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• W2582417926 abstract "Dent disease is a rare X-linked tubulopathy caused by mutations in the endosomal chloride-proton exchanger (ClC-5) resulting in defective receptor-mediated endocytosis and severe proximal tubule dysfunction. Bone marrow transplantation has recently been shown to preserve kidney function in cystinosis, a lysosomal storage disease causing proximal tubule dysfunction. Here we test the effects of bone marrow transplantation in Clcn5Y/- mice, a faithful model for Dent disease. Transplantation of wild-type bone marrow in Clcn5Y/- mice significantly improved proximal tubule dysfunction, with decreased low-molecular-weight proteinuria, glycosuria, calciuria, and polyuria four months after transplantation, compared to Clcn5Y/- mice transplanted with ClC-5 knockout bone marrow. Bone marrow–derived cells engrafted in the interstitium, surrounding proximal tubule cells, which showed a rescue of the apical expression of ClC-5 and megalin receptors. The improvement of proximal tubule dysfunction correlated with Clcn5 gene expression in kidneys of mice transplanted with wild-type bone marrow cells. Coculture of Clcn5Y/- proximal tubule cells with bone marrow–derived cells confirmed rescue of ClC-5 and megalin, resulting in improved endocytosis. Nanotubular extensions between the engrafted bone marrow–derived cells and proximal tubule cells were observed in vivo and in vitro. No rescue was found when the formation of the tunneling nanotubes was prevented by actin depolymerization or when cells were physically separated by transwell inserts. Thus, bone marrow transplantation may rescue the epithelial phenotype due to an inherited endosomal defect. Direct contacts between bone marrow–derived cells and diseased tubular cells play a key role in the rescue mechanism. Dent disease is a rare X-linked tubulopathy caused by mutations in the endosomal chloride-proton exchanger (ClC-5) resulting in defective receptor-mediated endocytosis and severe proximal tubule dysfunction. Bone marrow transplantation has recently been shown to preserve kidney function in cystinosis, a lysosomal storage disease causing proximal tubule dysfunction. Here we test the effects of bone marrow transplantation in Clcn5Y/- mice, a faithful model for Dent disease. Transplantation of wild-type bone marrow in Clcn5Y/- mice significantly improved proximal tubule dysfunction, with decreased low-molecular-weight proteinuria, glycosuria, calciuria, and polyuria four months after transplantation, compared to Clcn5Y/- mice transplanted with ClC-5 knockout bone marrow. Bone marrow–derived cells engrafted in the interstitium, surrounding proximal tubule cells, which showed a rescue of the apical expression of ClC-5 and megalin receptors. The improvement of proximal tubule dysfunction correlated with Clcn5 gene expression in kidneys of mice transplanted with wild-type bone marrow cells. Coculture of Clcn5Y/- proximal tubule cells with bone marrow–derived cells confirmed rescue of ClC-5 and megalin, resulting in improved endocytosis. Nanotubular extensions between the engrafted bone marrow–derived cells and proximal tubule cells were observed in vivo and in vitro. No rescue was found when the formation of the tunneling nanotubes was prevented by actin depolymerization or when cells were physically separated by transwell inserts. Thus, bone marrow transplantation may rescue the epithelial phenotype due to an inherited endosomal defect. Direct contacts between bone marrow–derived cells and diseased tubular cells play a key role in the rescue mechanism. The epithelial cells lining the proximal tubules (PTs) of the kidney constitute a paradigm of effective communication between the external environment and the endomembrane system.1De Matteis M.A. Luini A. Mendelian disorders of membrane trafficking.N Engl J Med. 2011; 365: 927-938Crossref PubMed Scopus (78) Google Scholar By processing incoming substances and recycling receptors and transporters at the apical membrane, the connecting endolysosomal system governs the terminal differentiation of PT cells and the recovery of essential substances that would otherwise be lost in the urine.2Eckardt K.U. Coresh J. Devuyst O. et al.Evolving importance of kidney disease: from subspecialty to global health burden.Lancet. 