Limits...
CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Reed AA, Loh NY, Terryn S, Lippiat JD, Partridge C, Galvanovskis J, Williams SE, Jouret F, Wu FT, Courtoy PJ, Nesbit MA, Rorsman P, Devuyst O, Ashcroft FM, Thakker RV - Am. J. Physiol. Renal Physiol. (2009)

Bottom Line: Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules.KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin.Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit, Nuffield Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford, United Kingdom.

ABSTRACT
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

Show MeSH

Related in: MedlinePlus

Endogenous Kif3b expression and albumin endocytosis in mouse proximal renal tubular primary cell cultures (mPTCs). A: endogenous expression of Kif3b in mPTC cultures from Clcn5Y/+ and Clcn5Y/− mice. The expression of Kif3b was determined by qPCR and was found to be significantly higher in the Clcn5Y/− mice and consistent with the observations from mouse whole kidney extracts (Fig. 5A). B: expression of human KIF3B (hKIF3B) in transfected mPTCs from Clcn5Y/+ (n = 3) and Clcn5Y/− (n = 3) mice was assessed by qPCR and this showed an ∼3,000-fold higher expression of the exogenous human KIF3B, compared with the endogenous expression of Kif3b [Kif3b(m)] and Kif3a. C: uptake of FITC-albumin in mPTC cultures from Clcn5Y/+ and Clcn5Y/− mice in mock-transfected cells, in the absence or presence of competition by excess unlabeled albumin and transferrin, or after energy depletion by deoxyglucose (DOG) and sodium azide (NaN3), or in KIF3B-transfected cells. In Clcn5Y/+ mPTCs, overexpression of hKIF3B decreases specific albumin endocytic uptake. In Clcn5Y/− mPTCs, albumin uptake is fully abrogated (to the level of metabolic inhibition and albumin competition) by hKIF3B overexpression. D: RT-PCR analyses of caveolin-1 (Cav-1) and caveolin-2 (Cav-2) in HEK293 cells, OK cells, Clcn5Y/+ and Clcn5Y/− whole mouse kidneys, and Clcn5Y/+ and Clcn5Y/− mPTCs. GAPDH and a water blank were used as positive and negative controls, respectively. Means ± 1 SE and P values were calculated by the Student's unpaired, 2-tailed t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2822520&req=5

Figure 6: Endogenous Kif3b expression and albumin endocytosis in mouse proximal renal tubular primary cell cultures (mPTCs). A: endogenous expression of Kif3b in mPTC cultures from Clcn5Y/+ and Clcn5Y/− mice. The expression of Kif3b was determined by qPCR and was found to be significantly higher in the Clcn5Y/− mice and consistent with the observations from mouse whole kidney extracts (Fig. 5A). B: expression of human KIF3B (hKIF3B) in transfected mPTCs from Clcn5Y/+ (n = 3) and Clcn5Y/− (n = 3) mice was assessed by qPCR and this showed an ∼3,000-fold higher expression of the exogenous human KIF3B, compared with the endogenous expression of Kif3b [Kif3b(m)] and Kif3a. C: uptake of FITC-albumin in mPTC cultures from Clcn5Y/+ and Clcn5Y/− mice in mock-transfected cells, in the absence or presence of competition by excess unlabeled albumin and transferrin, or after energy depletion by deoxyglucose (DOG) and sodium azide (NaN3), or in KIF3B-transfected cells. In Clcn5Y/+ mPTCs, overexpression of hKIF3B decreases specific albumin endocytic uptake. In Clcn5Y/− mPTCs, albumin uptake is fully abrogated (to the level of metabolic inhibition and albumin competition) by hKIF3B overexpression. D: RT-PCR analyses of caveolin-1 (Cav-1) and caveolin-2 (Cav-2) in HEK293 cells, OK cells, Clcn5Y/+ and Clcn5Y/− whole mouse kidneys, and Clcn5Y/+ and Clcn5Y/− mPTCs. GAPDH and a water blank were used as positive and negative controls, respectively. Means ± 1 SE and P values were calculated by the Student's unpaired, 2-tailed t-test.

