single-step method of acidguanidinium thiocyanate-phenol chloroform extraction[19]. Briefly, kidneys were homogenized in a denaturingsolution of 4 M guanidinium isothiocyanate, 0.5% sarcosyl,0.1 M b-mercaptoethanol in 25 mM sodium citrate (pH 7.0).After phenol/chloroform extraction, the RNA was precip-itated twice with isopropanol and washed with 70% etha-nol. The concentration and purity of RNA in each samplewas determined using optical density at 260 and 280 nm.Fifteen micrograms of total RNA from each sample waselectrophoresed 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 thequality of the RNA and to determine the location of the18S and 28S ribosomal RNA. After the gel was rinsed indistilled water, the gel was soaked in 50 mM NaOH for 10minutes, then the samples were transferred to a nylonmembrane (Boehringer Mannheim Products, Mannheim,Germany) using vacuum blotting (Model 785; Bio-RadLaboratory) in 103 SSC. Nucleic acids were cross-linked by

Fig. 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 thesame gel. Animals were examined after one day (N 5 4 animals in each group) and after 15 days (N 5 4 in Hi-salt group and N 5 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 correspondingLo-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 ribosomalRNA in the gel were represented by the arrows.

Fig. 2. Summary of mean relative steady-states levels of mRNA of THP ofthe Hi-salt (f), Lo-salt (M), Hi-F (u), and Hi-C (;) groups in this study.Rats ingesting the 8.0% NaCl diet had sustained increases in steady-statemRNA, 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 forfour days. *P , 0.05 than the corresponding Hi-salt group on that day ofstudy; ‡P , 0.05 than the other three groups on that day of study.

Ying and Sanders: Dietary salt controls production of THP1152

short ultraviolet irradiation (Stratagene, La Jolla, CA,USA). Membranes were prehybridized for 15 minutes at68°C in a standard hybridization solution (QuikHyb; Strat-agene). The membranes were hybridized at 68°C for 60minutes with a 2.2 kb cDNA probe labeled with a-32P-dCTP by random oligonucleotide priming (Prime-a-GeneLabeling system; Promega, Inc., WI, USA). Rat THPcDNA was kindly provided by Dr. George A. Scheele [20].The blots were washed twice in 23 SSC in 0.1% SDS for 15minutes at room temperature, followed by a single high-stringency wash using 0.13 SSC in 0.1% SDS for 20 to 30minutes at 60°C. Membranes were exposed to XAR-5 film(Kodak), which was developed after overnight exposure at280°C. The blots were then stripped in solution containing1 mM Tris-HCI, pH 8.0, 0.1 mM EDTA and 0.13 Den-hardt’s solution, at 75°C for two hours. The membraneswere then hybridized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe, which hadbeen labeled with 32P using the same method as thatdescribed above. The human GAPDH probe (plasmidpBR322) was obtained through American Type CultureCollection (Rockville, MD, USA). Autoradiography wasthen performed as described above. Autoradiographs werescanned using a densitometer (Model 620 Video Densitom-eter; Bio-Rad Laboratory). The density of the GAPDHband in the same lane was used to normalize mRNAloading. For quantification, the density of the THP bandswere individually divided by the density of band forGAPDH in the same lane.

Statistical analysis

All data were expressed as mean 6 standard error.Significant differences among data sets were determinedusing either unpaired t-test or one-way analysis of varianceusing multiple comparisons by Fisher’s protected leastsignificant difference method, where appropriate. A Pvalue of , 5% was considered significant.

RESULTS

Mean body wts, food intake, serum [Na1], urinary flowrate (UV̇), and urinary sodium excretion (UNaV̇) andpotassium excretion (UKV̇) rates of the groups of rats inthis study were shown in Table 1. One animal in the Lo-saltgroup died of unexplained causes. Mean body wts andserum sodium concentrations did not differ among thegroups of rats studied on the same days. As expected,compared to the Lo-salt and Mid-salt groups, rats on the8.0% NaCl diet exhibited higher mean UV̇, UNaV̇, andUKV̇ at each time of study (days 1, 4 and 15).

Mean relative steady-state mRNA levels of THP in-creased (P , 0.05) by the first day and remained elevatedthroughout the study, indicating a direct effect of dietarysalt on production of THP (Figs. 1 and 2). The effect offurosemide was also examined. Four days following addi-tion 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

Fig. 3. Western blot of membrane preparationsobtained from the medulla of rats on the threediets. All samples from the same day wereprocessed simultaneously and run on the samegel. Animals were examined on days 1, 4 and 15.A single band at approximately 100 kD wasidentified by the mouse monoclonal anti-THPantibody (#5B4E2).