2013; 382: 158-169Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar These substances include low-molecular-weight (LMW) proteins, which are filtered and then completely reabsorbed by apical endocytosis involving the multiligand receptors megalin and cubilin. After binding and internalization, ligands are delivered to endosomes and further to lysosomes for processing and degradation so that PT cells play a pivotal role in homeostasis.3Nielsen R. Christensen E.I. Birn H. Megalin and cubulin in PT protein reabsorption: from experimental models to human disease.Kidney Int. 2016; 89: 58-67Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar Alterations in these transport processes can lead to generalized PT dysfunction (renal Fanconi syndrome), characterized by urinary loss of solutes including LMW proteins, glucose, and phosphate and is often complicated by dehydration, electrolyte imbalance, rickets, and development of chronic kidney disease. Such PT dysfunction is typically encountered in congenital disorders of the endolysosomal compartment, including Dent disease and nephropathic cystinosis.4Devuyst O. Igarashi T. Renal Fanconi syndrome, Dent’s disease and Bartter’s syndrome.in: Thakker R.V. Whyte M.P. Eisman J.A. Igarashi T. Genetics of Bone Biology and Skeletal Disease. 2nd ed. Elsevier Inc., Amsterdam2015: 553-567Google Scholar Dent disease (MIM #300009) is a rare, X-linked disorder characterized by LMW proteinuria, generalized PT dysfunction, kidney stones, and renal failure.5Devuyst O. Thakker R.V. Dent’s disease.Orphanet J Rare Dis. 2010; 5: 28Crossref PubMed Scopus (145) Google Scholar The disease is caused by inactivating mutations of the CLCN5 gene that encodes ClC-5, an electrogenic Cl−/H+ exchanger that is located in the early endosomes of PT cells,6Devuyst O. Christie P.T. Courtoy P.J. et al.Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent's disease.Hum Mol Genet. 1999; 8: 247-257Crossref PubMed Scopus (251) Google Scholar where it regulates vesicular Cl− concentration, potentially affecting the activity of the vacuolar H+-ATPase and/or vesicle recycling.7Stauber T. Jentsch T.J. Chloride in vesicular trafficking and function.Annu Rev Physiol. 2013; 75: 453-477Crossref PubMed Scopus (144) Google Scholar Studies in Clcn5Y/− mice, which represent a well-established model of Dent disease, demonstrated that the functional loss of ClC-5 induced a severe loss of megalin and cubilin at the brush border membrane, with impaired endocytosis and lysosomal processing of internalized ligands in PT cells.8Piwon N. Gunther W. Schwake M. et al.ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent's disease.Nature. 2000; 408: 369-373Crossref PubMed Scopus (483) Google Scholar, 9Christensen E.I. Devuyst O. Dom G. et al.Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules.Proc Natl Acad Sci U S A. 2003; 100: 8472-8477Crossref PubMed Scopus (278) Google Scholar The defective endocytosis and apical transport processes explain why solutes are lost in the urine, often resulting in renal Fanconi syndrome. In the absence of therapy targeting the molecular defect, the current care of patients with Dent disease is solely supportive, focusing on the prevention of metabolic complications and nephrolithiasis. Recently, bone marrow (BM) and hematopoietic stem cell (HSC) transplantation have been shown to preserve kidney function in nephropathic cystinosis, the most frequent cause of renal Fanconi syndrome in children.10Syres K. Harrison F. Tadlock M. et al.Successful treatment of the murine model of cystinosis using bone marrow cell transplantation.Blood. 