Vitamin D3 ameliorates podocyte injury through the nephrin signalling pathway

Abstract

Renal podocytes form the main filtration barrier possessing unique phenotype maintained by proteins including podocalyxin and nephrin, which are modulated in pathological conditions. In diabetic nephropathy (DN), podocytes become structurally and functionally compromised. Nephrin, a structural backbone protein of the slit diaphragm, acts as regulator of podocyte intracellular signalling with renoprotective role. Vitamin D₃ through its receptor, VDR, provides renal protection in DN but limited data exist about its effect on podocytes. In this study, we used isolated rat glomeruli to assess podocalyxin and nephrin expression after treatment with the 1,25-dihydroxyvitamin D₃ analogue paricalcitol in the presence of normal and diabetic glucose levels. The role of 1,25-dihydroxyvitamin D₃ (calcitriol) and its analogue, paricalcitol, on podocyte morphology and survival was also investigated in the streptozotocin (STZ)-diabetic animal model. In our ex vivo model, glomeruli exhibited high glucose-mediated down-regulation of podocalyxin, and nephrin, while paricalcitol reversed the high glucose-induced decrease of nephrin and podocalyxin expression. Paricalcitol treatment enhanced VDR expression and promoted VDR and RXR co-localization in the nucleus. Our data also indicated that hyperglycaemia impaired survival of cultured glomeruli and suggested that the implemented nephrin down-regulation was reversed by paricalcitol treatment, initiating Akt signal transduction which may be involved in glomerular survival. Our findings were further verified in vivo, as in the STZ-diabetic animal model, calcitriol and paricalcitol treatment resulted in significant amelioration of hyperglycaemia and restoration of nephrin signalling, suggesting that calcitriol and paricalcitol may provide molecular bases for protection against loss of the permselective renal barrier in DN.

Keywords: Diabetic nephropathy; STZ; Vitamin D3; Vitamin D3 Receptor; glomerulus; high glucose; nephrin; paricalcitol; podocalyxin.

Figure 1

VDRA increased the expression levels of Nephrin, PODXL and VDR in isolated rat glomeruli. (A) Western blot analysis in isolated rat glomeruli cultured in 5 mM or 25 mM glucose in presence of 150 nM or 300 nM VDRA (paricalcitol) for (i) nephrin, (ii) PODXL and (iii) VDR. (B) Western blot analysis in isolated rat glomeruli cultured in 25 mM glucose or in 25 mM glucose in presence of VDRA for nephrin, PODXL and VDR. Western blot quantification was performed using ImageJ software, and the results were normalized to the tubulin positive control and then to isolated rat glomeruli cultured in 5 mM glucose. The values are presented as the mean ± S.D. of three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001 Student's t‐test).

Figure 2

VDRA induced nuclear translocation of VDR and co‐localization with RXR. Representative confocal microscopy images of triple staining for Nephrin (green), VDR (red), RXR (blue) and DAPI (grey) in isolated rat glomeruli cultured in 5 mM glucose, 25 mM glucose and in presence of 300 nM VDRA. Original magnifications, 63×.

Figure 3

Nephrin and PI3K are co‐immunoprecipitated in isolated rat glomeruli lysate. (A) Representative confocal microscopy images of double staining for Nephrin (green), PI3K (red) and DAPI (blue) in isolated rat glomeruli cultured in 5 mM glucose, 25 mM glucose and in presence of VDRA. Original magnifications, 63×. (B) Immunoprecipitation (IP) with anti‐nephrin in isolated rat glomeruli cultured in (i) 5 mM glucose, (ii) 5 mM glucose in presence of VDRA and (iii) 25 mM glucose in presence of VDRA. No interactions were found with anti‐CBP, serving as negative control. P, precipitate; S, supernatant.

Figure 4

Paricalcitol enhanced Akt phosphorylation. (A)Representative confocal microscopy images of double staining for nephrin (green), pAkt (red) and DAPI (blue) in isolated rat glomeruli cultured in 5 mM glucose, 25 mM glucose and in presence of VDRA. Original magnifications, 63×. (B) Western blot analysis of pAkt/Akt in isolated rat glomeruli cultured in 5 mM glucose, 25 mM glucose and in presence of VDRA. Western blot quantification was performed using ImageJ software, and the results were normalized to the tubulin positive control and then to isolated rat glomeruli cultured in 5 mM glucose. The values are presented as the mean ± S.D. of three independent experiments (*P < 0.05; **P < 0.01 Student's t‐test).

Figure 5

Determination of apoptosis in isolated rat glomeruli by TUNEL assay. (A) Representative fluorescent images of TUNEL‐positive cells in isolated rat glomeruli cultured in 5 mM glucose, 25 mM glucose and in presence of VDRA. Original magnifications, 63×. (B) Western blot analysis of cleaved PARP in isolated rat glomeruli cultured in 5 mM glucose, 25 mM glucose and in presence of VDRA. (C) Western blot analysis of activated caspase‐3 in isolated rat glomeruli cultured in 5 mM glucose, 25 mM glucose and in presence of VDRA. Western blot quantification was performed using ImageJ software, and the results were normalized to the tubulin positive control and then to isolated rat glomeruli cultured in 5 mM glucose. The values are presented as the mean ± S.D. of three independent experiments (*P < 0.05; **P < 0.01; ***P < 0,001 Student's t‐test).

Figure 6

STZ model animals. Rats were randomly divided into four groups: control (C) (n = 5), STZ diabetic (DN) (n = 5), STZ rats treated with 400 ng/kg paricalcitol daily (DN‐P) (n = 5) and STZ rats treated with 100 ng/kg calcitriol daily (DN‐C) (n = 5). (A) Blood glucose levels (mg/ml) of control (C) and STZ‐diabetic animals (DN). (B) (i) Kidney weight/Bodyweight, (ii) Representative images of kidneys of control, STZ and diabetic animals after in vivo treatment with paricalcitol (DN‐P) or calcitriol (DN‐C). (C) Volume of urine 24 hrs (D) (i) urine nitrogen (g/l), (ii) urine creatinine (g/l). (E) Representative images of sirius red staining of C, DN, DN‐P and DN‐C animals. Original magnifications, 40× and 10×.

Figure 7

In vivo assessment of nephrin and VDR expression levels. Western blot analysis in isolated rat glomeruli after 5 weeks in vivo treatment with paricalcitol or calcitriol for (A) nephrin and (B) VDR. Western blot quantification was performed using ImageJ software, and the results were normalized to the positive control and then to isolated rat glomeruli from control animals, respectively. The values are presented as the mean ± S.D. of three independent experiments (*P < 0.05; ***P < 0.001 Student's t‐test).

Figure 8

Molecular basis of VDRA beneficiary action through the nephrin signalling pathway. VDRA treatment enhances VDR expression and promotes VDR and RXR co‐localization in the nucleus, leading to increased nephrin expression, which participates in PI3K‐mediated Akt activation thus promoting glomerular survival.