WO2010142619A1 - Method for detecting glucose transporter gene expression - Google Patents

Method for detecting glucose transporter gene expression Download PDF

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WO2010142619A1
WO2010142619A1 PCT/EP2010/057871 EP2010057871W WO2010142619A1 WO 2010142619 A1 WO2010142619 A1 WO 2010142619A1 EP 2010057871 W EP2010057871 W EP 2010057871W WO 2010142619 A1 WO2010142619 A1 WO 2010142619A1
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cells
glucose transporter
sglt
urine
expression level
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Inventor
Andreas Albers
Beatrix Bahle
Maria Bobadilla
Barbara Ecabert
Sabine Lohmann
Céline PALLAUD
Jeremy Quirk
Thomas Schindler
Gabriela Suchankova
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F Hoffmann La Roche AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins

Definitions

  • the present invention provides a method for the measurement of glucose transporter gene expression in renal cells.
  • Glucose is the main source or energy for the mammalian cells and its entry is mediated via various transporters. About 7 facilitative (GLUT-I to -7) and 2 concentrative glucose transporters (SGLT-I and -2) have been identified. The facilitative glucose transporters allow the glucose entry into the cell interior due to the concentration gradient and the latter via the Na+- dependent electrochemical gradient. They have similar structural motifs with 12-14 putative transmembrane domains with a predicted protein size varying from 50 to 76kDa. Some of the facilitative glucose transporters (GLUT-I, -2, -4 and -5) and both the sodium glucose co- transporters (SGLT-I and -2) are expressed in the kidney. The transporters that are involved in the major transport of glucose in the kidney include GLUT-2 and SGLT-2.
  • the bulk of glucose that is filtered by the renal glomerulus is reabsorbed by the glucose transporters of the proximal convoluted tubular epithelium. 90% of the glucose is reabsorbed by the low-affinity/high capacity sodium glucose co transporter (SGLT) 2 located in the Sl segments of proximal tubule.
  • SGLT sodium glucose co transporter
  • Available methods for the detection of glucose transporter expression in renal cells either involve a renal biopsy or the in vitro cultivation of exfoliated renal cells isolated from urine prior to the determination of the SGLT expression level.
  • It is an object of the present invention to provide an in vitro method for determining the expression level of a glucose transporter protein in renal cells comprising:
  • step b) determining the expression level of the glucose transporter, wherein the isolated cells are not cultivated in vitro prior to step b).
  • the expression level of the glucose transporter protein is determined by RT-PCR.
  • the expression level of the glucose transporter protein is determined by RT-PCR using housekeeping genes selected from the group consisting of CD 13, MRPL19 (mitochondrial ribosomal protein L 19) and ALASl (amino levulinate, delta-, synthase
  • the glucose transporter protein is selected from the group consisting of GLUT-I, GLUT-2, SGLT-I and SGLT-2, preferably SGLT-2.
  • the renal cells are proximal tubular epithelial cells.
  • the cells are isolated from urine using a polypropylene membrane filter having a pore size of approximately 1.6 ⁇ m.
  • a suitable filter for the isolation of renal cells from urine is the ZRC GFTM filter which is commercially available from Zymo Research Corporation.
  • Techniques for the detection and quantification of gene expression of a glucose transporter gene include, but are not limited to northern blots, RT-PCR, real time quantitative PCR, primer extension, RNase protection, RNA expression profiling and related techniques. These techniques are well known to those of skill in the art see e.g. Sambrook J et al., Molecular Cloning: A
  • Techniques for the detection of protein expression of a glucose transporter include, but are not limited to immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • Fig. 1 shows SGLT-2 and CD 13 expression in T2D (Type 2 Diabetes) urine samples.
  • Urine from 10 (T2D) volunteers was collected. 30 ml urine was processed with the Zmyo Research kit to extract the RNA according the instructions of the manufacturer.
  • RNA Expression of CD 13 and SGLT-2 was analysed with Real-time PCR using the LightCycler® 480 Instrument II and
  • Fig. 2 shows the linearity of SGLT-2 quantification in urine samples spiked with SGLT-2 transfected Chinese hamster ovary (CHO) cells. RNA isolation was performed with the Zymo Research Kit. The linear measuring range for 0 to 50.000 CHO/SGLT-2 cells spiked in urine sediment are shown and corresponds to a cp-range from 31,19 (only urine) to 17,13.
  • RNA isolation from fresh clean-catch urine using a Urine RNA Isolation Kit (Zymo research Inc, Orange CA)
  • Clean-catch urine was freshly collected into a 200 ml beaker. Cells were resuspended by carefully swirling the beaker and filtered through the ZRC GF filter according to the manufacturer's instructions. A total of 30 ml urine was filtered. Cells retained by the filter were extracted using the Zymo Research extraction buffer. The cell extract was stored at -80°C before further processing.
  • RNA of SGLT-2 and 3 potential housekeeping genes were amplified in real-time reverse transcription - polymerase chain Taqman reaction using the following instruments, reagents and reaction conditions:
  • SGLT-2 was labeled by Dyel and BHQ2, Housekeeping genes were labeled by Dye2 and BHQ2.
  • Results are calculated based on relative crossing point values of SGLT-2 relative to potential housekeeping gene crossing points.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
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  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
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  • Biotechnology (AREA)
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  • Physics & Mathematics (AREA)
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Abstract

