An optimal serum-free defined condition for in vitro culture of kidney organoids.

Kidney organoid is an emerging topic of importance for research in kidney development and regeneration. Conventional culture systems for kidney organoids reported thus far use culture media containing serum, which may compromise our understanding and the potential clinical applicability of the organoid system. In our present study, we tested two serum-free culture conditions and compared their suitability for the maintenance and growth of kidney organoids in culture. One of the serum-free culture conditions was the combination of keratinocytes serum free medium (KSFM) with knockout serum replacement (KSR) (KSFM + KSR), and the other was the combination of knockout DMEM/F12 (KD/F12) and KSR (KD/F12 + KSR). With cell aggregates derived from E12.5 mouse embryonic kidneys, we found that KD/F12 + KSR was superior to KSFM + KSR in promoting the growth of the aggregate with expansion of Six2+ nephron progenitor cells (NPC) and elaborated ureteric branching morphogenesis. With KD/F12 + KSR, we found that lower concentrations of KSR at 5-10% were superior to a higher concentration (20%) in promoting the growth of aggregates without affecting the expression levels of NPC marker genes. We also found that NPC in aggregates retained their differentiation potential to develop nephron tubules through mesenchyme-to-epithelial transition (MET), after being maintained in culture under these conditions for up to 7 days. In conclusion, we have identified a defined serum-free culture condition suitable for the maintenance and growth of kidney organoids that retain the differentiation potential to develop nephron structures. This defined serum-free culture condition may serve as a useful platform for further investigation of kidney organoids in vitro.

In Vivo Vascularization Chamber for the Implantation of Embryonic Kidneys.

A major obstacle to the implantation of ex vivo engineered tissues is the incorporation of functional vascular supply to support the growth of new tissue and to minimize ischemic injury. Existing prevascularization systems, such as arteriovenous (AV) loop-based systems, require microsurgery, limiting their use to larger animals. We aimed to develop an implantable device that can be prevascularized to enable vascularization of tissues in small rodents, and test its application on the vascularization of embryonic kidneys. Implanting the chamber between the abdominal aorta and the inferior vena cava, we detected endothelial cells and vascular networks after 48 h of implantation. Loading the chamber with collagen I (C), Matrigel (M), or Matrigel + vascular endothelial growth factor) (MV) had a strong influence on vascularization speed: Chambers loaded with C took 7 days to vascularize, 4 days for chambers with M, and 2 days for chambers with MV. Implantation of E12.5 mouse embryonic kidneys into prevascularized chambers (C, MV) was followed with significant growth and ureteric branching over 22 days. In contrast, the growth of kidneys in non-prevascularized chambers was stunted. We concluded that our prevascularized chamber is a valuable tool for vascularizing implanted tissues and tissue-engineered constructs. Further optimization will be necessary to control the directional growth of vascular endothelial cells within the chamber and the vascularization grade. Impact Statement Vascularization of engineered tissue, or organoids, constructs is a major hurdle in tissue engineering. Failure of vascularization is associated with prolonged ischemia time and potential tissue damage due to hypoxic effects. The method presented, demonstrates the use of a novel chamber that allows rapid vascularization of native and engineered tissues. We hope that this technology helps to stimulate research in the field of tissue vascularization and enables researchers to generate larger engineered vascularized tissues.

Stepwise renal lineage differentiation of mouse embryonic stem cells tracing in vivo development.

The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0-2, A plus BMP-4 (4) during days 2-4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4-6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6-8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24(+) cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III.

Effect of Hypoxia on Branching Characteristics and Cell Subpopulations during Kidney Organ Culture.

