The excretion of uromodulin is modulated by the calcium-sensing receptor
Authors: Natsuko Tokonami, Eric Olinger, Huguette Debaix, Pascal Houillier, Olivier Devuyst
Journal: Kidney International (2018)

Uromodulin is produced in the thick ascending limb, but little is known about regulation of its excretion in urine. Using mouse and cellular models, we demonstrate that excretion of uromodulin by thick ascending limb cells is increased or decreased upon inactivation or activation of the calcium-sensing receptor (CaSR), respectively. These effects reflect changes in uromodulin trafficking and likely involve alterations in intracellular cyclic adenosine monophosphate (cAMP) levels. Administration of the CaSR agonist cinacalcet led to a rapid reduction of urinary uromodulin excretion in healthy subjects. Modulation of uromodulin excretion by the CaSR may be clinically relevant considering the increasing use of CaSR modulators. Uromodulin is produced in the thick ascending limb, but little is known about regulation of its excretion in urine. Using mouse and cellular models, we demonstrate that excretion of uromodulin by thick ascending limb cells is increased or decreased upon inactivation or activation of the calcium-sensing receptor (CaSR), respectively. These effects reflect changes in uromodulin trafficking and likely involve alterations in intracellular cyclic adenosine monophosphate (cAMP) levels. Administration of the CaSR agonist cinacalcet led to a rapid reduction of urinary uromodulin excretion in healthy subjects. Modulation of uromodulin excretion by the CaSR may be clinically relevant considering the increasing use of CaSR modulators. Uromodulin (Tamm-Horsfall protein), the most abundant protein excreted in normal urine, is synthesized in the cells lining the thick ascending limb (TAL) of the loop of Henle. Convergent studies indicate that uromodulin regulates salt transport and urinary concentration, protects against kidney stones and urinary tract infections, and plays roles in acute kidney injury and innate immunity. Furthermore, the UMOD gene coding for uromodulin has been associated with a spectrum of rare and common kidney diseases.1Devuyst O. Olinger E. Rampoldi L. Uromodulin: from physiology to rare and complex kidney disorders.Nat Rev Nephrol. 2017; 13: 525-544Crossref PubMed Scopus (160) Google Scholar Although the excretion of uromodulin in urine is known to fluctuate,2Olden M. Corre T. Hayward C. et al.Common variants in UMOD associate with urinary uromodulin levels: a meta-analysis.J Am Soc Nephrol. 2014; 25: 1869-1882Crossref PubMed Scopus (78) Google Scholar surprisingly little is known about the regulators of uromodulin abundance in the kidney and urine. The correlations between urinary levels of uromodulin and markers of tubular function3Pruijm M. Ponte B. Ackermann D. et al.Associations of urinary uromodulin with clinical characteristics and markers of tubular function in the general population.Clin J Am Soc Nephrol. 2016; 11: 70-80Crossref PubMed Scopus (63) Google Scholar, 4Troyanov S. Delmas-Frenette C. Bollée G. et al.clinical, genetic, and urinary factors associated with uromodulin excretion.Clin J Am Soc Nephrol. 2016; 11: 62-69Crossref PubMed Scopus (29) Google Scholar and the potential influence of arginine vasopressin5Bachmann S. Dawnay A.B. Bouby N. Bankir L. Tamm-Horsfall protein excretion during chronic alterations in urinary concentration and protein intake in the rat.Ren Physiol Biochem. 1991; 14: 236-245PubMed Google Scholar suggest that factors operating on the TAL may be involved. The CaSR is a G protein–coupled receptor expressed in the TAL, where it regulates paracellular Ca2+ reabsorption through mechanisms that include a dose-dependent inhibition of intracellular cAMP levels.6Loupy A. Ramakrishnan S.K. Wootla B. et al.PTH-independent regulation of blood calcium concentration by the calcium-sensing receptor.J Clin Invest. 