Identification of up-regulated Ras-like GTPase, Rap1b, by suppression subtractive hybridization

S. Lin, S. Chugh, X. Pan, E. I. Wallner, Jun Wada, Y. S. Kanwar

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Background. Diabetic nephropathy accounts for over 30% of the end-stage renal disease (ESRD). A number of defined mechanisms and molecules that are involved in its pathogenesis are known, while others remain to be identified. Methods. Suppression subtraction hybridization (SSH)-polymerase chain reaction (PCR) was employed to search for new genes that may be relevant to the pathogenesis of diabetic nephropathy during embryonic development, the time when the kidney is most susceptible to various forms of stress. A diabetic state was induced in pregnant mice at day-13 of gestation by administration of streptozotocin. The kidneys of newborn mice with blood glucose level >200 mg/dL were harvested, mRNA isolated and subjected to SSH-PCR. Several differentially expressed cDNA fragments with up-regulated expression were isolated. One of the cDNA fragments had homology with human Ras-like guanine 5′-triphosphate (GTPase), Rap1b gene. By utilizing the λZAP II mouse cDNA library and SMART™ RACE amplification, a full-length Rap1b cDNA was isolated. A recombinant protein was generated in pET15b bacterial expression system. An anti-Rap1b antibody was raised in rabbits by immunizing them with the fusion protein, and its specificity was confirmed by Western blot analysis. Results. Rap1b cDNA had an open reading frame of 552 bp with a predicted putative protein size of ∼21 kD. In vitro translation verified the authentication of the Rap1b cDNA clone. Northern blot analyses revealed a single ∼2.3 kb Rap1b mRNA transcript. Its expression was up-regulated in several tissues, including the kidney of newborn diabetic mice. The degree of up-regulation of Rap1b mRNA expression was proportional to the blood glucose levels. Western blot analyses confirmed the hyperglycemia-induced up-regulation of the Rap1b expression. In situ hybridization and immunofluorescence studies revealed that Rap1b was expressed in the inner medullary collecting tubules. During hyperglycemia, its expression was accentuated and extended into the outer medullary and cortical collecting tubules. Similar up-regulation of Rap1b was observed when embryonic kidneys, harvested at day-13 of gestation, were exposed to high glucose ambience. Conclusion. The data suggest that Rap1b, a GTP-binding protein that plays a critical role in various signaling intracellular events, is another molecule that may be relevant to the pathobiology of diabetic nephropathy.

Original languageEnglish
Pages (from-to)2129-2141
Number of pages13
JournalKidney International
Volume60
Issue number6
DOIs
Publication statusPublished - 2001

Fingerprint

ras Proteins
Complementary DNA
Diabetic Nephropathies
Kidney
Up-Regulation
Hyperglycemia
Messenger RNA
Blood Glucose
Western Blotting
Pregnancy
Polymerase Chain Reaction
GTP Phosphohydrolases
Guanine
Streptozocin
Gene Library
GTP-Binding Proteins
Recombinant Proteins
Northern Blotting
Open Reading Frames
Genes

Keywords

  • Diabetic nephropathy
  • End-stage renal disease
  • GTP binding protein
  • Neonate
  • Rap1b
  • Renal development

ASJC Scopus subject areas

  • Nephrology

Cite this

Identification of up-regulated Ras-like GTPase, Rap1b, by suppression subtractive hybridization. / Lin, S.; Chugh, S.; Pan, X.; Wallner, E. I.; Wada, Jun; Kanwar, Y. S.

In: Kidney International, Vol. 60, No. 6, 2001, p. 2129-2141.

Research output: Contribution to journalArticle

Lin, S. ; Chugh, S. ; Pan, X. ; Wallner, E. I. ; Wada, Jun ; Kanwar, Y. S. / Identification of up-regulated Ras-like GTPase, Rap1b, by suppression subtractive hybridization. In: Kidney International. 2001 ; Vol. 60, No. 6. pp. 2129-2141.
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AU - Wada, Jun

AU - Kanwar, Y. S.

PY - 2001

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N2 - Background. Diabetic nephropathy accounts for over 30% of the end-stage renal disease (ESRD). A number of defined mechanisms and molecules that are involved in its pathogenesis are known, while others remain to be identified. Methods. Suppression subtraction hybridization (SSH)-polymerase chain reaction (PCR) was employed to search for new genes that may be relevant to the pathogenesis of diabetic nephropathy during embryonic development, the time when the kidney is most susceptible to various forms of stress. A diabetic state was induced in pregnant mice at day-13 of gestation by administration of streptozotocin. The kidneys of newborn mice with blood glucose level >200 mg/dL were harvested, mRNA isolated and subjected to SSH-PCR. Several differentially expressed cDNA fragments with up-regulated expression were isolated. One of the cDNA fragments had homology with human Ras-like guanine 5′-triphosphate (GTPase), Rap1b gene. By utilizing the λZAP II mouse cDNA library and SMART™ RACE amplification, a full-length Rap1b cDNA was isolated. A recombinant protein was generated in pET15b bacterial expression system. An anti-Rap1b antibody was raised in rabbits by immunizing them with the fusion protein, and its specificity was confirmed by Western blot analysis. Results. Rap1b cDNA had an open reading frame of 552 bp with a predicted putative protein size of ∼21 kD. In vitro translation verified the authentication of the Rap1b cDNA clone. Northern blot analyses revealed a single ∼2.3 kb Rap1b mRNA transcript. Its expression was up-regulated in several tissues, including the kidney of newborn diabetic mice. The degree of up-regulation of Rap1b mRNA expression was proportional to the blood glucose levels. Western blot analyses confirmed the hyperglycemia-induced up-regulation of the Rap1b expression. In situ hybridization and immunofluorescence studies revealed that Rap1b was expressed in the inner medullary collecting tubules. During hyperglycemia, its expression was accentuated and extended into the outer medullary and cortical collecting tubules. Similar up-regulation of Rap1b was observed when embryonic kidneys, harvested at day-13 of gestation, were exposed to high glucose ambience. Conclusion. The data suggest that Rap1b, a GTP-binding protein that plays a critical role in various signaling intracellular events, is another molecule that may be relevant to the pathobiology of diabetic nephropathy.

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KW - Diabetic nephropathy

KW - End-stage renal disease

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KW - Neonate

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KW - Renal development

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