During early developmental stages, embryonic kidneys are not fully vascularized and are potentially exposed to hypoxic conditions, which is known to influence cell proliferation and survival, ureteric bud branching, and vascularization of the developing kidney. To optimize the culture conditions of in vitro cultured kidneys and gain further insight into the effect of hypoxia on kidney development, we exposed mouse embryonic kidneys isolated at E11.5, E12.5, and E13.5 to hypoxic and normal culture conditions and compared ureteric bud branching patterns, the growth of the progenitor subpopulation hoxb7+, and the expression patterns of progenitor and differentiation markers. Branching patterns were quantified using whole organ confocal imaging and gradient-vector-based analysis. In our model, hypoxia causes an earlier expression of UB tip cell markers, and a delay in stalk cell marker gene expression. The metanephric mesenchyme (MM) exhibited a later expression of differentiation marker FGF8, marking a delay in nephron formation. Hypoxia further delayed the expression of stroma cell progenitor markers, a delay in cortical differentiation markers, as well as an earlier expression of medullary and ureteral differentiation markers. We conclude that standard conditions do not apply universally and that tissue engineering strategies need to optimize suitable culture conditions for each application. We also conclude that adapting culture conditions to specific aspects of organ development in tissue engineering can help to improve individual stages of tissue generation.

Bioprinting Scaffolds for Vascular Tissues and Tissue Vascularization.

In recent years, tissue engineering has achieved significant advancements towards the repair of damaged tissues. Until this day, the vascularization of engineered tissues remains a challenge to the development of large-scale artificial tissue. Recent breakthroughs in biomaterials and three-dimensional (3D) printing have made it possible to manipulate two or more biomaterials with complementary mechanical and/or biological properties to create hybrid scaffolds that imitate natural tissues. Hydrogels have become essential biomaterials due to their tissue-like physical properties and their ability to include living cells and/or biological molecules. Furthermore, 3D printing, such as dispensing-based bioprinting, has progressed to the point where it can now be utilized to construct hybrid scaffolds with intricate structures. Current bioprinting approaches are still challenged by the need for the necessary biomimetic nano-resolution in combination with bioactive spatiotemporal signals. Moreover, the intricacies of multi-material bioprinting and hydrogel synthesis also pose a challenge to the construction of hybrid scaffolds. This manuscript presents a brief review of scaffold bioprinting to create vascularized tissues, covering the key features of vascular systems, scaffold-based bioprinting methods, and the materials and cell sources used. We will also present examples and discuss current limitations and potential future directions of the technology.

Knockdown of Nuclear lncRNAs by Locked Nucleic Acid (LNA) Gapmers in Nephron Progenitor Cells.

Despite recent advance in our understanding on the role of long noncoding RNAs (lncRNAs), the function of the vast majority of lncRNAs remains poorly understood. To characterize the function of lncRNAs, knockdown studies are essential. However, the conventional silencing methods for mRNA, such as RNA interference (RNAi), may not be as efficient against lncRNAs, partly due to the mismatch of the localization of lncRNAs and RNAi machinery. To circumvent such limitation, a new technique has recently been developed, i.e., locked nucleic acid (LNA) gapmers. This system utilizes RNase H that distributes evenly in both nucleus and cytoplasm and is expected to knock down lncRNAs of interest more consistently regardless of their localization in the cell. In this chapter, we describe the procedure with tips to silence lncRNAs by LNA gapmers, by using mouse nephron progenitor cells as an example.

Comprehensive analysis of chromatin signature and transcriptome uncovers functional lncRNAs expressed in nephron progenitor cells.

Emerging evidence from recent studies has unraveled the roles of long noncoding RNAs (lncRNAs) in the function of various tissues. However, little is known about the roles of lncRNAs in kidney development. In our present study, we aimed to identify functional lncRNAs in one of the three lineages of kidney progenitor cells, i.e., metanephric mesenchymal (MM) cells. We conducted comprehensive analyses of the chromatin signature and transcriptome by RNA-seq and ChIP-seq. We found seventeen lncRNAs that were expressed specifically in MM cells with an active chromatin signature, while remaining silenced in a bivalent chromatin state in non-MM cells. Out of these MM specific lncRNAs, we identified a lncRNA, Gm29418, in a distal enhancer region of Six2, a key regulatory gene of MM cells. We further identified three transcript variants of Gm29418 by Rapid Amplification of cDNA Ends (RACE), and confirmed that the transcription-start-sites (TSSs) of these variants were consistent with the result of Cap Analysis Gene Expression (CAGE). In support of the enhancer-like function of Gm29418 on Six2 expression, we found that knock-down of Gm29418 by two independent anti-sense locked nucleic acid (LNA) phosphorothioate gapmers suppressed Six2 mRNA expression levels in MM cells. We also found that over-expression of Gm29418 led to an increase in Six2 mRNA expression levels in a mouse MM cell line. In conclusion, we identified a lncRNA, Gm29418, in nephron progenitor cells that has an enhancer-like function on a key regulatory gene, Six2.