The present invention provides an in vitro for determining the expression level of a glucose transporter protein in renal cells comprising: a) isolation of renal cells from urine by filtration and b) determining the expression level of the glucose transporter, wherein the isolated cells are not cultivated in vitro prior to step b).

Description

METHOD FOR DETECTING GLUCOSE TRANSPORTER GENE EXPRESSION
The present invention provides a method for the measurement of glucose transporter gene expression in renal cells.
Glucose is the main source or energy for the mammalian cells and its entry is mediated via various transporters. About 7 facilitative (GLUT-I to -7) and 2 concentrative glucose transporters (SGLT-I and -2) have been identified. The facilitative glucose transporters allow the glucose entry into the cell interior due to the concentration gradient and the latter via the Na+- dependent electrochemical gradient. They have similar structural motifs with 12-14 putative transmembrane domains with a predicted protein size varying from 50 to 76kDa. Some of the facilitative glucose transporters (GLUT-I, -2, -4 and -5) and both the sodium glucose co- transporters (SGLT-I and -2) are expressed in the kidney. The transporters that are involved in the major transport of glucose in the kidney include GLUT-2 and SGLT-2.
The bulk of glucose that is filtered by the renal glomerulus is reabsorbed by the glucose transporters of the proximal convoluted tubular epithelium. 90% of the glucose is reabsorbed by the low-affinity/high capacity sodium glucose co transporter (SGLT) 2 located in the Sl segments of proximal tubule. Available methods for the detection of glucose transporter expression in renal cells either involve a renal biopsy or the in vitro cultivation of exfoliated renal cells isolated from urine prior to the determination of the SGLT expression level.
It is an object of the present invention to provide an in vitro method for determining the expression level of a glucose transporter protein in renal cells comprising:
a) isolation of renal cells from urine by filtration and
b) determining the expression level of the glucose transporter, wherein the isolated cells are not cultivated in vitro prior to step b).
In a preferred embodiment, the expression level of the glucose transporter protein is determined by RT-PCR.
In another preferred embodiment, the expression level of the glucose transporter protein is determined by RT-PCR using housekeeping genes selected from the group consisting of CD 13, MRPL19 (mitochondrial ribosomal protein L 19) and ALASl (amino levulinate, delta-, synthase In a further preferred embodiment, the glucose transporter protein is selected from the group consisting of GLUT-I, GLUT-2, SGLT-I and SGLT-2, preferably SGLT-2.
In another preferred embodiment, the renal cells are proximal tubular epithelial cells.
In yet another preferred embodiment, the cells are isolated from urine using a polypropylene membrane filter having a pore size of approximately 1.6 μm. A suitable filter for the isolation of renal cells from urine is the ZRC GF™ filter which is commercially available from Zymo Research Corporation.
Techniques for the detection and quantification of gene expression of a glucose transporter gene include, but are not limited to northern blots, RT-PCR, real time quantitative PCR, primer extension, RNase protection, RNA expression profiling and related techniques. These techniques are well known to those of skill in the art see e.g. Sambrook J et al., Molecular Cloning: A
Laboratory Manual, Third Edition (Cold Spring Harbor Press, Cold Spring Harbor, 2000).
Techniques for the detection of protein expression of a glucose transporter include, but are not limited to immunohistochemistry (IHC).
Short description of the figures:
Fig. 1 shows SGLT-2 and CD 13 expression in T2D (Type 2 Diabetes) urine samples. Urine from 10 (T2D) volunteers was collected. 30 ml urine was processed with the Zmyo Research kit to extract the RNA according the instructions of the manufacturer. RNA Expression of CD 13 and SGLT-2 was analysed with Real-time PCR using the LightCycler® 480 Instrument II and
Fig. 2 shows the linearity of SGLT-2 quantification in urine samples spiked with SGLT-2 transfected Chinese hamster ovary (CHO) cells. RNA isolation was performed with the Zymo Research Kit. The linear measuring range for 0 to 50.000 CHO/SGLT-2 cells spiked in urine sediment are shown and corresponds to a cp-range from 31,19 (only urine) to 17,13.
Example:
Isolation of Cells from Urine and subsequent isolation of RNA from cells:
RNA isolation from fresh clean-catch urine using a Urine RNA Isolation Kit (Zymo research Inc, Orange CA)
Clean-catch urine was freshly collected into a 200 ml beaker. Cells were resuspended by carefully swirling the beaker and filtered through the ZRC GF filter according to the manufacturer's instructions. A total of 30 ml urine was filtered. Cells retained by the filter were extracted using the Zymo Research extraction buffer. The cell extract was stored at -80°C before further processing.
RNA was isolated from the thawed cell extract. After adding one volume 95% ethanol to the cell extract the solution was mixed and applied to a Zymo-spin IC column and processed according to the manufacturers instructions. In brief, the column was washed with RNA wash buffer and RNA was eluted using 20 mL RNA Elution Buffer. The RNA was stored at -800C.
Amplification and detection of SGLT-2 RNA from total RNA isolated from urinary cells
RNA of SGLT-2 and 3 potential housekeeping genes (CD 13, MRPL 19, ALASl) were amplified in real-time reverse transcription - polymerase chain Taqman reaction using the following instruments, reagents and reaction conditions:
• Instrument: LightCycler® 480 II: Serien 25200, Software version: LCS480 1.5.0.39
• Primers and probes sequences:
Figure imgf000004_0001
SGLT-2 was labeled by Dyel and BHQ2, Housekeeping genes were labeled by Dye2 and BHQ2.
Mastermix:
Mn2+ (25mM Manganacetat), Ident. No. 05 384 567. 5x RNA Mix TaqMan RNA Amp Kit, Ident. No. 05 384 583
Preparation of the Amplification Mix:
Figure imgf000004_0002
Figure imgf000005_0001
Amplification conditions and settings on Lightcycler:
Figure imgf000005_0002
Result calculation:
Results are calculated based on relative crossing point values of SGLT-2 relative to potential housekeeping gene crossing points.
While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Claims