2009; 114: 2542-2552Crossref PubMed Scopus (81) Google Scholar, 11Yeagy B.A. Harrison F. Gubler M.C. et al.Kidney preservation by bone marrow cell transplantation in hereditary nephropathy.Kidney Int. 2011; 79: 1198-1206Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Cystinosis (MIM #219800) is caused by autosomal recessive mutations in the CTNS gene, which encodes cystinosin, a lysosomal cystine-H+ cotransporter exporting cystine into the cytosol. Functional loss of cystinosin results in lysosomal accumulation of cystine crystals leading to progressive dysfunction of multiple organs including the kidney. In children with nephropathic cystinosis, the most severe and frequent form of cystinosis, PT dysfunction and renal Fanconi syndrome develop before 1 year of age, often followed by chronic kidney disease.12Gahl W.A. Thoene J.G. Schneider J.A. Cystinosis.N Engl J Med. 2002; 347: 111-121Crossref PubMed Scopus (531) Google Scholar Healthy BM-derived cells decreased cystine levels in the kidneys of diseased mice and improved several serum and urinary parameters in relation to the level of engraftment.11Yeagy B.A. Harrison F. Gubler M.C. et al.Kidney preservation by bone marrow cell transplantation in hereditary nephropathy.Kidney Int. 2011; 79: 1198-1206Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Subsequently, in vitro studies suggested that vesicular cross-correction mediated by transfer of cystinosin-bearing lysosomes could sustain the correction.13Harrison F. Yeagy B.A. Rocca C.J. et al.Hematopoietic stem cell gene therapy for the multisystemic lysosomal storage disorder cystinosis.Mol Ther. 2013; 21: 433-444Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 14Naphade S. Sharma J. Gaide Chevronnay H.P. et al.Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes.Stem Cells. 2015; 33: 301-309Crossref PubMed Scopus (71) Google Scholar Whether BM transplantation could improve the epithelial phenotype in other congenital defects of the endolysosomal pathway, in particular those that are not associated with lysosomal storage, is unknown. Furthermore, the mechanism of rescue, without evidence of target tissue differentiation and replacement of damaged cells, remains a major issue in stem cell transplantation.15Pinkernell K. Cellular therapies: what is still missing?.Kidney Int. 2011; 79: 1161-1163Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar Based on the encouraging results in cystinosis, we tested the effects of BM transplantation in the well-established Clcn5Y/− mouse model of Dent disease and used a coculture system to analyze the potential mechanisms involved. To test whether BM transplantation improves the PT dysfunction associated with Dent disease, we transplanted 10-week-old ClC-5 knockout (KO) male (Clcn5Y/−) mice with wild-type (WT) (GFP)–expressing BM cells (“treated”). As controls, Clcn5Y/− mice were transplanted with KO-BM cells from Clcn5Y/− mice (“negative controls”) and age- and sex-matched WT littermates (Clcn5Y/+) were transplanted with WT-BM cells (“positive controls”) (Figure 1a ). Urine and plasma were collected at baseline (1 week before transplantation) and then 10 weeks and 16 weeks after transplantation. Because recipients were lethally irradiated per protocol, BM transplantation resulted in full multilineage hematopoietic chimerism in peripheral blood in all mice, as shown by a nearly 100% myeloid and B-cell chimerism (Figure 1b). As previously described,9Christensen E.I. Devuyst O. Dom G. et al.Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules.Proc Natl Acad Sci U S A. 2003; 100: 8472-8477Crossref PubMed Scopus (278) Google Scholar, 16Wang S.S. Devuyst O. Courtoy P.J. et al.Mice lacking renal chloride channel, CLC-5, are a model for Dent's disease, a nephrolithiasis disorder associated with defective receptor-mediated endocytosis.Hum Mol Genet. 2000; 9: 2937-2945Crossref PubMed Scopus (272) Google Scholar Clcn5Y/− mice exhibited features of PT dysfunction including polyuria, glycosuria, hypercalciuria, and LMW proteinuria (detection of 16-kD Clara cell protein [CC16]) compared with WT littermates at baseline (Table 1).Table 1Urine and plasma parameters at baseline and in transplanted Clcn5Y/− and Clcn5Y/+ miceBaselineClcn5Y/−Clcn5Y/+P value(Negative controls)(n = 8–10)(Treated)(n = 13–15)(Positive controls)(n = 6–9)Clcn5Y/− versus Clcn5Y/+Diuresis (ml/12 h)1.98 ± 0.26aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).2.04 ± 0.21aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).1.20 ± 0.19<0.01U CC16 (mg/g