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Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF₁₆₅b) mRNA and protein

¹Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, University of Bristol, Bristol BS2 8EJ; ³Childrens' and ⁴Academic Renal Unit, University of Bristol, and ⁵Bristol Urological Institute, Southmead Hospital, Bristol BS10 5NB, United Kingdom; and ²Institute of Nephrology, First Teaching Hospital, University of Beijing, Beijing 10034, Peoples' Republic of China

Submitted 22 September 2003 ; accepted in final form 21 November 2003

Despite production by podocytes of the proangiogenic molecule vascular endothelial growth factor-A (VEGF), the glomeruli are not sites of angiogenesis. We recently described mRNA expression of an inhibitory splice variant of VEGF (VEGF₁₆₅b) in normal kidney (Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, Peat D, Gillatt D, and Harper SJ. Cancer Res 62: 4123–4131, 2002). Available anti-VEGF antibodies do not distinguish stimulatory from inhibitory VEGF families. To assess the production of VEGF₁₆₅ (stimulatory) and VEGF₁₆₅b (inhibitory) isoforms by human podocytes, we examined both primary cultured and conditionally immortalized human podocytes using family- and isoform-specific RT-PCR. In addition, VEGF protein production was analyzed in podocytes, using isoform-specific double-strand small-interference RNAs (siRNA). RT-PCR demonstrated the production of VEGF₁₈₉ mRNA by podocytes of both phenotypes. In contrast, on differentiation there was a splicing change from VEGF₁₆₅ to VEGF₁₆₅b mRNA. In addition, VEGF protein in the supernatant of conditionally immortalized, differentiated podocytes was reduced by VEGF₁₆₅b siRNA to 20 ± 11% of the level of mock-transfected cells (P < 0.01). No reduction was seen with mismatch siRNA. Moreover, there was no reduction in VEGF protein concentration in the supernatant of primary cultured, dedifferentiated human podocytes (109 ± 8% of mismatch siRNA, P > 0.1). In conclusion, differentiated but not dedifferentiated human podocytes secrete significant amounts of VEGF₁₆₅b protein. It is possible that this may explain the paradox of high VEGF production in the glomerulus but no angiogenesis. Furthermore, the existence of this splicing switch in relation to podocyte phenotype suggests that alternative splicing of the VEGF pre-RNA is a regulated process that is open to manipulation and therefore could be a target for novel cancer therapies.

angiogenesis; small-interference RNA; splicing

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