2012; 122: 3355-3367Crossref PubMed Scopus (143) Google Scholar, 7Riccardi D. Valenti G. Localization and function of the renal calcium-sensing receptor.Nat Rev Nephrol. 2016; 12: 414-425Crossref PubMed Scopus (88) Google Scholar Inactivating or activating mutations of the CASR gene lead to rare hypercalcemia or hypocalcemia disorders, respectively.8Toka H.R. Pollak M.R. Houillier P. Calcium sensing in the renal tubule.Physiology (Bethesda). 2015; 30: 317-326PubMed Google Scholar The latter can be associated with Bartter-like syndrome, supporting the role of CaSR in maintaining the TAL function.9Vargas-Poussou R. Huang C. Hulin P. et al.Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome.J Am Soc Nephrol. 2002; 13: 2259-2266Crossref PubMed Scopus (287) Google Scholar Whether the renal CaSR regulates uromodulin production and/or excretion by TAL cells is unknown. Here we used mouse models with inactivating and activating mutations in the Casr gene, combined with pharmacologic agents and physiological stimuli, to demonstrate that chronic or acute modulation of the CaSR influences the urinary excretion of uromodulin. We first verified that, in mouse kidney, Umod and Casr mRNAs are most abundantly expressed in the TAL (Figure 1a), with distinct apical (uromodulin) and basolateral (CaSR) distribution in that segment (Figure 1b). Immunoblotting on primary cultures of mouse TAL (mTAL) cells obtained from TAL segments10Glaudemans B. Terryn S. Gölz N. et al.A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing.Pflugers Arch. 2014; 466: 343-356Crossref PubMed Scopus (18) Google Scholar demonstrated that they endogenously express uromodulin and CaSR, NKCC2, and ROMK and excrete uromodulin into the apical medium (Figure 1c). To examine whether the CaSR modulates uromodulin excretion in vivo, we analyzed mice with activating (CasrNuf/Nuf) and inactivating (CasrBCH002/+) mutations in Casr (Supplementary Figure S1A and S1B). The activating Casr mutation p.Leu723Gln in CasrNuf/Nuf mice (Supplementary Figure S1C and S1E)11Hough T.A. Bogani D. Cheeseman M.T. et al.Activating calcium-sensing receptor mutation in the mouse is associated with cataracts and ectopic calcification.Proc Natl Acad Sci U S A. 2004; 101: 13566-13571Crossref PubMed Scopus (120) Google Scholar results in significantly decreased plasma Ca2+ and Mg2+ levels, elevated plasma phosphorus levels, and lower urinary Ca2+, Mg2+, and phosphorus levels compared with Casr+/+ mice (Supplementary Table S1). The CasrNuf/Nuf mice excreted significantly less uromodulin in urine than did Casr+/+ mice, despite similar uromodulin protein (Figure 1d and Supplementary Figure S2C) and slightly increased Umod mRNA levels in the kidneys (Supplementary Figure S2A). The lower urine levels were reflected by a less pronounced apical and more diffuse intracellular staining for uromodulin in TAL cells of the CasrNuf/Nuf kidneys (Figure 1e). The Casr p.Ile859Asn inactivating mutation is causing early death in homozygous CasrBCH002/BCH002 mice, likely because of decreased CaSR expression (Supplementary Figure S1D and S1F) and severe hyperparathyroidism and hypercalcemia.12Sabrautzki S. Rubio-Aliaga I. Hans W. et al.New mouse models for metabolic bone diseases generated by genome-wide ENU mutagenesis.Mamm Genome. 