Claims
1. An in vitro method for determining the expression level of a glucose transporter protein in renal cells comprising:
a) isolation of renal cells from urine by filtration and
b) determining the expression level of the glucose transporter, wherein the isolated cells are not cultivated in vitro prior to step b).
2. The method of claim 1, wherein the expression level is determined by RT-PCR.
3. The method of claim 2, wherein the expression level is determined by RT-PCR using housekeeping genes selected from the group consisting of CD 13, MRPL 19, and ALASl.
4. The method of claim 1 - 3, wherein the glucose transporter protein is selected from the group consisting of GLUT-I, GLUT-2, SGLT-I and SGLT-2.
5. The method of claim 4, wherein the glucose transporter protein is SGLT-2.
6. The method of claim 1 - 5, wherein the renal cells are proximal tubular epithelial cells.
7. The method claim 1 - 6, wherein the cells are isolated from urine using a polypropylene membrane filter having a pore size of about 1.6 μm.
8. The methods substantially as hereinbefore described, especially with reference to the foregoing examples.
PCT/EP2010/057871 2009-06-10 2010-06-07 Method for detecting glucose transporter gene expression Ceased WO2010142619A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP09162425 2009-06-10
EP09162425.4 2009-06-10

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
RAHMOUNE HASSAN ET AL: "Glucose transporters in human renal proximal tubular cells isolated from the urine of patients with non-insulin-dependent diabetes", DIABETES, vol. 54, no. 12, December 2005 (2005-12-01), pages 3427 - 3434, XP002596589, ISSN: 0012-1797 *
SAMBROOK J ET AL.: "Molecular Cloning: A Laboratory Manual", vol. 3RD.ED., 2000, COLD SPRING HARBOR PRESS, COLD SPRING HARBOR
TABATABAI N M ET AL: "Enhanced expressions of sodium-glucose cotransporters in the kidneys of diabetic Zucker rats", DIABETES RESEARCH AND CLINICAL PRACTICE 200901 IE LNKD- DOI:10.1016/J.DIABRES.2008.11.003, vol. 83, no. 1, January 2009 (2009-01-01), pages E27 - E30, XP002596590, ISSN: 0168-8227 *
VESTRI S ET AL: "Changes in sodium or glucose filtration rate modulate expression of glucose transporters in renal proximal tubular cells of rat.", THE JOURNAL OF MEMBRANE BIOLOGY 15 JUL 2001 LNKD- PUBMED:11447502, vol. 182, no. 2, 15 July 2001 (2001-07-15), pages 105 - 112, XP002596591, ISSN: 0022-2631 *

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