Effect of fluid shear stress on in vitro cultured ureteric bud cells.

Most kidney cells are continuously exposed to fluid shear stress (FSS) from either blood flow or urine flow. Recent studies suggest that changes in FSS could contribute to the function and injury of these kidney cells. However, it is unclear whether FSS influences kidney development when urinary flow starts in the embryonic kidneys. In this study, we evaluated the influence of FSS on in vitro cultured ureteric bud (UB) cells by using a pumpless microfluidic device, which offers the convenience of conducting parallel cell culture experiments while also eliminating the need for cumbersome electronic driven equipment and intricate techniques. We first validated the function of the device by both mathematical model and experimental measurements. UB cells dissected from E15.5 mouse embryonic kidneys were cultured in the pumpless microfluidic device and subjected to FSS in the range of 0.4-0.6 dyn mm-2 for 48 h (dynamic). Control UB cells were similarly cultured in the device and maintained under a no-flow condition (static). We found from our present study that the exposure to FSS for up to 48 h led to an increase in mRNA expression levels of UB tip cell marker genes (Wnt11, Ret, Etv4) with a decrease in stalk cell marker genes (Wnt7b, Tacstd2). In further support of the enrichment of UB tip cell population in response to FSS, we also found that exposure to FSS led to a remarkable reduction in the binding of lectin Dolichos Biflorus Agglutinin. In conclusion, results of our present study show that exposure to FSS led to an enrichment in UB tip cell populations, which could contribute to the development and function of the embryonic kidney when urine flow starts at around embryonic age E15.5 in mouse. Since UB tip cells are known to be the proliferative progenitor cells that contribute to the branching morphogenesis of the collecting system in the kidney, our finding could imply an important link between the FSS from the initiation of urine flow and the development and function of the kidney.

In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells.

A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-ß-catenin pathway or RA. The activation of WNT-ß-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys.

Correction of the mineralization defect in hyp mice treated with protease inhibitors CA074 and pepstatin.

Increased expression of several osteoblastic proteases and MEPE (a bone matrix protein) occurs in X-linked hypophosphatemic rickets (hyp). This is associated with an increased release of a protease-resistant MEPE peptide (ASARM peptide), a potent inhibitor of mineralization. Cathepsin B cleaves MEPE releasing ASARM peptide and hyp osteoblast/osteocyte cells hypersecrete cathepsin D, an activator of cathepsin B. Our aims were to determine whether cathepsin inhibitors correct the mineralization defect in vivo and whether hyp-bone ASARM peptide levels are reduced after protease treatment. Normal littermates and hyp mice (n = 6) were injected intraperitoneally once a day for 4 weeks with pepstatin, CAO74 or vehicle. Animals were then sacrificed and bones plus serum removed for comprehensive analysis. All hyp mice groups (treated and untreated) remained hypophosphatemic with serum 1,25 vitamin D3 inappropriately normal. Serum PTH was significantly elevated in all hyp mice groups relative to normal mice (P = 0.0017). Untreated hyp mice had six-fold elevated levels of serum alkaline-phosphatase and two-fold elevated levels of ASARM peptides relative to normal mice (P < 0.001). In contrast, serum alkaline phosphatase and serum ASARM peptides were significantly reduced (normalized) in hyp mice treated with CA074 or pepstatin. Serum FGF23 levels remained high in all hyp animal groups (P < 0.0001). Hyp mice treated with protease inhibitors showed dramatic reductions in unmineralized osteoid (femurs) compared to control hyp mice (Goldner staining). Also, hyp animals treated with protease inhibitors showed marked and significant improvements in growth plate width (42%), osteoid thickness (40%) and cortical area (40%) (P < 0.002). The mineralization apposition rate, bone formation rate and mineralization surface were normalized by protease-treatment. High-resolution pQCT mineral histomorphometry measurements and uCT also confirmed a marked mineralization improvement. Finally, the growth plate and cortical bone of hyp femurs contained a massive accumulation of osteoblast-derived ASARM peptide(s) that was reduced in hyp animals treated with CA074 or pepstatin. This study confirms in vivo administration of cathepsin inhibitors improves bone mineralization in hyp mice. This may be due to a protease inhibitor mediated decrease in proteolytic degradation of the extracellular matrix and a reduced release of ASARM peptides (potent mineralization inhibitors).