creat)62.3 ± 4.366.7 ± 5.3NDNDU glucose (mg/12 h)1.21 ± 0.15aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).1.44 ± 0.14aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).0.49 ± 0.06<0.0001U Ca2+ (mg/g creat)216 ± 29aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).196 ± 12aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).83 ± 5<0.0001BUN (mg/dl)24.5 ± 1.224.3 ± 1.125.1 ± 1.7NSWeek 10Clcn5Y/− + KO-BM(negative controls)Clcn5Y/− + GFP-BM(treated)Clcn5Y/+ + GFP-BM(positive controls)P valueDiuresis (ml/12 h)4.07 ± 0.64aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).4.04 ± 0.84aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).1.31 ± 0.190.0001U CC16 (mg/g creat)66.6 ± 5.553.9 ± 6.0ND0.087U glucose (mg/12 h)1.07 ± 0.18aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).1.07 ± 0.16aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).0.40 ± 0.080.0006U Ca2+ (mg/g creat)160 ± 26132 ± 13111 ± 360.13BUN (mg/dl)26.8 ± 1.725.0 ± 1.324.1 ± 1.20.54Week 16Clcn5Y/− + KO-BM(negative controls)Clcn5Y/− + GFP-BM(treated)Clcn5Y/+ + GFP-BM(positive controls)P valueDiuresis (ml/12 h)6.04 ± 1.52aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).3.36 ± 0.92aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).1.03 ± 0.240.0002U CC16 (mg/g creat)71.2 ± 3.546.9 ± 5.1bP < 0.05 versus Clcn5Y/– + knockout bone marrow.ND0.0031U glucose (mg/12 h)0.87 ± 0.10aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).0.81 ± 0.12aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).0.36 ± 0.070.0023U Ca2+ (mg/g creat)146 ± 21aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).131 ± 10aP < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).99 ± 310.0126BUN (mg/dl)24.6 ± 1.424.7 ± 0.923.4 ± 1.50.70BM, bone marrow; BUN, blood urea nitrogen; CC16, Clara cell protein of 16 kDa; creat, creatinine; GFP, green fluorescent protein; KO, knockout; ND, not detected; NS, not significant; P, plasma; U, urine.Baseline parameters: Mann-Whitney test (Clcn5Y/− vs Clcn5Y/+); weeks 10 and 16: 1-way analysis of variance nonparametric (Kruskal-Wallis test) for all parameters except U CC16 (Mann-Whitney test).a P < 0.05 versus Clcn5Y/+ (baseline) + green fluorescent protein-labeled bone marrow (weeks 10 and 16).b P < 0.05 versus Clcn5Y/– + knockout bone marrow. Open table in a new tab BM, bone marrow; BUN, blood urea nitrogen; CC16, Clara cell protein of 16 kDa; creat, creatinine; GFP, green fluorescent protein; KO, knockout; ND, not detected; NS, not significant; P, plasma; U, urine. Baseline parameters: Mann-Whitney test (Clcn5Y/− vs Clcn5Y/+); weeks 10 and 16: 1-way analysis of variance nonparametric (Kruskal-Wallis test) for all parameters except U CC16 (Mann-Whitney test). In order to assess the potential effect of BM transplantation, we analyzed markers of PT dysfunction over time in treated versus negative control Clcn5Y/− mice (Figure 2, Table 1). Over the 16-week observation period, the negative controls (Clcn5Y/− mice transplanted with KO-BM) showed a significant increase in diuresis and urinary excretion of CC16, pointing toward progression of the disease over time. In contrast, the transplanted Clcn5Y/− mice showed no significant change in diuresis and a significant reduction (∼30% decrease) in the urinary excretion of CC16. Similarly, glycosuria and calciuria decreased significantly in transplanted animals, whereas these parameters were unchanged in negative controls. These data show a significant improvement of PT dysfunction in mice transplanted with WT BM cells compared with negative controls. The biological effect of BM transplantation was reflected by the engraftment of BM-derived cells into Clcn5Y/− kidneys (Figure 3). The Clcn5Y/− mice displayed a significantly higher recruitment of GFP+ BM–derived cells into the kidneys than Clcn5Y/+ mice, at the mRNA and protein levels and confirmed by immunofluorescence for GFP (Figure 3a,b). Because filtered LMW proteins are reabsorbed by receptor-mediated endocytosis in PT cells, the reduced urinary loss of CC16 in transplanted mice suggests that the endocytic machinery is at least partially recovered. To test this hypothesis, we investigated