Uromodulin is expressed in the distal convoluted tubule, where it is critical for regulation of the sodium chloride cotransporter NCC
2018; Elsevier BV; Volume: 94; Issue: 4 Linguagem: Inglês
10.1016/j.kint.2018.04.021
ISSN1523-1755
AutoresNatsuko Tokonami, Tomoaki Takata, Jan Beyeler, Iris Ehrbar, Ayumi Yoshifuji, Erik Christensen, Johannes Loffing, Olivier Devuyst, Eric Olinger,
Tópico(s)Ion Channels and Receptors
ResumoUromodulin, the most abundant protein in normal urine, is essentially produced by the cells lining the thick ascending limb. There it regulates the activity of the cotransporter NKCC2 and is involved in sodium chloride handling and blood pressure regulation. Conflicting reports suggested that uromodulin may also be expressed in the distal convoluted tubule (DCT) where its role remains unknown. Using microdissection studies combined with fluorescent in situ hybridization and co-immunostaining analyses, we found a significant expression of uromodulin in mouse and human DCT at approximately 10% of thick ascending limb expression levels, but restricted to the early part of the DCT (DCT1). Genetic deletion of Umod in mouse was reflected by a major shift in NCC activity from the DCT1 to the downstream DCT2 segment, paralleled by a compensatory expansion of DCT2. By increasing the distal sodium chloride and calcium ion load with chronic furosemide administration, an intrinsic compensatory defect in the DCT from Umod-/- compared to wild type mice was found manifested as sodium wasting and hypercalciuria. In line, co-expression studies in HEK cells suggested a facilitating role for uromodulin in NCC phosphorylation, possibly via SPAK-OSR1 modulation. These experiments demonstrate a significant expression of uromodulin in the early part of mouse and human DCT. Thus, biosynthesis of uromodulin in the DCT1 is critical for its function, structure and plasticity, suggesting novel links between uromodulin, blood pressure control and risk of kidney stones. Uromodulin, the most abundant protein in normal urine, is essentially produced by the cells lining the thick ascending limb. There it regulates the activity of the cotransporter NKCC2 and is involved in sodium chloride handling and blood pressure regulation. Conflicting reports suggested that uromodulin may also be expressed in the distal convoluted tubule (DCT) where its role remains unknown. Using microdissection studies combined with fluorescent in situ hybridization and co-immunostaining analyses, we found a significant expression of uromodulin in mouse and human DCT at approximately 10% of thick ascending limb expression levels, but restricted to the early part of the DCT (DCT1). Genetic deletion of Umod in mouse was reflected by a major shift in NCC activity from the DCT1 to the downstream DCT2 segment, paralleled by a compensatory expansion of DCT2. By increasing the distal sodium chloride and calcium ion load with chronic furosemide administration, an intrinsic compensatory defect in the DCT from Umod-/- compared to wild type mice was found manifested as sodium wasting and hypercalciuria. In line, co-expression studies in HEK cells suggested a facilitating role for uromodulin in NCC phosphorylation, possibly via SPAK-OSR1 modulation. These experiments demonstrate a significant expression of uromodulin in the early part of mouse and human DCT. Thus, biosynthesis of uromodulin in the DCT1 is critical for its function, structure and plasticity, suggesting novel links between uromodulin, blood pressure control and risk of kidney stones. Uromodulin, the most abundant protein in healthy urine, is exclusively expressed in the kidney,1Pennica D. Kohr W.J. Kuang W.J. et al.Identification of human uromodulin as the Tamm-Horsfall urinary glycoprotein.Science. 1987; 236: 83-88Crossref PubMed Scopus (217) Google Scholar with transcription levels among the highest in this organ.2Uhlen M. Fagerberg L. Hallstrom B.M. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (7240) Google Scholar Early studies in mammalian species, including humans, established that uromodulin is essentially synthesized in the epithelial cells lining the thick ascending limb (TAL) of the loop of Henle.3Bachmann S. Metzger R. Bunnemann B. Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney.Histochemistry. 1990; 94: 517-523Crossref PubMed Scopus (102) Google Scholar, 4Sikri K.L. Foster C.L. MacHugh N. Marshall R.D. Localization of Tamm-Horsfall glycoprotein in the human kidney using immuno-fluorescence and immuno-electron microscopical techniques.J Anat. 1981; 132: 597-605PubMed Google Scholar Uromodulin is a glycosylphosphatidylinositol–anchored protein targeted to the apical membrane, where it is cleaved by the serine protease hepsin. In the lumen, uromodulin monomers assemble via their zona pellucida domain to form a dense matrix of high-molecular-weight polymers constituting the hyaline casts.5Devuyst 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 The biological importance of uromodulin is supported by genetic evidence. Rare mutations in the UMOD gene encoding uromodulin lead to defective trafficking of mutant uromodulin, with endoplasmic reticulum (ER) stress causing autosomal dominant tubulointerstitial kidney disease.6Eckardt K.U. Alper S.L. Antignac C. et al.Autosomal dominant tubulointerstitial kidney disease: diagnosis, classification, and management–A KDIGO consensus report.Kidney Int. 2015; 88: 676-683Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 7Piret S.E. Olinger E. Reed A.A. et al.Mouse model for inherited renal fibrosis associated with endoplasmic reticulum stress.Dis Model Mech. 2017; 10: 773-786Crossref PubMed Scopus (28) Google Scholar Genome-wide association studies have revealed that common variants in the promoter of UMOD are associated with the risk of chronic kidney disease, calcium stones, and hypertension in the general population.8Pattaro C. Teumer A. Gorski M. et al.Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.Nat Commun. 2016; 7: 10023Crossref PubMed Scopus (300) Google Scholar, 9Gudbjartsson D.F. Holm H. Indridason O.S. et al.Association of variants at UMOD with chronic kidney disease and kidney stones-role of age and comorbid diseases.PLoS Genet. 2010; 6: e1001039Crossref PubMed Scopus (154) Google Scholar, 10Padmanabhan S. Melander O. Johnson T. et al.Genome-wide association study of blood pressure extremes identifies variant near UMOD associated with hypertension.PLoS Genet. 2010; 6: e1001177Crossref PubMed Scopus (282) Google Scholar The physiological roles of uromodulin, which have been demonstrated in Umod−/− mice, include protection against intrarenal/tubular calcium oxalate crystallization11Mo L. Huang H.Y. Zhu X.H. et al.Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation.Kidney Int. 2004; 66: 1159-1166Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar and regulation of the activity and surface abundance of the Na+/K+/2Cl- cotransporter NKCC2, and renal outer medullary potassium channel ROMK in the TAL, hence the sodium avidity and urinary concentrating ability of the kidney.12Mutig K. Kahl T. Saritas T. et al.Activation of the bumetanide-sensitive Na+,K+,2Cl- cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner.J Biol Chem. 2011; 286: 30200-30210Crossref PubMed Scopus (129) Google Scholar, 13Renigunta A. Renigunta V. Saritas T. et al.Tamm-Horsfall glycoprotein interacts with renal outer medullary potassium channel ROMK2 and regulates its function.J Biol Chem. 2011; 286: 2224-2235Crossref PubMed Scopus (88) Google Scholar, 14Graham L.A. Padmanabhan S. Fraser N.J. et al.Validation of uromodulin as a candidate gene for human essential hypertension.Hypertension. 2014; 63: 551-558Crossref PubMed Scopus (86) Google Scholar The latter hypothesis is supported by the fact that the UMOD variants associated with hypertension drive higher uromodulin expression and are accompanied by higher NKCC2 activity in humans.15Trudu M. Janas S. Lanzani C. et al.Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression.Nat Med. 2013; 19: 1655-1660Crossref PubMed Scopus (249) Google Scholar If the TAL is established as the major site of uromodulin production, there are conflicting reports about a potential expression of uromodulin gene and protein in downstream segments. Radio-labeled in situ hybridization detected Umod mRNA signals in the rat TAL, with an abrupt ending at the transition to the distal convoluted tubule (DCT).3Bachmann S. Metzger R. Bunnemann B. Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney.Histochemistry. 1990; 94: 517-523Crossref PubMed Scopus (102) Google Scholar Consistent with this finding, immunofluorescent and electron microscopy in rat kidney detected uromodulin only in TAL cells, excepting the macula densa.16Hoyer J.R. Sisson S.P. Vernier R.L. Tamm-Horsfall glycoprotein: ultrastructural immunoperoxidase localization in rat kidney.Lab Invest. 1979; 41: 168-173PubMed Google Scholar, 17Bachmann S. Koeppen-Hagemann I. Kriz W. Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry.Histochemistry. 1985; 83: 531-538Crossref PubMed Scopus (91) Google Scholar In contrast, uromodulin protein was reported in the DCT of hamster,18Sikri K.L. Foster C.L. Bloomfield F.J. Marshall R.D. Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm-Horsfall glycoprotein in adult hamster kidney.Biochem J. 1979; 181: 525-532Crossref PubMed Scopus (67) Google Scholar rat,19Sikri K.L. Alexander D.P. Foster C.L. Localization of Tamm-Horsfall glycoprotein in the normal rat kidney and the effect of adrenalectomy on its localization in the hamster and rat kidney.J Anat. 1982; 135: 29-45PubMed Google Scholar mouse,20de Baaij J.H. Groot Koerkamp M.J. Lavrijsen M. et al.Elucidation of the distal convoluted tubule transcriptome identifies new candidate genes involved in renal Mg(2+) handling.Am J Physiol Renal Physiol. 2013; 305: F1563-F1573Crossref PubMed Scopus (41) Google Scholar and human4Sikri K.L. Foster C.L. MacHugh N. Marshall R.D. Localization of Tamm-Horsfall glycoprotein in the human kidney using immuno-fluorescence and immuno-electron microscopical techniques.J Anat. 1981; 132: 597-605PubMed Google Scholar kidneys. However, these results were inconclusive because the positive immunostaining signal could reflect luminal uromodulin adhering on the apical region of tubular cells lining segments downstream of the production site in the TAL. In fact, studies in human kidney showed that, in contrast to the plasmalemmal and cytoplasmic staining for uromodulin in TAL cells, only the very luminal plasmalemma was stained in DCT cells.21Peach R.J. Day W.A. Ellingsen P.J. McGiven A.R. Ultrastructural localization of Tamm-Horsfall protein in human kidney using immunogold electron microscopy.Histochem J. 1988; 20: 156-164Crossref PubMed Scopus (44) Google Scholar More recently, transcriptomic data showed some level of uromodulin mRNA in the DCT in mouse,22Cheval L. Pierrat F. Dossat C. et al.Atlas of gene expression in the mouse kidney: new features of glomerular parietal cells.Physiol Genomics. 2011; 43: 161-173Crossref PubMed Scopus (48) Google Scholar rat,23Lee J.W. Chou C.L. Knepper M.A. Deep sequencing in microdissected renal tubules identifies nephron segment-specific transcriptomes.J Am Soc Nephrol. 2015; 26: 2669-2677Crossref PubMed Scopus (344) Google Scholar and human24Chabardes-Garonne D. Mejean A. Aude J.C. et al.A panoramic view of gene expression in the human kidney.Proc Natl Acad Sci U S A. 2003; 100: 13710-13715Crossref PubMed Scopus (147) Google Scholar kidneys—again difficult to interpret owing to potential contamination by the very high expression levels in adjacent TAL segments. In this study we combined multilevel analyses of uromodulin mRNA and protein expression along the TAL and the DCT and in-depth testing of mouse models to demonstrate a specific expression of uromodulin in the early part of the DCT, with a distinct role in Na+/Cl− cotransporter (NCC) activation and local electrolyte handling. We first analyzed the abundance of Umod transcripts by quantitative reverse transcription–polymerase chain reaction (RT-qPCR) using well-characterized tubular segments of mouse kidney obtained by microdissection.25Glaudemans B. Terryn S. Golz 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 highest Umod transcript levels were detected in the TAL, followed by the DCT that showed ∼10% of TAL expression levels (24.3 ± 3.4 vs. 2.1 ± 0.8 in DCT; expressed as 2ˆ(CtGapdh-CtUmod) × 106). Umod transcripts were undetectable in proximal tubules and collecting duct fractions (Figure 1a). We excluded TAL contamination in DCT fractions by showing virtual absence of Slc12a1f transcripts, encoding NKCC2F, the most abundant isoform expressed in the outer medulla (Figure 1b).26Castrop H. Schnermann J. Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance.Am J Physiol Renal Physiol. 2008; 295: F859-F866Crossref PubMed Scopus (66) Google Scholar We confirmed the presence of Umod transcripts in a pure DCT1 fraction by using fluorescence-assisted microdissection. The transgenic mouse line PV-EGFP expresses enhanced green fluorescence protein (EGFP) under the promoter of the Pvalb gene, encoding parvalbumin selectively in DCT1 cells.27Meyer A.H. Katona I. Blatow M. et al.In vivo labeling of parvalbumin-positive interneurons and analysis of electrical coupling in identified neurons.J Neurosci. 2002; 22: 7055-7064Crossref PubMed Google Scholar, 28Picard N. Trompf K. Yang C.L. et al.Protein phosphatase 1 inhibitor-1 deficiency reduces phosphorylation of renal NaCl cotransporter and causes arterial hypotension.J Am Soc Nephrol. 2014; 25: 511-522Crossref PubMed Scopus (51) Google Scholar Colocalization of EGFP with NCC confirmed its localization in DCT1 versus DCT2 segments (Figure 1ci, ii). Robust Umod transcript levels were detected in pure DCT1 fractions, characterized by virtually absent Slc12a1 (NKCC2) transcripts and high levels of Slc12a3 (NCC) transcripts (Slc12a3: 5.2 ± 0.6; Umod: 1.4 ± 0.3; Slc12a1: 0.001 ± 0.0003; expressed as 2ˆ(CtGapdh-Cttarget gene) × 10ˆ6) (Figure 1ciii). Analysis of open-source transcriptomics databases confirmed that Umod transcripts in DCT accounted for 3.8% and 2.6% of cortical TAL levels in CD1 mice and Sprague-Dawley rats, respectively (Supplementary Figure S1A, B).22Cheval L. Pierrat F. Dossat C. et al.Atlas of gene expression in the mouse kidney: new features of glomerular parietal cells.Physiol Genomics. 2011; 43: 161-173Crossref PubMed Scopus (48) Google Scholar, 23Lee J.W. Chou C.L. Knepper M.A. Deep sequencing in microdissected renal tubules identifies nephron segment-specific transcriptomes.J Am Soc Nephrol. 2015; 26: 2669-2677Crossref PubMed Scopus (344) Google Scholar Multiplex fluorescent in situ hybridization probes were designed for Umod, Slc12a1 (NKCC2), and Slc12a3 (NCC), the specificity of the Umod probe being verified on Umod−/− kidneys (Supplementary Figure S2). Hybridization on mouse kidney revealed 2 clusters of Umod-positive tubules (green) in the cortex, differing in their amount of Umod dots (Figure 2a). The high Umod mRNA tubules (arrows) were positive for Slc12a1 (red) and negative for Slc12a3 (cyan), identifying them as TALs. In contrast, the low Umod mRNA tubules (arrowheads) were negative for Slc12a1 (red) and positive for Slc12a3 (cyan), identifying them as DCTs. No Umod mRNA expression outside of TAL or DCT was detected. The distinct Umod mRNA expression patterns are clearly visible in TAL (Figure 2b) and DCT (Figure 2c) segments, with an abrupt transition in Umod mRNA abundance from TAL to DCT1 (Figure 2d). Semi-automated quantification of fluorescent dots, representing single Umod mRNA molecules,29Wang F. Flanagan J. Su N. et al.RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.J Mol Diagn. 2012; 14: 22-29Abstract Full Text Full Text PDF PubMed Scopus (1453) Google Scholar indicated that an average DCT tubule expresses ∼15% of the Umod mRNA expressed in a cortical TAL (138.6 ± 8.0 vs. 20.8 ± 2.0 in DCT, arbitrary units representing dots normalized to tubule area) (Figure 2e). This finding is in close agreement with the expression values detected by RT-qPCR on microdissected tubules. An increasing body of evidence suggests the TAL being heterogeneous along the corticomedullary axis.30Bleich M. Wulfmeyer V.C. Himmerkus N. Milatz S. Heterogeneity of tight junctions in the thick ascending limb.Ann N Y Acad Sci. 2017; 1405: 5-15Crossref PubMed Scopus (8) Google Scholar Transcriptomics data suggested higher Umod expression in cortical TAL (cTAL) compared with medullary TAL (mTAL) in rodent kidney (1.4- and 7.8-fold