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Complications

Glucose Transporters in Human Renal Proximal Tubular Cells Isolated From the Urine of Patients With Non–Insulin-Dependent Diabetes

  1. Hassan Rahmoune,
  2. Paul W. Thompson,
  3. Joanna M. Ward,
  4. Chari D. Smith,
  5. Guizhu Hong and
  6. John Brown
  1. From the Clinical Pharmacology Unit, GlaxoSmithKline, Translational Medicine and Technology, Human Biomarkers Centre, Addenbrooke’s Hospital, Cambridge, U.K
  1. Address correspondence and reprint requests to Hassan Rahmoune, PhD, GlaxoSmithKline, Clinical Pharmacology Unit, Human Biomarkers Centre, Translational Medicine and Technology, Addenbrooke’s Hospital, Cambridge, CB2 2GG, U.K. E-mail: hassan_2_rahmoune{at}gsk.com
Diabetes 2005 Dec; 54(12): 3427-3434. https://doi.org/10.2337/diabetes.54.12.3427
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  • FIG. 1.
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    FIG. 1.

    Phase-contrast light micrograph of human primary cells isolated from fresh urine in primary culture after 24 h in culture (A). After, adhering to the collagen after 2–3 days, the cells are adhering to the collagen-I (B). Colonies of cells are formed after a week (C) and formed a confluent monolayer of cuboidal cells with cobblestone-like appearance. D: The cells after passage 2. Original magnification: ×20.

  • FIG. 2.
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    FIG. 2.

    Confocal microscopy of the human exfoliated primary cells (bar = 10 μm). Proximal cell marker: CD13-aminopeptidase-N (green) and distal and loop of Henle cell marker: Tamm-Horsfall protein (red). These markers indicate the presence of distal and proximal cells in the exfoliated primary cell culture.

  • FIG. 3.
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    FIG. 3.

    HEPTEC immunocychemistry characterization prior (A and C) and post (B and D) immuno-magnetic cell separation. TRITC-phalloidin (red) staining actin filaments (A–D) and CD13-aminopeptidase-N (green) enrichment 50% (A) to 90% (B). CD90-FITC (green) a fibroblast-like expression decreasing from ∼10% (C) to <5% post immunomagnetic cell separation (D).

  • FIG. 4.
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    FIG. 4.

    Typical human exfoliated primary cells of two healthy volunteers (V1 and V2). Positive cell counting by confocal microscopy at passage 4 prior (A) to CD13-immunomagnetic cell separation and post separation at passage 6 (B). Five fields on each of two separate coverslips were randomly selected and counted. An average of 30.2 cells per field were counted (range from 19 to 54 cells), and the counts were averaged and converted to percentages.

  • FIG. 5.
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    FIG. 5.

    Glucose transporter mRNA expression in HEPTECs isolated from healthy and type 2 diabetic subjects. A: SGLT2 (n = 4), B: GLUT2 (n = 4), and C: GLUT1 (n = 5). The glucose transporters were analyzed by real-time RT-PCR and normalized to GAPDH. Values are means ± SE (*P < 0.05).

  • FIG. 6.
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    FIG. 6.

    Immunoblot representing SGLT2 and GLUT2 protein expression in HEPTECs isolated from healthy (H) and type 2 diabetic (D) volunteers’ fresh urine (n = 4). The predicted band of 75-kDa molecular weight representing the SGLT2 level of protein expression in HEPTECs isolated from type 2 diabetes increases up to three times compared with healthy volunteers. GLUT2 (55 kDa) level of protein expression in HEPTECs isolated from type 2 diabetes increases up to 10 times compared with healthy volunteers. The level of glucose transporter protein expression is determined from densitometry relative to GAPDH. Values are means ± SE (n = 4; *P < 0.05).

  • FIG. 7.
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    FIG. 7.

    Glucose uptake assay using the AMG uptake, in triplicates, as a tracer to assess HEPTEC glucose transport in healthy and type 2 diabetic subjects (n = 3) as described by Takamotoa et al. (28). AMG uptake HEPTECs isolated from type 2 diabetes increases up to threefold compared with healthy volunteers. AMG cellular accumulation was presented as means ± SE (n = 3; *P < 0.05)

Tables

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  • TABLE 1

    Expression of relevant cellular markers in human exfoliated primary cells and HEPTECs

    Cellular markermRNA*Protein*Cell type
    Cytokeratin-7ND+++High in distal cells and bladder cells and low in proximal cells
    Aquaporin-2ND++Collecting duct
    ENaCα++++++Collecting duct and distal cells
    ENaCβ/γ++NDCollecting duct and distal cells
    PPARγ/α/δ+++PPARγ onlyCollecting duct and distal cells/proximal cells
    NaCl cotransporter+++NDProximal cells/connecting tubules
    Aquaporin-2ND+++Collecting duct
    L1-CAM (cell adhesion molecule)ND+++Collecting duct
    Aminopeptidase-N (CD13)ND+++Proximal cells
    Sodium-glucose transporters (SGLT2, GLUT1, and GLUT2)++++++Proximal cells
    Alkaline phosphataseND+++Proximal cells
    Uroplakin IIIND+++Bladder cells
    Mineralocortcoid receptor+++NDConnecting tubules/distal cells/collecting duct
    • Expression of cellular markers of human primary exfoliated cells: 1) bladder/urethra (uroplakin III/cytokeratin 7), 2) proximal tubular cells (CD13 aminopepetidase-N/SGLT2, 3) renal distal tubular/collecting duct cells (aldosterone receptor, cytokeratin-7, aquaporin-2, and ENaCα) by PCR and/or immunoblotting. Marker level of presence (+ + +).

    • *

      * mRNA (Taqman) and proteins were measured and expressed as relative abundance to gapdh. +, weak expression; ++ high expression; +++ very high expression; ND, not been done.

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Glucose Transporters in Human Renal Proximal Tubular Cells Isolated From the Urine of Patients With Non–Insulin-Dependent Diabetes
Hassan Rahmoune, Paul W. Thompson, Joanna M. Ward, Chari D. Smith, Guizhu Hong, John Brown
Diabetes Dec 2005, 54 (12) 3427-3434; DOI: 10.2337/diabetes.54.12.3427

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Glucose Transporters in Human Renal Proximal Tubular Cells Isolated From the Urine of Patients With Non–Insulin-Dependent Diabetes
Hassan Rahmoune, Paul W. Thompson, Joanna M. Ward, Chari D. Smith, Guizhu Hong, John Brown
Diabetes Dec 2005, 54 (12) 3427-3434; DOI: 10.2337/diabetes.54.12.3427
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