the expression level of megalin in the kidneys (Figure 4). Contrasting with the selective loss of megalin expression observed in control Clcn5Y/− versus Clcn5Y/+ kidneys, a strong rescue of megalin expression was detected by Western blot in Clcn5Y/− kidneys of mice transplanted with WT BM cells (Figure 4a). The rescue of megalin was specific because the expression of aquaporin-1 and the endosome catalyst Rab5 remained unchanged in all treatment groups. Of note, BM transplantation had no effect on protein expression in any genotype. The rescue of megalin was confirmed by immunofluorescence staining of the kidneys in BM-transplanted mice (Figure 4b). Whereas almost no megalin staining could be detected in Clcn5Y/− kidneys transplanted with KO-BM cells, an appreciable rescue of megalin was observed in the brush border of PT cells in Clcn5Y/− kidneys transplanted with WT BM cells. In parallel, a significant rescue of ClC-5 expression was observed in Clcn5Y/− kidneys of mice transplanted with WT BM cells, as shown by immunoblot (Figure 4a) and real-time polymerase chain reaction (qPCR) (Figure 4c). The rescued ClC-5 in treated Clcn5Y/− kidneys was confirmed by immunofluorescence staining at the brush border of PT cells (Figure 4b) and at high magnification in subapical vesicles reminiscent of endosomes (Supplementary Figure S1A). The staining for ClC-5 was partially overlapping with that of megalin in PT cells (Supplementary Figure S2). Of note, the degree of LMW proteinuria (monitored by the urinary loss of CC16) in the treated Clcn5Y/− mice inversely correlated with the level of ClC-5 mRNA rescued in the kidneys (Figure 4c). To further investigate the mechanisms driving the improvement in PT dysfunction, we analyzed the location and identity of the BM-derived cells in Clcn5Y/− kidneys. Besides the fact that more BM-derived cells engrafted to the Clcn5Y/− versus Clcn5Y/+ kidneys (Figure 3), the engrafted cells also showed a distinct distribution pattern. In Clcn5Y/− kidneys, the transplanted cells were found to be clustering around PT profiles, in close contact with PT cells, whereas in Clcn5Y/+ kidneys, the transplanted cells were interspersed in the interstitium without clear contact with PT sections (Figure 5a ). The different locations were confirmed by automated confocal image analysis, showing a lower average distance of GFP+ cells from PT cells in Clcn5Y/− versus Clcn5Y/+ kidneys, respectively (Figure 5a). The nature of the engrafted cells was then assessed (Figure 5b). All transplanted GFP+ cells were found in the interstitium. Because we transplanted a whole BM cell suspension (i.e., a mixture of HSCs, mesenchymal stromal cells, differentiated lymphocytes, and myeloid cells as well as their immature precursors), the engrafted cells may theoretically derive from mesenchymal stromal cells present in the suspension. Such cells do not migrate into healthy tissues, but on irradiation injury, increased recruitment into peripheral organs has been observed.17François S. Bensidhoum M. Mouiseddine M. et al.Local irradiation not only induces homing of human mesenchymal stem cells at exposed sites but promotes their widespread engraftment to multiple organs: a study of their quantitative distribution after irradiation damage.Stem Cells. 2006; 24: 1020-1029Crossref PubMed Scopus (325) Google Scholar In case of acute injury, mesenchymal stromal cells have been recruited in the kidneys where they may persist for a long time.18Morigi M. Imberti B. Zoja C. et al.Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure.J Am Soc Nephrol. 2004; 15: 1794-1804Crossref PubMed Scopus (664) Google Scholar However, with their characteristic protrusions, the GFP-expressing cells phenotypically resembled dendritic cells (DCs) or macrophages rather than MSC-derived cells, which would be spindle-shaped and compact. As DCs and macrophages further possess overlapping functions during steady state in the kidney, these cells are often referred to as mononuclear phagocytes.19Weisheit C.K. Engel D.R. Kurts C. Dendritic cells and macrophages: sentinels in the kidney.Clin J Am Soc Nephrol. 