higher expression in mouse and rat cTAL, respectively) (Supplementary Figure S1) and a trend in the same direction in human kidney.24Chabardes-Garonne D. Mejean A. Aude J.C. et al.A panoramic view of gene expression in the human kidney.Proc Natl Acad Sci U S A. 2003; 100: 13710-13715Crossref PubMed Scopus (147) Google Scholar Our analysis of mTAL and cTAL fractions, validated by enrichment of splicing isoforms of Slc12a1 (Figure 3a), revealed 1.5-fold higher Umod transcript levels in the cTAL compared with mTAL (41.2 ± 3.2 vs. 27.0 ± 2.6 in mTAL, expressed as 2ˆ(CtGapdh-CtUmod) × 10ˆ6) (Figure 3b). In situ hybridization showed a corticomedullary gradient for Umod expression, with cortical TALs displaying a stronger fluorescent labeling compared with mTALs (Figure 3c, d). Semi-automated quantification of fluorescent dots indicates that an average cTAL (as defined by close spatial relation with a glomerulus) expresses roughly 1.6-fold the amount of Umod mRNA expressed in mTAL (138.6 ± 8.0 vs. 87.2 ± 3.2 in mTAL, arbitrary units representing dots normalized to tubule area) (Figure 2e), similar to the RT-qPCR data. To investigate whether the Umod mRNA expression in DCT is mirrored by uromodulin protein localization, we performed immunofluorescent stainings for uromodulin and NCC in serial sections from mouse kidneys (Figure 4a). A cluster of tubules displaying a signal for uromodulin (green) and for NCC (red; arrowheads) was identified (Figure 4a, upper panels). The uromodulin signal was weaker in the NCC-positive tubules (DCT) compared with that observed in TAL segments (Figure 4a, lower panels). Furthermore, a consistent uromodulin expression was detected in EGFP-expressing DCT1 tubules in sections from PV-EGFP mouse kidneys, with confirmation of different staining intensity between TAL and DCT segments (Figure 4b). Confocal imaging showed uromodulin located at the apical plasma membrane in DCT1 tubules (arrow), similar to the TAL distribution pattern (arrowhead) (Figure 4c). Coimmunostaining for uromodulin (green) and NCC (red) in human kidney confirmed the partial overlap of expression (arrow) (Figure 4d), in line with mRNA data.24Chabardes-Garonne D. Mejean A. Aude J.C. et al.A panoramic view of gene expression in the human kidney.Proc Natl Acad Sci U S A. 2003; 100: 13710-13715Crossref PubMed Scopus (147) Google Scholar Uromodulin was also detected by immunoperoxidase in parvalbumin-expressing human DCT1 (Figure 4e).Figure 4Immunofluorescent localization of uromodulin along the distal convoluted tubule (DCT). (a) Upper panels: Representative immunofluorescence staining for uromodulin (green) and Na+/Cl− cotransporter (NCC, red) on serial paraffin-embedded sections from mouse kidney, showing consistent but weaker staining of uromodulin in NCC-positive tubules (arrowheads) compared with putative thick ascending limbs (TALs) (arrows). Lower panels: Higher magnification of a transition from a TAL to an NCC-positive DCT1 and highlighting the accompanying decrease in uromodulin intensity. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI). (b) Representative immunofluorescence staining for uromodulin (red) on paraffin-embedded kidney sections from PVALB-EGFP mice, confirming consistent but weaker staining of uromodulin in enhanced green fluorescent protein (EGFP, green) positive DCT1 tubules. (c) Apical localization of uromodulin (red) in DCT1, as shown on immunofluorescent confocal imaging on paraffin-embedded PVALB-EGFP kidneys (arrow). A putative TAL is shown for comparison (arrowhead). Nuclei are counterstained with DAPI. (d) Representative immunofluorescence staining for uromodulin (green) and NCC (red) on paraffin-embedded sections from normal human kidney showing partial overlap of signal (arrow). (e) Immunoperoxidase labeling for uromodulin and parvalbumin on normal human kidney serial sections showing partial overlap of signal in DCT1. (f) Representative immunofluorescence staining for uromodulin (green) and NCC (red) on paraffin-embedded kidney sections from Umod+/+ and UmodC125R/+ mice. Nuclei are counterstained with DAPI. G, glomerulus. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because missense mutations in UMOD are causing an ER accumulation of mutant uromodulin in autosomal dominant tubulointerstitial kidney disease,6Eckardt K.U. Alper S.L. Antignac C. et al.Autosomal dominant tubulointerstitial kidney disease: diagnosis, classification, and management–A KDIGO consensus report.Kidney Int. 2015; 88: 676-683Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 7Piret S.E. Olinger E. Reed A.A. et al.Mouse model for inherited renal fibrosis associated with endoplasmic reticulum stress.Dis Model Mech. 2017; 10: 773-786Crossref PubMed Scopus (28) Google Scholar we assessed whether uromodulin is also accumulating inside the DCT of the UmodC125R/+ mouse model.7Piret S.E. Olinger E. Reed A.A. et al.Mouse model for inherited renal fibrosis associated with endoplasmic reticulum stress.Dis Model Mech. 2017; 10: 773-786Crossref PubMed Scopus (28) Google Scholar Immunofluorescent analyses showed that, in addition to the TAL, uromodulin is accumulating in the cytoplasm of DCT cells in UmodC125R/+ kidneys, contrasting with its apical expression in Umod+/+ mice (Figure 4f). These data provide more evidence for the biosynthesis of uromodulin in the DCT. The DCT is divided into the functionally distinct DCT1 and downstream DCT2.31Subramanya A.R. Ellison D.H. Distal convoluted tubule.Clin J Am Soc Nephrol. 2014; 9: 2147-2163Crossref PubMed Scopus (134) Google Scholar, 32Loffing J. Loffing-Cueni D. Valderrabano V. et al.Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron.Am J Physiol Renal Physiol. 2001; 281: F1021-F1027Crossref PubMed Scopus (292) Google Scholar A cluster of tubules identified as DCT by in situ hybridization were devoid of Umod expression, whereas others showed consistent Umod expression (Figure 5a). Furthermore, some DCTs in the UmodC125R/+ kidneys were not accumulating uromodulin (Figure 4f). To assess whether uromodulin expression is restricted to DCT1, we performed a triple staining with uromodulin (green), NCC (cyan), and parvalbumin (red) (Figure 5b). The DCT1 sections were defined as NCC- and parvalbumin-positive, whereas NCC-positive and parvalbumin-negative tubules were defined as DCT2. A strong expression gradient for uromodulin was quantified along the DCT, with DCT2 expression intensity being ∼15% of that detected in DCT1 (DCT2: 14.9% ± 2.4% of DCT1 uromodulin expression, assessed as uromodulin fluorescent intensity normalized to tubule area) (Figure 5c). In line with this finding, fluorescent costaining analyses between uromodulin and TRPV5 (ECaC1), an apical Ca2+ channel expressed in the DCT2 and the downstream connecting tubule but not in DCT1,32Loffing J. Loffing-Cueni D. Valderrabano V. et al.Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron.Am J Physiol Renal Physiol. 2001; 281: F1021-F1027Crossref PubMed Scopus (292) Google Scholar showed no significant co-distribution (Figure 5d). These data show that a significant expression of uromodulin is also detected in the DCT, being largely restricted to the initial portion of that segment (DCT1). Uromodulin is not detected elsewhere than in the TAL and DCT. The DCT is critical for the transcellular reabsorption of NaCl, mediated by NCC, and the fine-tuning of urinary Ca2+ and Mg2+ excretion.31Subramanya A.R. Ellison D.H. Distal convoluted tubule.Clin J Am Soc Nephrol. 2014; 9: 2147-2163Crossref PubMed Scopus (134) Google Scholar Absence of uromodulin in Umod−/− mice is known to affect apical transport systems in the TAL, causing adaptations in the DCT (i.e., NCC hyperactivation and DCT hypertrophy).12Mutig K. Kahl T. Saritas T. et al.Activation of the bumetanide-sensitive Na+,K+,2Cl- cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner.J Biol Chem. 2011; 286: 30200-30210Crossref PubMed Scopus (129) Google Scholar To substantiate our findings of uromodulin expression in DCT1, we dissected direct effects of uromodulin on DCT function from secondary compensatory mechanisms by using a chronic (5-day) furosemide treatment coupled with acute hydrochlorothiazide (HCTZ) administration in Umod−/− and Umod+/+ mice. At baseline, Umod−/− mice showed a slight increase in total NCC expression