2012; 23: 416-430Crossref PubMed Scopus (27) Google Scholar Heterozygous CasrBCH002/+ mice showed significantly increased blood Ca2+ and decreased plasma phosphorus levels (Supplementary Table S1) and significantly higher levels of uromodulin in urine (Figure 1f), reflected by an enhanced apical staining for uromodulin in TAL cells (Figure 1g) compared with Casr +/+ mice. Of note, uromodulin mRNA and protein expression levels (Supplementary Figure S2B and S2D) were unchanged in CasrBCH002/+ versus Casr+/+ kidneys. We directly tested the effect of CaSR activation on uromodulin secretion by using well-established pharmacologic agents on mTAL cells. Treatment of mTAL cells with the CaSR agonist (calcimimetic) calindol (10 nM to 1 μM, 16 hours) induced a strong dose-dependent decrease in transepithelial voltage (Figure 2a), paralleled by a decrease in the excretion of uromodulin in the apical medium (Figure 2b) in the absence of detectable change in the expression of uromodulin in cell lysates (Supplementary Figure S3A). Short-term treatment (4 hours) of mTAL cells with 100 nM calindol significantly decreased the transepithelial voltage (Figure 2c) and the apical excretion of uromodulin (Figure 2d) in the absence of changes of uromodulin, NKCC2, and ROMK in corresponding cell lysates (Supplementary Figure S3B). Activation of the CaSR by high extracellular Ca2+ (3 mM, 6 hours) lowered transepithelial voltage (Figure 2e) and also resulted in a strong reduction of uromodulin excretion (Figure 2f) in the absence of changes in protein levels of uromodulin, NKCC2, and ROMK in cell lysates (Supplementary Figure S3D). The effects of extracellular Ca2+ on voltage and uromodulin excretion were abolished upon co-incubation with the specific CaSR antagonist NPS2143 (1 μM) (Figure 2e and f), which had no effect on uromodulin excretion in the absence of high basolateral Ca2+ concentrations (Supplementary Figure S3C). The potential link between CaSR signaling, uromodulin excretion, and intracellular cAMP levels is supported by the fact that calindol (which decreases uromodulin excretion: Figure 2b and d) prevented the 1-desamino-8d-arginine vasopressin (dDAVP)-induced rise of intracellular cAMP levels in mTAL cells (Figure 2g), whereas acute exposure of mTAL cells to dDAVP (100 nM, 4 hours) (Figure 2h) and to cAMP (50 μM, 4 hours) (Figure 2i) increased uromodulin excretion. To verify the translational value of the link between CaSR and uromodulin excretion, we tested the effect of a single dose (60 mg) of the calcimimetic drug cinacalcet in healthy subjects (Figure 2j and k). As expected, administration of cinacalcet induced a rapid increase (+266%) of urinary calcium excretion that was paralleled by a decrease (–18%) in urinary excretion of uromodulin. The data presented here demonstrate for the first time that urinary excretion of uromodulin is modulated by the CaSR operating in the basolateral membrane of the TAL. The use of primary mTAL cells with endogenous expression of uromodulin highlights the value of this system to investigate the biology of uromodulin. The link between CaSR activity and excretion of uromodulin is evidenced by (i) decreased or increased urinary uromodulin levels in mice harboring activating or inactivating mutations of the Casr, respectively; (ii) decreased uromodulin excretion after activation of the CaSR in mTAL cells by a specific CaSR agonist; (iii) a strong reduction in uromodulin excretion after activation of the CaSR in mTAL cells by high extracellular Ca2+; (iv) abolition of the effects of high extracellular Ca2+ upon treatment of mTAL cells with the CaSR antagonist NPS2143; and (v) a significant decrease in uromodulin excretion after a single dose of cinacalcet in healthy subjects. The fact that the changes in uromodulin excretion are reflected by modifications of its intracellular distribution rather than changes in total mRNA and protein levels suggests that the CaSR may regulate the trafficking of uromodulin. Activation of the CaSR decreases intracellular cAMP levels, resulting from both the inhibition of its production and the stimulation of its degradation.7Riccardi D. Valenti G. Localization and function of the renal calcium-sensing receptor.Nat Rev Nephrol. 