Controlled tubulogenesis from dispersed ureteric bud-derived cells using a micropatterned gel.

Developmental engineering is a potential option for neo-organogenesis of complex organs such as the kidney. The application of this principle requires the ability to construct a tubular structure from dispersed renal progenitor cells with defined size and geometry. In this present study we report the generation of tubular structures from dispersed ureteric bud cells in vitro by using a micropatterned gel. Dispersed CMUB-1 cells, a mouse ureteric bud-derived cell line, or mIMCD cells, a mouse collecting duct-derived cell line, were suspended in collagen I and seeded into an agarose-based micropatterned gel. We found that within 24-36 h of incubation, the cells developed a tubular structure that conformed to the geometry of the micropattern of the gel. The lumen formation of the tubular structure was confirmed by immunohistochemical staining and observed by confocal microscopy. We found that higher concentrations of collagen I negatively influenced the efficiency of tubular formation. Tubule formation in CMUB-1, but not mIMCD, cells was positively influenced by the addition of aldosterone (10, 50 and 200 µg/ml), FGF (50 and 100 µg/ml) and fibronectin (10 and 50 µg/ml) to the growth medium. We further demonstrated the functionality of the generated tubes by in vitro budding, which was induced by growth factors, such as glial cell-derived neurotrophic factor (GDNF) or fibroblast growth factor 7 (FGF7), in the presence of beads soaked with the activin A inhibitor follistatin. Our current study thus demonstrates the possibility of constructing a functional tubular structure from dispersed ureteric bud cells in vitro in a controlled manner. Copyright © 2014 John Wiley & Sons, Ltd.

Maintenance of Mouse Nephron Progenitor Cells in Aggregates with Gamma-Secretase Inhibitor.

Knowledge on how to maintain and expand nephron progenitor cells (NPC) in vitro is important to provide a potentially valuable source for kidney replacement therapies. In our present study, we examined the possibility of optimizing NPC maintenance in the "re-aggregate" system. We found that Six2-expressing (Six2(+))-NPC could be maintained in aggregates reconstituted with dispersed cells from E12.5 mouse embryonic kidneys for at least up to 21 days in culture. The maintenance of Six2(+)-NPC required the presence of ureteric bud cells. The number of Six2(+)-NPC increased by more than 20-fold at day 21, but plateaued after day 14. In an attempt to further sustain NPC proliferation by passage subculture, we found that the new (P1) aggregates reconstituted from the original (P0) aggregates failed to maintain NPC. However, based on the similarity between P1 aggregates and aggregates derived from E15.5 embryonic kidneys, we suspected that the differentiated NPC in P1 aggregates may interfere with NPC maintenance. In support of this notion, we found that preventing NPC differentiation by DAPT, a γ-secretase inhibitor that inhibits Notch signaling pathway, was effective to maintain and expand Six2(+)-NPC in P1 aggregates by up to 65-fold. The Six2(+)-NPC in P1 aggregates retained their potential to epithelialize upon exposure to Wnt signal. In conclusion, we demonstrated in our present study that the "re-aggregation" system can be useful for in vitro maintenance of NPC when combined with γ-secretase inhibitor.