in total kidney membranes, in line with previous report.12Mutig K. Kahl T. Saritas T. et al.Activation of the bumetanide-sensitive Na+,K+,2Cl- cotransporter (NKCC2) is facilitated by Tamm-Horsfall protein in a chloride-sensitive manner.J Biol Chem. 2011; 286: 30200-30210Crossref PubMed Scopus (129) Google Scholar The phosphorylated NCC (pNCC) (at T53 and T58) were both increased, reflecting ∼1.5-fold increased pNCC/total NCC ratios in Umod−/− compared with Umod+/+ mice (Figure 6a, b ). Chronic furosemide infusion, which increases distal Na+ delivery and triggers DCT activity,33Kaissling B. Bachmann S. Kriz W. Structural adaptation of the distal convoluted tubule to prolonged furosemide treatment.Am J Physiol. 1985; 248: F374-F381PubMed Google Scholar led to a dramatic increase of total and phosphorylated NCC in both groups (+270% vs. 284% for total NCC; +978% vs. +1063% for T53 pNCC; and +685% vs. +675% for T58 pNCC in Umod+/+ and Umod−/− mice, respectively, relative to Umod+/+ baseline levels) (Figure 6a, b). Although the absolute levels of total NCC and pNCC were similar between the 2 genotypes after furosemide, the fold change of pNCC (between baseline and furosemide infusion) was markedly reduced in Umod−/− mice (Figure 6c). Because uromodulin is mostly expressed in DCT1, we investigated the relative contribution of DCT1 versus DCT2 in the changes observed. Immunostaining revealed a ∼2-fold decrease of T53 pNCC in DCT1 tubules of Umod−/− versus Umod+/+ mice at baseline (14.0 ± 0.9 vs. 25.6 ± 1.2, respectively; P < 0.0001; arbitrary units of fluorescent intensity per tubule area), paralleled by stronger T53 pNCC in DCT2 (20.8 ± 0.9 vs. 14.6 ± 0.7 respectively; P < 0.0001) (Figure 6d [upper panels] and Figure 6e [left panels]). Chronic furosemide infusion equalized the T53 pNCC signals in the DCT1 (28.1 ± 1.4 vs. 29.9 ± 1.4, respectively), whereas T53 pNCC was further increased in DCT2 of Umod−/− versus Umod+/+ mice (33.4 ± 1.8 vs. 18.6 ± 1.7, respectively; P < 0.0001) (Figure 6d [lower panels] and Figure 6e [right panels]). To detect structural adaptations, we investigated the renal fractional volume occupied by T53 pNCC-positive DCT1 versus DCT2 segments (Supplementary Figure S3).34Jensen E.C. Quantitative analysis of histological staining and fluorescence using ImageJ.Anat Rec (Hoboken). 2013; 296: 378-381Crossref PubMed Scopus (569) Google Scholar At baseline the renal fractional volume occupied by pNCC-positive DCT1 was slightly lower in Umod−/− versus Umod+/+ mice (1.6% ± 0.2% vs. 2.0% ± 0.2% of renal volume, respectively; P = 0.09). Conversely, the renal fractional volume occupied by pNCC-positive DCT2 was higher in Umod−/− versus Umod+/+ mice (3.5% ± 0.4% vs. 1.5% ± 0.2% of renal volume, respectively; P< 0.001) (Figure 6f, left panels), in line with quantification data based on TRPV5 stainings (Supplementary Figure S4B). After chronic furosemide infusion, the renal fractional volume occupied by pNCC-positive DCT1 was significantly lower in Umod−/− versus Umod+/+ mice (2.1% ± 0.3% vs. 3.2 ± 0.3% of renal volume, respectively; P = 0.015), whereas the renal fractional volume occupied by pNCC-positive DCT2 was higher (3.7% ± 0.3% vs. 2.5% ± 0.4% of renal volume, respectively; P = 0.025) (Figure 6f, right panel), also in line with TRPV5 staining data (Supplementary Figure S4B). Although chronic furosemide infusion triggers a significant compensatory increase in DCT1 and DCT2 fractional volume in Umod+/+ mice, this response is markedly blunted in Umod−/− mice (Figure 6f, right panel; Supplementary Figure S4). To test the DCT function at baseline and in the situation of increased NaCl delivery, we monitored NaCl, as well as Ca2+ and Mg2+ handling, after acute HCTZ injection at baseline and after chronic furosemide treatment in mice. The parameters of Umod−/− and Umod+/+ mice were similar at baseline or after vehicle (0.9% NaCl) injection (Tables 1 and 2; Supplementary Table S1). Chronic furosemide infusion led to a slight urinary Na+ wasting in Umod−/− but not in Umod+/+ mice (Figure 7ai, Table 2; Supplementary Figure S5A), suggesting im
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