Ying and Sanders: Dietary salt controls production of THP 1153

the low-salt diet. However, supplementation of rats on thehigh-salt diet with chlorothiazide (Hi-C) produced nofurther increases (Fig. 2).

Using Western blotting with a monoclonal antibody(#5B4E2) that recognized rat THP (Methods section), anincrease in THP protein expression in the medulla was seenafter one day on 8.0% NaCl diet and persisted throughoutthe study (Figs. 3 and 4). Differences in protein expressionbetween animals on 0.3% NaCl diet (Lo-salt) and those on1.0% NaCl diet (Mid-salt) were also evident.

DISCUSSION

Production of THP occurs exclusively in the medullaryand cortical portions of the thick ascending limb of the loopof Henle (TALH) [5, 21]. THP belongs to a unique class ofglycophospholipid-anchored proteins [22]. This post-trans-lational modification directs THP to the apical surface ofcells of the TALH, but the protein remains embedded inthe outer leaflet of the lipid bilayer and extends into thetubular lumen [22]. The unique physicochemical propertiesand anatomic distribution suggest a role for THP in salttransport activity in the TALH. Our current study demon-strated that a high-salt diet increased steady-state levels ofmRNA, along with protein expression, of THP in thekidney. The increase in mRNA occurred by the first day onthe high-salt diet and remained persistently elevatedthrough the 15-day course of the study. Finally, twice dailyadministration of the loop diuretic, furosemide, but notchlorothiazide, a diuretic that inhibits salt transport in theearly distal convoluted tubule, to rats on the high-salt dietproduced further increases in steady-state mRNA levels ofTHP. Thus, although considered a constitutively producedprotein, our findings showed that this process was modifiedunder certain physiological conditions. While our presentstudy did not demonstrate the physiologic role of THP, anincrease in THP expression in the TALH, as seen in thisstudy, along with the increase in luminal [NaCl] that occursin rats during high-salt intake [23], directly promote self-aggregation of THP [24–26]. Formation of a highly nega-tively charged, hydrophobic, gelatinous barrier may de-crease transepithelial electrolyte flux in the TALH.

The effect of changes in dietary salt on THP productionwas not examined previously. Results of our experimentsthat used furosemide agreed with several investigators [18,27], but disagreed with others [9]. Using Western blotting,Ecelbarger and associates clearly showed an increase inTHP in kidneys of rats exposed to furosemide [18]. Also,Wirdnam and Milne demonstrated that furosemide stimu-lated release of THP directly from cortical slices [27]. Incontrast to these studies and our current findings, however,Bachmann et al showed that THP excretion did not changeone to three weeks following institution of furosemide, 12mg/day, administered by osmotic minipumps [9]. Althoughour study administered 5 mg/kg per day by intraperitonealinjection, Ecelbarger and associates [18] administered 10mg/day by osmotic minipumps over five days. Thus, differ-ences in the doses of furosemide used in these studies doesnot appear to explain the apparent discrepancy, which was

Fig. 4. Quantification of protein expression in the medulla of rats on thevarious 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 5 4 rats), compared to rats on a 1.0% NaCl (Mid-salt, N 5 4)and 0.3% NaCl (Lo-salt, N 5 4) at each of the time points studied. Inaddition, the amount of THP in preparations from the Mid-salt groupswas greater (‡P , 0.05) than that obtained from the Lo-salt groups at eachtime point examined.

Ying and Sanders: Dietary salt controls production of THP1154

not determined. Certainly, THP expression in the kidneymay not correlate with protein excretion, which is techni-cally difficult to assay in the urine [28]. In our present study,the combined effect of furosemide and a high-salt diet wasgreater than the effect of dietary salt alone.

In summary, the present study demonstrates that anincrease in dietary salt facilitated production of THP inrats. The data also show that furosemide augments thiseffect and provides additional confirmation of previousstudies [18, 27] that showed an effect of furosemide onTHP. Our findings suggest an involvement of THP in thephysiological function of the TALH. In addition, THP playsan important role in pathological conditions that includescast nephropathy (“myeloma kidney”) and nephrolithiasis.Cast nephropathy develops when nephrotoxic immuno-globulin light chains bind to THP to produce a poorlysoluble complex that precipitates in the lumen of the distalnephron and blocks flow of tubule fluid [10–15]. Intrarenalexpression of THP is particularly important in the patho-genesis of this lesion. The role of THP in nephrolithiasis iscomplex [16, 29, 30]. While THP from healthy volunteersserves as an inhibitor of calcium oxalate formation, THPfrom patients with recurrent stone formation augmentscalcium oxalate aggregation [16]. In addition, a diet high insodium has been suggested to facilitate renal stone forma-tion [31, 32]. By facilitating intrarenal expression of THP, ahigh-salt diet, with or without furosemide, may producedetrimental effects on renal function in patients withmultiple myeloma and facilitate renal stone formation inpredisposed individuals.