Effective induction of cells expressing GABAergic neuronal markers from mouse embryonic stem cell.

Successful derivations of specific neuronal and glial cells from embryonic stem cells have enormous potential for cell therapies and regenerative medicine. However, the low efficiency, the complexity of induction method, and the need for purification represent obstacles that make their application impractical. In this study, we found that PDGFRα(+) cells derived from mouse embryonic stem cells (mESC) can serve as a useful source from which to induce cells that express γ-aminobutyric-acid (GABA)-releasing (GABAergic) neuronal markers. PDGFRα(+) cells were induced from mESC on collagen IV-coated plates in mesenchymal stem cell (MSC) culture medium with limited exposure to retinoic acid, sorted by fluorescence-activated cell sorter and maintained in MSC culture medium containing Y-27632, a Rho-associated kinase inhibitor. We found that supplementation of vascular endothelial growth factor, fibroblast growth factor-basic, and sodium azide (NaN3) to MSC culture medium effectively differentiated PDGFRα(+) cells into cells that express GABAergic neuronal markers, such as Pax2, Dlx2, GAD67 NCAM, and tubulin-ßIII, while markers for oligodendrocyte (Sox2) and astrocyte (Glast) were suppressed. Immunostaining for GABA showed the majority (86 ± 5%) of the induced cells were GABA-positive. We also found that the PDGFRα(+) cells retained such differentiation potential even after more than ten passages and cryopreservation. In summary, this study presents a simple and highly efficient method of inducing cells that express GABAergic neuronal markers from mESC. Together with its ease of maintenance in vitro, PDGFRα(+) cells derived from mESC may serve as a useful source for such purpose.

Lithium induces c-Ret expression in mouse inner medullary collecting duct cells.

We found in our present study that lithium (Li(+)) induced the expression of endogenous c-Ret, a tyrosine kinase receptor, in murine inner medullary collecting duct (mIMCD-3) cells. Delineation of the promoter region required for the effect of Li(+) identified a positive regulatory element within 180bp upstream of the transcription initiation site. This region contained three putative GC-rich Sp1 binding sites found to be essential for c-Ret induction by Li(+). The effect of Li(+) was mediated through glycogen synthase kinase 3ß (GSK-3ß) inhibition, although there was no biding site for T cell factor/lymphoid enhancer factor (TCF/LEF) in the 180bp. We found that Li(+) activated the mammalian target of rapamycin (mTOR) pathway via GSK-3ß in these cells, and the effect of Li(+) to induce c-Ret was amenable to the inhibitory effect of the mTOR inhibitor, rapamycin. We also found that alterations in both cellular ß-catenin levels and mTOR activities affected the effect of Li(+) on c-Ret transcription in a cooperative manner. In summary, our results show that Li(+) can induce c-Ret expression in mIMCD-3 cells through both ß-catenin- and mTOR-dependent pathways downstream of GSK-3ß inhibition, which act synergistically on the GC-rich Sp1 binding elements in the promoter region.

Altered cathepsin D metabolism in PHEX antisense human osteoblast cells.

X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX gene in osteoblast cells, leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism, but also by locally released osteoblastic mineralization inhibitory factor(s), referred to as minhibin. In our present study, we found that suppression of PHEX expression by PHEX antisense in human osteoblast cells caused an increase in cathepsin D expression at protein, but not mRNA, levels. This was associated with a decrease in cathepsin D degradation and an increased cathepsin D release into culture media. Our results also showed that lowering cathepsin D activity in antisense cell conditioned media abolished their inhibitory effect on osteoblast cell calcification, suggesting the involvement of cathepsin D in mediating the minhibin activity of the antisense cell conditioned media.

Increased cathepsin D release by Hyp mouse osteoblast cells.