Mentions: To study further the role of the CLC-5 and KIF3B interaction in albumin endocytosis, primary cell cultures of mPTCs, which are polarized and differentiated (13), were prepared from the kidneys of Clcn5Y/+ and Clcn5Y/− mice, as described previously (51). qPCR analysis using RNA from these mPTCs revealed that endogenous Kif3b expression was significantly higher in the mPTCs of Clcn5Y/− mice (Fig. 6A). This finding is consistent with the observations of increased Kif3b expression in whole kidney extracts from Clcn5Y/− mice (Fig. 5, A–D). Fluorescently labeled albumin endocytosis in the absence or presence of excess unlabeled albumin or transferrin was investigated in Clcn5Y/+ and Clcn5Y/− mPTCs that were either untransfected or transfected with human GFP-tagged KIF3B (Figs. 6, B and C). The transfection efficiency of human KIF3B (hKIF3B) in mPTCs, as assessed by GFP expression, was ∼40% in mPTC cultures from both Clcn5Y/+ and Clcn5Y/− mice (data not shown), and hKIF3B expression, as assessed by qPCR, in the transfected mPTCs was ∼3,000-fold higher than the endogenous mouse Kif3b and Kif3a (Fig. 6B). Studies of albumin uptake, in these polarized cells, revealed a strong decrease in mPTCs of Clcn5Y/− mice compared with mPTCs of Clcn5Y/+ mice (Fig. 6C). The residual uptake of FITC-BSA in Clcn5Y/− cells may indicate that Clc-5 strongly promotes but is not absolutely essential for receptor-mediated endocytosis. Alternatively, these observations may reflect a Clc-5-independent endocytic pathway, such as caveolin-dependent endocytosis. Indeed, caveolin-1 and caveolin-2 were shown to be expressed in HEK293 and OK cells, mouse kidney, and mPTCs (Fig. 6D), and expression of caveolin-2 has been reported to be increased in Clcn5Y/− proximal tubules (57). In both Clcn5Y/+ and Clcn5Y/− mPTCs, fluorescently labeled albumin endocytosis was reduced by competition with excess unlabeled albumin and transferrin. These findings are similar to those obtained using the polarized OK cells (Figs. 4, J and K), and consistent with a role of CLC-5 and KIF3B interaction in facilitating endocytosis via the megalin-cubilin receptor complex (54, 57). Thus, an excess of albumin will block the available binding sites on megalin and cubilin and an excess of transferrin will block the available binding sites on cubilin, and both will therefore decrease endocytosis of fluorescently labeled albumin. In addition, this endocytosis by the mPTCs of Clcn5Y/+ and Clcn5Y/− mice was essentially abrogated by treatment with DOG and sodium azide (NaN3), which block the production of ATP from glycolysis and oxidative phosphorylation, respectively, and hence lead to energy depletion in the cells (50). In Clcn5Y/+ mPTCs, overexpression of hKIF3B induced a strong decrease in the uptake of FITC-BSA, but not complete abolishment, as evidenced by the residual level of endocytosis that was reduced by competition with albumin and transferrin excess, or metabolic inhibition (Fig. 6C). However, in Clcn5Y/− mPTCs, hKIF3B overexpression completely abrogated endocytosis down to the level achieved by energy depletion (Fig. 6C). This further suppression of albumin endocytosis by hKIF3B overexpression in the CLC-5-deficient mPTCs indicates that the level of Clc-5 expression influences the effects of KIF3B on albumin endocytosis, and this is consistent with the role of KIF3B in the intracellular trafficking of other membranous organelles (61), such as caveolae, which involves the Kinesin-2 complex (41). Thus, the greater inhibitory effect on FITC-BSA endocytosis observed on hKIF3B overexpression in the absence of Clc-5 indicates that the effects on endocytosis of KIF3B alterations are dependent on Clc-5 expression.


CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Reed AA, Loh NY, Terryn S, Lippiat JD, Partridge C, Galvanovskis J, Williams SE, Jouret F, Wu FT, Courtoy PJ, Nesbit MA, Rorsman P, Devuyst O, Ashcroft FM, Thakker RV - Am. J. Physiol. Renal Physiol. (2009)

Endogenous Kif3b expression and albumin endocytosis in mouse proximal renal tubular primary cell cultures (mPTCs). A: endogenous expression of Kif3b in mPTC cultures from Clcn5Y/+ and Clcn5Y/− mice. The expression of Kif3b was determined by qPCR and was found to be significantly higher in the Clcn5Y/− mice and consistent with the observations from mouse whole kidney extracts (Fig. 5A). B: expression of human KIF3B (hKIF3B) in transfected mPTCs from Clcn5Y/+ (n = 3) and Clcn5Y/− (n = 3) mice was assessed by qPCR and this showed an ∼3,000-fold higher expression of the exogenous human KIF3B, compared with the endogenous expression of Kif3b [Kif3b(m)] and Kif3a. C: uptake of FITC-albumin in mPTC cultures from Clcn5Y/+ and Clcn5Y/− mice in mock-transfected cells, in the absence or presence of competition by excess unlabeled albumin and transferrin, or after energy depletion by deoxyglucose (DOG) and sodium azide (NaN3), or in KIF3B-transfected cells. In Clcn5Y/+ mPTCs, overexpression of hKIF3B decreases specific albumin endocytic uptake. In Clcn5Y/− mPTCs, albumin uptake is fully abrogated (to the level of metabolic inhibition and albumin competition) by hKIF3B overexpression. D: RT-PCR analyses of caveolin-1 (Cav-1) and caveolin-2 (Cav-2) in HEK293 cells, OK cells, Clcn5Y/+ and Clcn5Y/− whole mouse kidneys, and Clcn5Y/+ and Clcn5Y/− mPTCs. GAPDH and a water blank were used as positive and negative controls, respectively. Means ± 1 SE and P values were calculated by the Student's unpaired, 2-tailed t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2822520&req=5

