Supplementary Material for: Vitamin D Receptor Activators Upregulate and Rescue Podocalyxin Expression in High Glucose-Treated Human Podocytes

Background: Vitamin D is beneficial in human and
experimental chronic kidney disease, the leading cause of which is
diabetic nephropathy. Vitamin D through its receptor, VDR, provides
renal protection in diabetic nephropathy, but limited data exist about
its effect on podocytes. Renal podocytes form the main filtration
barrier possessing a unique phenotype maintained by proteins including
podocalyxin and nephrin, the expression of which is suppressed in
pathological conditions. Methods: We used immortalized
human podocytes (human glomerular epithelial cells, HGEC) to assess
podocalyxin and nephrin expression after treatment with
1,25-dihydroxyvitamin D3 (calcitriol) and its analogue
paricalcitol. The involvement of VDR was investigated by silencing with
hVDR-siRNA and ChIP analysis. Results: HGEC exhibit high
glucose-mediated downregulation of podocalyxin and nephrin, loss of
which has been linked with loss of the permselective renal barrier and
proteinuria. Calcitriol and paricalcitol reversed high glucose-induced
decrease of nephrin and significantly enhanced podocalyxin expression in
podocytes cultured in high glucose. HGEC express VDR and retinoid X
receptor (RXR). In the presence of calcitriol and paricalcitol, VDR
expression was upregulated and VDR colocalized with RXR in the nucleus.
VDR knockdown abolished the protective action of calcitriol and
paricalcitol on podocalyxin expression indicating that podocalyxin
activation of expression is partly mediated by VDR. Furthermore, VDR
specifically regulates podocalyxin expression by bounding to a site
upstream of the podocalyxin promoter. Conclusion: Vitamin D
analogues maintain and, furthermore, re-activate the expression of
specialized components of podocytes including podocalyxin, hence they
provide protection against loss of the permselective renal barrier, with
molecular mechanisms elucidated herein.