The X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism but also by factor(s) locally released by osteoblast cells (ObCs). The identity of these ObC-derived pathogenic factors remains unclear. In our present study, we report our finding of a prominent protein in the culture media derived from ObC of the hypophosphatemic (Hyp) mice, a murine homolog of human XLH, which was identified as the murine procathepsin D (Cat D). By metabolic labeling studies, we further confirmed that Hyp mouse ObCs released greater amount of Cat D into culture media. This increased Cat D release by Hyp mouse ObCs was unlikely to be due to nonspecific cell damage or heterogeneous cell population and was found to be associated with an increased Cat D expression at the protein level, possibly due to a reduced Cat D degradation. However, we were not able to detect a direct effect of PHEX protein on Cat D cleavage. In support of the involvement of Cat D in mediating the inhibitory effect of Hyp mouse ObC-conditioned media on ObC calcification, we found that exposure to Cat D inhibited ObC (45)Ca incorporation and that inhibition of Cat D abolished the inhibitory effect of Hyp mouse-conditioned media on ObC calcification. In conclusion, results from our present study showed that Hyp mouse ObCs release a greater amount of Cat D, which may contribute to the inhibitory effect of Hyp mouse ObC-conditioned media on ObC mineralization.

Effects of PHEX antisense in human osteoblast cells.

X-linked hypophosphatemia (XLH) is an X-linked dominant disorder that is characterized by rachitic bone disease and hypophosphatemia due to renal phosphate transport defect. The candidate gene for XLH, PHEX, has recently been identified and found to share high homology with endopeptidases. PHEX is expressed in various tissues, including bones, and the available evidence today indicates that bones can release abnormal humoral factors that affect bone mineralization and proximal tubule phosphate transport in XLH. It was, therefore, hypothesized that the inactivating mutations of PHEX in bone may lead to the release of humoral factors and contribute to the phenotypic expression of the disease. To test this possibility, clones of MG-63 cells, a human osteoblast cell line, were produced and stably transfected with PHEX-antisense vectors, resulting in a decrease in PHEX expression at mRNA and protein levels. It was found that these antisense-transfected cells had impaired mineralization, with a decrease in 45Ca incorporation and calcification nodule formation. It was also found that the conditioned culture media collected from these antisense-transfected cells exhibited inhibitory activities on 45Ca incorporation by the nontransfected MG-63 cells and 32P uptake by the opossum kidney proximal tubular cells. The results of the study, therefore, provide strong evidence that supports the link between PHEX mutations and the pathogenesis of XLH.

Hypertonicity increases CLC-5 expression in mouse medullary thick ascending limb cells.

Genetic studies indicated that mutations of the chloride channel CLC-5 in the kidney are responsible for a group of clinical disorders, collectively called Dent's disease. In the kidney, CLC-5 was found to be expressed in the proximal tubule, medullary thick ascending limb (mTAL) of loop of Henle, and intercalated cells of the collecting tubule. In proximal tubular cells, CLC-5 was found to play an important role in receptor-mediated endocytosis. However, the functional roles of CLC-5 in mTAL and collecting tubules remain unclear. Because mTAL is normally exposed to a hypertonic environment, we aimed to examine the effect of hypertonicity on CLC-5 expression in this nephron segment. Our studies revealed that exposure to hypertonicity (up to 550 mosM) increased CLC-5 mRNA and protein levels in a murine mTAL cell line (MTAL) but not in an opossum kidney (OK) proximal tubular cell line. A similar effect was also found in mouse kidneys, where CLC-5 expression was enhanced in renal medulla, but not cortex, after 48 h of water deprivation. We also tested the effect of hypertonicity on endocytotic activity and found that exposure to hypertonicity caused a significant decrease in cellular uptake of FITC-labeled albumin in OK but not in MTAL cells. Our results suggest that CLC-5 expression is upregulated by hypertonicity in mTAL cells but not in proximal tubular cells. We speculate that the increased CLC-5 levels in mTAL may serve to maintain the endocytotic activity in a hypertonic environment.