Figure 6: Endogenous Kif3b expression and albumin endocytosis in mouse proximal renal tubular primary cell cultures (mPTCs). A: endogenous expression of Kif3b in mPTC cultures from Clcn5Y/+ and Clcn5Y/− mice. The expression of Kif3b was determined by qPCR and was found to be significantly higher in the Clcn5Y/− mice and consistent with the observations from mouse whole kidney extracts (Fig. 5A). B: expression of human KIF3B (hKIF3B) in transfected mPTCs from Clcn5Y/+ (n = 3) and Clcn5Y/− (n = 3) mice was assessed by qPCR and this showed an ∼3,000-fold higher expression of the exogenous human KIF3B, compared with the endogenous expression of Kif3b [Kif3b(m)] and Kif3a. C: uptake of FITC-albumin in mPTC cultures from Clcn5Y/+ and Clcn5Y/− mice in mock-transfected cells, in the absence or presence of competition by excess unlabeled albumin and transferrin, or after energy depletion by deoxyglucose (DOG) and sodium azide (NaN3), or in KIF3B-transfected cells. In Clcn5Y/+ mPTCs, overexpression of hKIF3B decreases specific albumin endocytic uptake. In Clcn5Y/− mPTCs, albumin uptake is fully abrogated (to the level of metabolic inhibition and albumin competition) by hKIF3B overexpression. D: RT-PCR analyses of caveolin-1 (Cav-1) and caveolin-2 (Cav-2) in HEK293 cells, OK cells, Clcn5Y/+ and Clcn5Y/− whole mouse kidneys, and Clcn5Y/+ and Clcn5Y/− mPTCs. GAPDH and a water blank were used as positive and negative controls, respectively. Means ± 1 SE and P values were calculated by the Student's unpaired, 2-tailed t-test.
Mentions: To study further the role of the CLC-5 and KIF3B interaction in albumin endocytosis, primary cell cultures of mPTCs, which are polarized and differentiated (13), were prepared from the kidneys of Clcn5Y/+ and Clcn5Y/− mice, as described previously (51). qPCR analysis using RNA from these mPTCs revealed that endogenous Kif3b expression was significantly higher in the mPTCs of Clcn5Y/− mice (Fig. 6A). This finding is consistent with the observations of increased Kif3b expression in whole kidney extracts from Clcn5Y/− mice (Fig. 5, A–D). Fluorescently labeled albumin endocytosis in the absence or presence of excess unlabeled albumin or transferrin was investigated in Clcn5Y/+ and Clcn5Y/− mPTCs that were either untransfected or transfected with human GFP-tagged KIF3B (Figs. 6, B and C). The transfection efficiency of human KIF3B (hKIF3B) in mPTCs, as assessed by GFP expression, was ∼40% in mPTC cultures from both Clcn5Y/+ and Clcn5Y/− mice (data not shown), and hKIF3B expression, as assessed by qPCR, in the transfected mPTCs was ∼3,000-fold higher than the endogenous mouse Kif3b and Kif3a (Fig. 6B). Studies of albumin uptake, in these polarized cells, revealed a strong decrease in mPTCs of Clcn5Y/− mice compared with mPTCs of Clcn5Y/+ mice (Fig. 6C). The residual uptake of FITC-BSA in Clcn5Y/− cells may indicate that Clc-5 strongly promotes but is not absolutely essential for receptor-mediated endocytosis. Alternatively, these observations may reflect a Clc-5-independent endocytic pathway, such as caveolin-dependent endocytosis. Indeed, caveolin-1 and caveolin-2 were shown to be expressed in HEK293 and OK cells, mouse kidney, and mPTCs (Fig. 6D), and expression of caveolin-2 has been reported to be increased in Clcn5Y/− proximal tubules (57). In both Clcn5Y/+ and Clcn5Y/− mPTCs, fluorescently labeled albumin endocytosis was reduced by competition with excess unlabeled albumin and transferrin. These findings are similar to those obtained using the polarized OK cells (Figs. 4, J and K), and consistent with a role of CLC-5 and KIF3B interaction in facilitating endocytosis via the megalin-cubilin receptor complex (54, 57). Thus, an excess of albumin will block the available binding sites on megalin and cubilin and an excess of transferrin will block the available binding sites on cubilin, and both will therefore decrease endocytosis of fluorescently labeled albumin. In addition, this endocytosis by the mPTCs of Clcn5Y/+ and Clcn5Y/− mice was essentially abrogated by treatment with DOG and sodium azide (NaN3), which block the production of ATP from glycolysis and oxidative phosphorylation, respectively, and hence lead to energy depletion in the cells (50). In Clcn5Y/+ mPTCs, overexpression of hKIF3B induced a strong decrease in the uptake of FITC-BSA, but not complete abolishment, as evidenced by the residual level of endocytosis that was reduced by competition with albumin and transferrin excess, or metabolic inhibition (Fig. 6C). However, in Clcn5Y/− mPTCs, hKIF3B overexpression completely abrogated endocytosis down to the level achieved by energy depletion (Fig. 6C). This further suppression of albumin endocytosis by hKIF3B overexpression in the CLC-5-deficient mPTCs indicates that the level of Clc-5 expression influences the effects of KIF3B on albumin endocytosis, and this is consistent with the role of KIF3B in the intracellular trafficking of other membranous organelles (61), such as caveolae, which involves the Kinesin-2 complex (41). Thus, the greater inhibitory effect on FITC-BSA endocytosis observed on hKIF3B overexpression in the absence of Clc-5 indicates that the effects on endocytosis of KIF3B alterations are dependent on Clc-5 expression.

Bottom Line: Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules.KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin.Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit, Nuffield Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford, United Kingdom.

ABSTRACT
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

Show MeSH
Related in: MedlinePlus