2015; 10: 1841-1851Crossref PubMed Scopus (59) Google Scholar We confirmed this identity by staining the kidney sections for CD45, CD11c (a DC marker), and F4/80 (a macrophage marker) (Figure 5b). Costaining of these markers with GFP allowed us to conclude that these cells originated from the hematopoietic lineage. Conversely, there was no overlap between the GFP cells and the fibroblast marker α-smooth muscle actin (Figure 5b). Furthermore, the engraftment efficiency of WT GFP+ BM cells and KO-BM cells in Clcn5Y/− kidneys was similar, as shown in Supplementary Figure S3. In order to substantiate the rescue of PT function as observed in vivo, we established a coculture system based on primary cultures of mouse PT cells (mPTCs) obtained from microdissected PT segments of Clcn5Y/− kidneys.20Terryn S. Jouret F. Vandenabeele F. et al.A primary culture of mouse proximal tubular cells established on collagen-coated membranes.Am J Physiol Renal Physiol. 2007; 293: F476-F485Crossref PubMed Scopus (120) Google Scholar These cells were then cocultivated for 2 days with BM-derived DCs/macrophages (BM-DC/MΦ). These BM-DC/MΦ were generated by stimulating either WT (GFP+) or KO-BM cells for 7 days with granulocyte-macrophage colony-stimulating factor (30 ng/ml), resulting in a mixed population of adherent phagocytic cells (Figure 6a ). Fully differentiated BM-DC/MΦ expressed CD45 and F4/80 (Figure 6b) as well as ClC-5 and megalin (Supplementary Figure S1B). During the differentiation process of freshly isolated BM cells into BM-DC/MΦ, gradual upregulation of mRNA encoding for ClC-5 (Clcn5), for the macrophage/DC markers F4/80 (Adgre1), CD45 (Ptprc), and CD11c (Itgax), and for the tunneling nanotube markers myosin Va (Myo5a) and M-Sec or TNFaip2 (Tnfaip2) was observed (Figure 6c). Immunofluorescence staining revealed that, compared with Clcn5Y/− mPTCs cocultivated with KO-BM-DC/MΦ, coculture of Clcn5Y/− mPTCs with WT BM-DC/MΦ showed rescue of megalin and ClC-5 expression, particularly evident in mPTCs close to GFP+ cells (Figure 7a , upper panel). The recovery of megalin and ClC-5 expression was confirmed by Western blot (Figure 7a, lower panel), with a functional counterpart based on albumin (Alexa 647 Fluor bovine serum albumin [BSA]) uptake by the Clcn5Y/− mPTCs (Figure 7b). Compared with the very low level observed in Clcn5Y/− mPTCs cocultivated with KO-BM-DC/MΦ, there was a significant, ∼50%, rescue of albumin uptake in Clcn5Y/− mPTCs cocultivated with WT BM-DC/MΦ, although this was still lower than the uptake by Clcn5Y/+ mPTCs cocultivated with WT BM-DC/MΦ (Figure 7b). We finally investigated potential rescue mechanisms of PT dysfunction by the transplanted BM cells. Visualization of GFP provided evidence, both in vivo (Figure 8a–d ) and in vitro (Figure 8e–g), for the existence of cellular prolongations between the GFP+ BM-derived cells and PT cells from the Clcn5Y/− kidneys. These cellular prolongations resemble the tunneling nanotubes that form intracellular bridges supporting the microtubule-mediated transfer of intracellular components, as previously described in BM transplantation experiments.14Naphade S. Sharma J. Gaide Chevronnay H.P. et al.Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes.Stem Cells. 2015; 33: 301-309Crossref PubMed Scopus (71) Google Scholar, 21Rustom A. Saffrich R. Markovic I. et al.Nanotubular highways for intercellular organelle transport.Science. 2004; 303: 1007-1010Crossref PubMed Scopus (1263) Google Scholar Immunostaining experiments revealed that the GFP+ BM-derived cells established multiple contacts with PT cells in the Clcn5Y/− kidneys (Figure 8b) and that the nanotubes crossed the collagen type IV–positive tubular basement membrane to establish direct contact with PT cells (Figure 8c,d). In coculture experiments, multiple tunneling nanotubes positive for tubulin were identified between GFP+ BM BM-DC/MΦ cells and mPTCs derived from Clcn5Y/− mice (Figure 8e–g). A few GFP+ structures were also detected in PT cells in transplanted kidneys and in mPTCs in coculture (Supplementary Figure S4).Figure 8Nanotubes mediate interactions between bone marrow (BM)−derived cells and proximal tubule (PT) cells. (a–d) Kidney