2016; 12: 414-425Crossref PubMed Scopus (88) Google Scholar, 13Hofer A.M. Brown E.M. Extracellular calcium sensing and signalling.Nat Rev Mol Cell Biol. 2003; 4: 530-538Crossref PubMed Scopus (520) Google Scholar The cAMP/protein kinase A pathway is involved in the trafficking of many proteins in tubular cells, including NKCC2 in the TAL and aquaporin-2 and epithelial sodium channel in the collecting ducts. Our data show that mTAL cells respond to dDAVP and cAMP by increasing the apical excretion of uromodulin; conversely, activation of the CaSR by calindol decreases uromodulin excretion and prevents the dDAVP-induced rise in cAMP in mTAL cells. Taken together, these observations support a role for CaSR-dependent changes in intracellular cAMP levels in uromodulin trafficking. The specific production of uromodulin in TAL cells and the association of urine levels of uromodulin with markers of tubular function3Pruijm M. Ponte B. Ackermann D. et al.Associations of urinary uromodulin with clinical characteristics and markers of tubular function in the general population.Clin J Am Soc Nephrol. 2016; 11: 70-80Crossref PubMed Scopus (63) Google Scholar, 4Troyanov S. Delmas-Frenette C. Bollée G. et al.clinical, genetic, and urinary factors associated with uromodulin excretion.Clin J Am Soc Nephrol. 2016; 11: 62-69Crossref PubMed Scopus (29) Google Scholar and genetic variants in KCNJ1/ROMK2Olden M. Corre T. Hayward C. et al.Common variants in UMOD associate with urinary uromodulin levels: a meta-analysis.J Am Soc Nephrol. 2014; 25: 1869-1882Crossref PubMed Scopus (78) Google Scholar suggest a cell-autonomous regulation. In fact, changes of uromodulin excretion induced by CaSR modulators in mTAL cells were paralleled by changes in the transepithelial voltage. The latter is predominantly K+ dependent, because the luminal recycling of K+ via the apical ROMK channel generates the lumen-positive voltage in TAL. Extracellular Ca2+ was shown to generate cytochrome P-450 metabolites that inhibit ROMK activity, likely via CaSR signaling.14Wang W.H. Lu M. Hebert S.C. Cytochrome P-450 metabolites mediate extracellular Ca(2+)-induced inhibition of apical K+ channels in the TAL.Am J Physiol. 1996; 271: C103-C111Crossref PubMed Google Scholar Future studies should decipher the mechanisms linking CaSR, ROMK, and uromodulin secretion, including a potential role of the serine protease hepsin.15Brunati M. Perucca S. Han L. et al.The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin.Elife. 2015; 4: e08887Crossref PubMed Scopus (66) Google Scholar In summary, the activation of CaSR modulates the excretion of uromodulin in the urine, probably through posttranslational control of uromodulin trafficking and cAMP levels in TAL cells. The possibility of modulating urinary levels of uromodulin may be clinically relevant, considering the multifaceted role of this protein in health and disease and the increasing use of pharmacologic modulators of the CaSR. Studies were conducted in C57BL6J mice and CasrNuf/Nuf (11) and CasrBCH002/+12Sabrautzki S. Rubio-Aliaga I. Hans W. et al.New mouse models for metabolic bone diseases generated by genome-wide ENU mutagenesis.Mamm Genome. 2012; 23: 416-430Crossref PubMed Scopus (27) Google Scholar mice, using well-established protocols.16Bernascone I. Janas S. Ikehata M. et al.A transgenic mouse model for uromodulin-associated kidney diseases shows specific tubulo-interstitial damage, urinary concentrating defect and renal failure.Hum Mol Genet. 2010; 19: 2998-3010Crossref PubMed Scopus (66) Google Scholar Healthy subjects gave informed consent to test the effect of a single dose of cinacalcet, 60 mg, on the excretion of uromodulin and calcium in urine. Urine and blood parameters were determined as described.16Bernascone I. Janas S. Ikehata M. et al.A transgenic mouse model for uromodulin-associated kidney diseases shows specific tubulo-interstitial damage, urinary concentrating defect and renal failure.Hum Mol Genet. 