ACKNOWLEDGMENTS

This work was supported by the Office of Research and Development,Medical Research Service, Department of Veterans Affairs, and NationalInstitutes of Health grant DK-46199. Portions of this manuscript havebeen published in abstract form (J Am Soc Nephrol 8:607A, 1997). Theauthors especially thank Dr. George A. Scheele for his very helpfuldiscussions and providing the cDNA for rat THP.

Reprint requests to Paul W. Sanders, M.D., Division of Nephrology/Department of Medicine, 642 Lyons-Harrison Research Building, Universityof Alabama at Birmingham, Birmingham, Alabama 35294-0007, USA.E-mail: paul sanders@nrtc.dom.uab.edu

APPENDIX

Abbreviations used in this paper are: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hi-C, animals maintained on 8.0% NaCl chowand given chlorothiazide; Hi-F, animals maintained on 8.0% NaCl chowand given furosemide; Hi-salt, animals maintained on 8.0% NaCl chow;Lo-salt, animals maintained on 0.3% NaCl chow; Mid-salt, animalsmaintained on 1.0% NaCl chow; TALH, thick ascending limb of the loopof Henle; THP, Tamm-Horsfall glycoprotein.

REFERENCES

1. TAMM I, HORSFALL FL JR: Characterization and separation of aninhibitor of viral hemagglutination present in urine. Proc Soc Exp BiolMed 74:108–114, 1950

2. MUCHMORE AV, DECKER JM: Uromodulin: A unique 85 kD immu-nosuppressive glycoprotein isolated from urine of pregnant women.Science (Wash) 229:479–481, 1985

3. PENNICA D, KOHR WJ, KUANG W-J, GLAISTER D, AGGARWAL BB,CHEN EY, GOEDDEL DV: Identification of human uromodulin as theTamm-Horsfall urinary glycoprotein. Science (Wash DC) 236:83–88,1987

4. HESSION C, DECKER JM, SHERBLOM AP, KUMAR S, YUE CC, MAT-TALIANO RJ, TIZARD R, KAWASHIMA E, SCHMEISSNER U, HELETKY S,CHOW EP, BURNE CA, SHAW A, MUCHMORE AV: Uromodulin(Tamm-Horsfall glycoprotein): A renal ligand for lymphokines. Sci-ence (Wash) 237:1479–1484, 1987

5. BACHMANN S, METZGER R, BUNNEMANN B: Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle’sloop in rat kidney. Histochemistry 94:517–523, 1990

6. BONDY C, CHIN E, SMITH BL, PRESTON GM, AGRE P: Developmentalgene expression and tissue distribution of the CHIP28 water-channelprotein. Proc Natl Acad Sci USA 90:4500–4504, 1993

7. ECELBARGER CA, TERRIS J, FRINDT G, ECHEVARRIA M, MARPLES D,NIELSEN S, KNEPPER MA: Aquaporin-3 water channel localization andregulation in the rat kidney. Am J Physiol 269(Renal Fluid ElectrolytePhysiol 38):F663–F672, 1995

8. MAUNSBACH AB, MARPLES D, CHIN E, NING G, BONDY CA, AGRE P,NIELSEN S: Aquaporin-1 water channel expression in human kidney.J Am Soc Nephrol 8:1–14, 1997

9. BACHMANN S, DAWNAY ABSJ, BOUBY N, BANKIR L: Tamm-Horsfallprotein excretion during chronic alterations in urinary concentrationand protein intake in the rat. Renal Physiol Biochem 14:236–245, 1991

10. HUANG Z-Q, KIRK KA, CONNELLY KG, SANDERS PW: Bence Jonesproteins bind to a common peptide segment of Tamm-Horsfallglycoprotein to promote heterotypic aggregation. J Clin Invest 92:2975–2983, 1993

11. HUANG Z-Q, SANDERS PW: Biochemical interaction of Tamm-Hors-fall glycoprotein with Ig light chains. Lab Invest 73:810–817, 1995

12. SANDERS PW, HERRERA GA, CHEN A, BOOKER BB, GALLA JH:Differential nephrotoxicity of low molecular weight proteins includingBence Jones proteins in the perfused rat nephron in vivo. J Clin Invest82:2086–2096, 1988