sections of Clcn5Y/− mice transplanted with green fluorescent protein (GFP)+ cells showing staining for GFP (green), 4′,6-diamidino-2-phenylindole (DAPI) (blue), and megalin or collagen IV (red). Arrowheads indicate the nanotubular prolongations of the engrafted GFP+ cells contouring the basal membrane of a PT (b) and penetrating to collagen IV–positive basement membrane to establish a direct contact with PT cells (c,d). The asterisk indicates the lumen of the PT. (e–g) Staining of GFP (green), tubulin (red), and DAPI (blue) and merge (g) in mouse PT cells (mPTCs) derived from Clcn5Y/− mice cocultivated with GFP+ BM, BM-DC/MΦ cells. Multiple tunneling nanotubes positive for tubulin are identified, establishing connections between BM-derived cells and mPTCs. (h) Uptake of Alexa 647-bovine serum albumin (BSA) (red channel) in cocultures of mPTCs derived from Clcn5Y/+ and Clcn5Y/− mice with GFP+ BM–derived cells in the presence of the actin depolymerizing agent Latrunculin B or vehicle (control). Incubation with Latrunculin B inhibits the formation of nanotubes from the GFP+ BM-DC/MΦ, with a lack of rescue of the endocytic uptake of Alexa-BSA in Clcn5Y/− mPTCs. *P < 0.05 versus control (Clcn5Y/+ + GFP-BM without Latrunculin B), §P < 0.05 versus Clcn5Y/− + GFP-BM without Latrunculin B). (i) Western blot for megalin and ClC-5 in lysates of mPTCs derived from Clcn5Y/+ and Clcn5Y/− mice, which were cocultured with WT GFP+ or KO-BM–derived cells in the presence or not of Latrunculin B and the quantification of optical densities. Treatment with Latrunculin B almost abolished the rescue of megalin and ClC-5 in the cocultures of Clcn5Y/− mPTCs and wild-type (WT)-BM–derived cells. Representative of 2 experiments. **P < 0.01 versus control. (j) Uptake of Alexa 647-BSA in mPTCs derived from Clcn5Y/+ and Clcn5Y/− kidneys seeded onto the top of collagen-coated transwell filters, with either WT- or KO-BM-DC/MΦ on the basolateral side. Quantification of BSA-positive vesicles per cell reveals no rescue of the endocytic uptake in Clcn5Y/+ mPTCs cultured with WT BM-derived cells in the basal compartment of the transwell filters. **P < 0.01 versus Clcn5Y/+.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To explore the role of these nanotubular formations in the rescue mechanism, we blocked their formation by treating the mPTCs with the actin-depolymerizing agent latrunculin B.21Rustom A. Saffrich R. Markovic I. et al.Nanotubular highways for intercellular organelle transport.Science. 2004; 303: 1007-1010Crossref PubMed Scopus (1263) Google Scholar This treatment prevented the development of cellular prolongations (Figure 8h), as well as the rescue of megalin and ClC-5 expression (Figure 8i) and the recovery of endocytosis activity (Alexa 647-BSA uptake) in cocultures of GFP+ BM-derived cells with Clcn5Y/− mPTCs (Figure 8h). Furthermore, when the Clcn5Y/− mPTCs were separated from the GFP+ BM-derived cells by using a transwell porous insert, we could not observe any rescue of the endocytic activity (Figure 8j). Taken together, these data suggest that the establishment of a direct contact between transplanted BM-derived cells and diseased PT cells, via tunneling nanotubes, plays a key role in the rescue. The results presented here demonstrate for the first time that BM transplantation may rescue the epithelial phenotype caused by a genetic defect in an endosomal transporter. Transplantation of WT Clcn5Y/+ BM cells to Clcn5Y/− mice significantly reduced the progression of LMW proteinuria, glycosuria, calciuria, and polyuria compare" @default.
• W2582417926 created "2017-02-03" @default.
• W2582417926 creator A5014815876 @default.
• W2582417926 creator A5015840072 @default.
• W2582417926 creator A5020182729 @default.
• W2582417926 creator A5030184276 @default.
• W2582417926 creator A5033792420 @default.
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• W2582417926 date "2017-04-01" @default.
• W2582417926 modified "2023-10-12" @default.
• W2582417926 title "Bone marrow transplantation improves proximal tubule dysfunction in a mouse model of Dent disease" @default.
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