2010; 19: 2998-3010Crossref PubMed Scopus (66) Google Scholar, 17Youhanna S. Weber J. Beaujean V. et al.Determination of uromodulin in human urine: influence of storage and processing.Nephrol Dial Transplant. 2014; 29: 136-145Crossref PubMed Scopus (68) Google Scholar Primary cell cultures of mTAL cells were obtained and cultured as described.10Glaudemans B. Terryn S. Gölz N. et al.A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing.Pflugers Arch. 2014; 466: 343-356Crossref PubMed Scopus (18) Google Scholar Confluent mTAL monolayers were exposed (basolateral side) to calindol,18Kessler A. Faure H. Petrel C. et al.N2-benzyl-N1-(1-(1-naphthyl)ethyl)-3-phenylpropane-1,2-diamines and conformationally restrained indole analogues: development of calindol as a new calcimimetic acting at the calcium sensing receptor.Bioorg Med Chem Lett. 2004; 14: 3345-3349Crossref PubMed Scopus (62) Google Scholar NPS2143,19Gowen M. Stroup G.B. Dodds R.A. et al.Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats.J Clin Invest. 2000; 105: 1595-1604Crossref PubMed Scopus (251) Google Scholar dDAVP, and 3-isobutyl-1-methylxanthine (IBMX) alone or with cAMP, as detailed in the supplementary material. Electrophysiology was performed as described.10Glaudemans B. Terryn S. Gölz N. et al.A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing.Pflugers Arch. 2014; 466: 343-356Crossref PubMed Scopus (18) Google Scholar The cAMP levels were determined with the DetectX Cyclic AMP enzyme immunoassay kit (Arbor Assays, Ann Arbor, MI). Gene and protein expression analyses were performed using primers, conditions, and antibodies listed in the Supplementary Material. Data are presented as mean ± SEM. Two-tailed unpaired Student t-test, Mann-Whitney test, or analysis of variance with Tukey's multiple comparison tests were used for the statistical analysis, as indicated. All the authors declared no competing interests. We thank Professor Jan Loffing for ROMK antibodies; Professor Carsten Wagner for Casr mutant mice; and Dr. Tanguy Corre, Professor Murielle Bochud, Professor Caroline Hayward, Dr. Moriz Kirschmann, Ms. Beatrice Paola Festa, and Professor Rajesh V. Thakker for helpful discussions and advice. These studies were supported by an Advanced Postdoc Mobility Fellowship from Swiss National Science Foundation (P300P3_158521) and a Forschundingskredit Postdoc from the University of Zurich (to NT); the Fonds National de la Recherche Luxembourg (6903109) and University Research Programme "Integrative Human Physiology, ZIHP" of the University of Zurich (to EO); the Swiss National Centre of Competence in Research Kidney Control of Homeostasis (NCCR Kidney.CH) program, the Swiss National Science Foundation (31003A_169850), and the Rare Disease Initiative Zurich (Radiz), a clinical research priority program of the University of Zurich, Switzerland (to OD). Download .pdf (.08 MB) Help with pdf files Supplementary Materials and methods Download .pdf (.14 MB) Help with pdf files Figure S1Description of the Casr mutant mice. Download .pdf (.1 MB) Help with pdf files Figure S2Transcriptional and protein expression profiles in kidneys of the Casr mutant mice. Download .pdf (.16 MB) Help with pdf files Figure S3Effect of calcium-sensing receptor modulators, 1-desamino-8d-arginine vasopressin (dDAVP), and cyclic adenosine monophosphate (cAMP) on uromodulin expression in mTAL cells. Download .pdf (.06 MB) Help with pdf files Table S1Urine and blood parameters in Casr mutant mice. Download .pdf (.04 MB) Help with pdf files Table S2List of primers. Corrigendum to Tokonami N, Olinger E, Debaix H, Houillier P, Devuyst O. The excretion of uromodulin is modulated by the calcium-sensing receptor. Kidney Int. 2018;94:882–886Kidney InternationalVol. 100Issue 1PreviewIn relation to the above-stated Brief Report, the authors wish to provide additional acknowledgments detailing the source of mouse lines used in the study. Full-Text PDF Open Access