13. SANDERS PW, BOOKER BB, BISHOP JB, CHEUNG HC: Mechanisms ofintranephronal proteinaceous cast formation by low molecular weightproteins. J Clin Invest 85:570–576, 1990

14. SANDERS PW, BOOKER BB: Pathobiology of cast nephropathy fromhuman Bence Jones proteins. J Clin Invest 89:630–639, 1992

15. START DA, SILVA FG, DAVIS LD, D’AGATI V, PIRANI CL: Myelomacast nephropathy: Immunohistochemical and lectin studies. ModernPathol 1:336–347, 1988

16. HESS B, ZIPPERLE L, JAEGER P: Citrate and calcium effects onTamm-Horsfall glycoprotein as a modifier of calcium oxalate crystalaggregation. Am J Physiol 265(Renal Fluid Electrolyte Physiol 34):F784–F791, 1993

17. CHEN PY, SANDERS PW: L-arginine abrogates salt-sensitive hyperten-sion in Dahl/Rapp rats. J Clin Invest 88:1559–1567, 1991

18. ECELBARGER CA, TERRIS J, HOYER JR, NIELSEN S, WADE JB,KNEPPER MA: Localization and regulation of the rat renal Na1-K1-2Cl2 cotransporter, BSC-1. Am J Physiol 271(Renal Fluid ElectrolytePhysiol 40):F619–F628, 1996

19. CHOMCZYNSKI P, SACCHI N: Single-step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction. AnalBiochem 162:156–159, 1987

20. FUKUOKA S-I, FREEDMAN SD, YU H, SUKHATME VP, SCHEELE GA:GP-2/THP gene family encodes self-binding glycosylphosphatidyli-nositol-anchored proteins in apical secretory compartments of pan-creas and kidney. Proc Natl Acad Sci USA 89:1189–1193, 1992

21. BACHMANN S, KOEPPEN-HAGEMANN I, KRIZ W: Ultrastructural local-ization of Tamm-Horsfall glycoprotein (THP) in rat kidney as re-vealed by protein A-gold immunocytochemistry. Histochemistry 83:531–538, 1985

22. RINDLER MJ, NAIK SS, LI N, HOOPS TC, PERALDI M-N: Uromodulin(Tamm-Horsfall glycoprotein/uromucoid) is a phosphatidylinositol-linked membrane protein. J Biol Chem 265:20784–20789, 1990

Ying and Sanders: Dietary salt controls production of THP 1155

23. GALLA JH, BOOKER BB, LUKE RG: Dietary NaCl intake alterschloride reabsorption in the loop segment of superficial nephrons.Presented at the American Society of Nephrology. Washington D.C.,1986

24. MCQUEEN EG, ENGEL GB: Factors determining the aggregation ofurinary mucoprotein. J Clin Pathol (Lond) 19:392–396, 1966

25. STEVENSON FK, CLEAVE AJ, KENT PW: The effect of ions on theviscometric and ultracentrifugal behaviour of Tamm-Horsfall glyco-protein. Biochim Biophys Acta 236:59–66, 1971

26. WIGGINS RC: Uromucoid (Tamm-Horsfall glycoprotein) forms differ-ent polymeric arrangements on a filter surface under different physi-cochemical conditions. Clin Chim Acta 162:329–340, 1987

27. WIRDNAM PK, MILNE RDG: Tamm-Horsfall glycoprotein releasefrom rat kidney cortex slices in vitro. Clin Sci 67:529–534, 1984

28. GRANT AMS, NEUBERGER A: The development of a radioimmunoas-say for the measurement of urinary Tamm-Horsfall glycoprotein inthe presence of sodium dodecyl sulphate. Clin Sci 44:163–179, 1973

29. HESS B, NAKAGAWA Y, COE FL: Inhibition of calcium oxalatemonohydrate crystal aggregation by urine proteins. Am J Physiol257(Renal Fluid Electrolyte Physiol 26):F99–F106, 1989

30. HESS B, NAKAGAWA Y, COE FL: Molecular abnormality of Tamm-Horsfall glycoprotein in calcium oxalate nephrolithiasis. Am J Physiol260(Renal Fluid Electrolyte Physiol 29):F569–F578, 1991

31. CIRILLO M, LAURENZI M, PANARELLI W, STAMLER J: Urinary sodiumto potassium ratio and urinary stone disease. Kidney Int 46:1133–1139,1994

32. STAMLER J, CIRILLO M: Dietary salt and renal stone disease. Lancet349:506–507, 1997

Ying and Sanders: Dietary salt controls production of THP1156