A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors

Peter R. Corridon, George J. Rhodes, Ellen C. Leonard, David Basile, Vincent H. Gattone, Robert Bacallao, Simon J. Atkinson

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Gene therapy has been proposed as a novel alternative to treat kidney disease. This goal has been hindered by the inability to reliably deliver transgenes to target cells throughout the kidney, while minimizing injury. Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys. We show, using intravital fluorescence two-photon microscopy, that fluorescent albumin and dextrans injected into the renal vein under defined conditions of hydrodynamic pressure distribute broadly throughout the kidney in live animals. We found injection parameters that result in no kidney injury as determined by intravital microscopy, histology, and serum creatinine measurements. Plasmids, baculovirus, and adenovirus vectors, designed to express EGFP, EGFP-actin, EGFP-occludin, EGFP-tubulin, tdTomato-H2B, or RFP-actin fusion proteins, were introduced into live kidneys in a similar fashion. Gene expression was then observed in live and ex vivo kidneys using two-photon imaging and confocal laser scanning microscopy. We recorded widespread fluorescent protein expression lasting more than 1 mo after introduction of transgenes. Plasmid and adenovirus vectors provided gene transfer efficiencies ranging from 50 to 90%, compared with 10-50% using baculovirus. Using plas-mids and adenovirus, fluorescent protein expression was observed 1) in proximal and distal tubule epithelial cells; 2) within glomeruli; and 3) within the peritubular interstitium. In isolated kidneys, fluorescent protein expression was observed from the cortex to the papilla. These results provide a robust approach for gene delivery and the study of protein function in live mammal kidneys.

Original languageEnglish
JournalAmerican Journal of Physiology - Renal Physiology
Volume304
Issue number9
DOIs
StatePublished - 2013

Fingerprint

Plasmids
Kidney
Gene Expression
Transgenes
Adenoviridae
Renal Veins
Baculoviridae
Hydrodynamics
Proteins
Photons
Actins
Occludin
Injections
Wounds and Injuries
Kidney Diseases
Tubulin
Dextrans
Confocal Microscopy
Genetic Therapy
Genes

Keywords

  • Confocal laser scanning microscopy
  • Fluorescent protein expression in kidney cortex and medulla
  • Hydrodynamic plasmid and adenovirus transgene delivery
  • Intravital two-photon fluorescence microscopy

ASJC Scopus subject areas

  • Physiology
  • Urology

Cite this

A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors. / Corridon, Peter R.; Rhodes, George J.; Leonard, Ellen C.; Basile, David; Gattone, Vincent H.; Bacallao, Robert; Atkinson, Simon J.

In: American Journal of Physiology - Renal Physiology, Vol. 304, No. 9, 2013.

Research output: Contribution to journalArticle

Corridon, Peter R. ; Rhodes, George J. ; Leonard, Ellen C. ; Basile, David ; Gattone, Vincent H. ; Bacallao, Robert ; Atkinson, Simon J. / A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors. In: American Journal of Physiology - Renal Physiology. 2013 ; Vol. 304, No. 9.
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