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Updated: Aug 30, 2026
Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions
Published on: July 2, 2020
Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor
Tai-Gen Cui1, Rebecca R Foster, Moin Saleem
1Microvascula Research Laboratories, Dept. of Physiology, Preclinical Veterinary School, Univ. of Bristol, Southwell St., Bristol BS2 8EJ, UK. Dave.Bates@bris.ac.uk
Abstract:
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.
Insights
Differentiated human podocytes produce inhibitory vascular endothelial growth factor-165b (VEGF165b) protein, explaining the lack of glomerular angiogenesis despite VEGF production. This suggests VEGF alternative splicing is a target for cancer therapies.
Area of Science:
- Nephrology
- Molecular Biology
- Cancer Biology
Background:
- Podocytes produce proangiogenic vascular endothelial growth factor-A (VEGF), yet glomeruli lack angiogenesis.
- Existing anti-VEGF antibodies cannot differentiate between stimulatory and inhibitory VEGF isoforms.
- VEGF165b, an inhibitory VEGF splice variant, is expressed in normal kidney tissue.
Purpose of the Study:
- To determine if human podocytes produce both stimulatory (VEGF165) and inhibitory (VEGF165b) VEGF isoforms.
- To investigate the role of podocyte differentiation in regulating VEGF isoform production.
- To explore the therapeutic potential of targeting VEGF alternative splicing in cancer.
Main Methods:
- Family- and isoform-specific RT-PCR was used to analyze VEGF mRNA expression in primary and immortalized human podocytes.
- Isoform-specific small-interference RNAs (siRNA) were employed to assess VEGF protein production.
- VEGF protein levels in podocyte supernatants were quantified following siRNA treatment.
Main Results:
- RT-PCR confirmed VEGF189 mRNA production in both podocyte phenotypes.
- Differentiated podocytes exhibited a shift in splicing from VEGF165 to VEGF165b mRNA.
- VEGF165b siRNA significantly reduced VEGF protein in differentiated podocyte supernatants (to 20% of control, P < 0.01), while dedifferentiated cells showed no significant change.
Conclusions:
- Differentiated human podocytes secrete substantial amounts of inhibitory VEGF165b protein.
- VEGF165b secretion may explain the absence of glomerular angiogenesis despite high VEGF levels.
- VEGF alternative splicing is a regulated process in podocytes, presenting a potential target for novel cancer therapies.
