Protective Role of the Podocyte IL-15 / STAT5 Pathway in Focal Segmental Glomerulosclerosis

Abstract

Introduction: During glomerular diseases, podocyte-specific pathways can modulate the intensity of histological disease and prognosis. The therapeutic targeting of these pathways could thus improve the management and prognosis of kidney diseases. The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway, classically described in immune cells, has been recently described in detail in intrinsic kidney cells.

Methods: We describe STAT5 expression in human kidney biopsies from patients with focal segmental glomerulosclerosis (FSGS) and studied mice with a podocyte-specific Stat5 deletion in experimental glomerular diseases.

Results: Here, we show, for the first time, that STAT5 is activated in human podocytes in FSGS. In addition, podocyte-specific Stat5 inactivation aggravates the structural and functional alterations in a mouse model of FSGS. This could be due, at least in part, to an inhibition of autophagic flux. Finally, interleukin 15 (IL-15), a classical activator of STAT5 in immune cells, increases STAT5 phosphorylation in human podocytes, and its administration alleviates glomerular injury in vivo by maintaining autophagic flux in podocytes.

Conclusion: Activating podocyte STAT5 with commercially available IL-15 represents a potential new therapeutic avenue for FSGS.

Keywords: IL-15; autophagy; cell signaling; cytokines; focal segmental glomerulosclerosis; podocyte.

Graphical abstract

Figure 1

STAT5 activation in podocytes in human focal segmental glomerulosclerosis. Representative images of phospho-STAT5B immunostaining in human kidney biopsies from patients with HIVAN, non-HIVAN collapsing, noncollapsing FSGS and pristine 3 months posttransplant kidney biopsy as a control. Scale bar, 50μm. Higher magnifications are shown in the insets. Each microphotograph corresponds to an individual patient’s biopsy. Arrows represent specific nuclear staining. FSGS, focal segmental glomerulosclerosis.

Figure 2

Podocyte STAT5 deficiency aggravates adriamycin-induced albuminuria and glomerular injury. (a) Western blot analysis of STAT5 expression in primary cultured podocytes from glomeruli isolated from 10-week-old Stat5^lox/lox and Nphs2.cre-Stat5^lox/lox mice. Beta-actin was used as loading control. Quantification of western blot bands for STAT5 normalized to Beta-actin band intensity. ∗∗∗P < 0.001 (b) Urinary albumin-to-creatinine ratio in Stat5^lox/lox and Nphs2.cre-Stat5^lox/lox mice at basal state and 7 days after adriamycin injection. Individual values are shown, and the bars correspond to means. ∗P < 0.05 and ∗∗∗P < 0.001 (c) Representative images of Masson’s trichrome stained sections (upper panels, scale bar: 20μm), transmission electron micrographs (second row panels, scale bar 2μm), nephrin staining (third row panels, scale bar, 20 μM), CD3 staining (fourth row panels, scale bar 20μm) and F4/80 staining (fifth row panels, scale bar 20 μm) of the renal cortex from Stat5^lox/lox and Nphs2.cre-Stat5^lox/lox mice 7 days after the injection of adriamycin. Arrow indicates glomerulosclerosis observed in optical microscopy and ∗ indicates podocyte feet effacement and glomerular basement membrane enlargement observed with EM in Nphs2.cre-Stat5^lox/lox mice. (d) Quantification of nephrin immunostaining, CD3 and F4/80 positive cells in kidneys from Nphs2.cre-Stat5^lox/lox mice compared to Stat5^lox/lox mice at day 7 after adriamycin injection. Individual values are shown, and the bars correspond to means. ∗P < 0.05. ∗∗P < 0.01.

Figure 3

Podocyte STAT5 deficiency aggravates proteinuria and glomerular lesions in anti-glomerular basement membrane experimental glomerulonephritis. (a) Urine protein-to-creatinine ratio at day 0, 4, and 9 after anti-GBM serum injection in Stat5^lox/lox and Nphs2.cre-Stat5^lox/lox (n = 7 to 9 mice per group). Data are mean±SEM. ∗P < 0.05. (b) Plasma BUN in Stat5^lox/lox and Nphs2.cre-Stat5^lox/lox mice after vehicle or anti-GBM injection. Individual values are shown, and the bars correspond to the means. ∗∗∗P < 0.001, (c) Representative images of Masson’s trichrome- (upper panel), Martius Scarlett Blue-stained sections showing glomerular fibrin deposits (middle panel) and transmission electron micrographs (lower panel) of renal cortex from 10–12-week-old anti-GBM-glomerulonephritis-induced Stat5^lox/lox and Nphs2.cre-Stat5^lox/lox mice and controls. Scale bar, 20 μM for upper panel, 20 μm for middle panel and 2 μm for lower panel (d) Quantification of tubulointerstitial damage in kidneys from Stat5^lox/lox and Nphs2.cre-Stat5^lox/lox mice at day 9 after anti-GBM serum injection. Individual values are shown, and the bars correspond to the means. ∗P < 0.05. (e) Quantification of fibrin glomerular deposits in kidneys from Stat5^lox/lox and Nphs2.cre-Stat5^lox/lox mice at day 9 after anti-GBM serum injection. Individual values are shown, and the bars correspond to the means. ∗P < 0.05.

Figure 4

Interleukin-15 activates STAT5 in podocytes and alleviates glomerular injury. (a)Western blot analysis of the expression of phospho-STAT5B and STAT5 in human podocyte cell line after IL-15 stimulation at different time points. (b) Scheme representing experiment details (c) RT-qPCR quantification of the expression of Stat5a and Stat5b in the kidneys from 12-week-old BALB/C mice on day 7 after adriamycin injection with or without IL-15 treatment. Individual values are shown, and the bars correspond to the means. ∗∗P < 0.01. (d) Western blot analysis of the expression of phospho-STAT5 and STAT5 in the kidneys from 12-week-old BALB/C mice at day 7 after adriamycin injection with or without IL-15 treatment and its quantification. Individual values are shown, and the bars correspond to the means. (e) Urine albumin-to-creatinine ratio at day 0, day 5, and day 7 after adriamycin injection in BALB/C mice treated or not with IL-15. The data represent means±SEM. ∗P < 0.05. ∗∗P < 0.01. (f) Representative images of Masson trichrome (upper panel; scale bar: 20μM) and transmission electron micrographs (lower panel; scale bar: 2 μm) of kidneys from IL-15 treated and nontreated mice at day 7 after adriamycin treatment. ∗shows podocyte feet effacement and glomerular basement membrane thickening.

Figure 5

Modulation of the autophagic flux by STAT5 and IL-15 in vitro and in vivo. (a) Scheme of CRISPR-Cas9 mediated STAT5B knock out in human podocytes. (b) Western blot analysis of the abundance of STAT5B in human podocytes with or without STAT5B deficiency. The stain-free gel serves as normalization. (c) Western blot analysis of the abundance of LC3B-II in human podocytes with or without STAT5B deficiency. Tubulin expression serves as normalization. Podocytes were treated or not treated with bafilomycin A1 (BafA1; 100 nM) for 4 hours before culture arrest. (d) Immunofluorescence of Podocalyxin (PODXL; red), a podocyte marker, and P62/SQSTM1 (green) in glomeruli from wild-type mice at day 7 after adriamycin injection with or without IL-15 treatment showing the accumulation of P62 in podocytes during adriamycin model and decreased by IL-15 treatment. Nuclei were counterstained with Hoechst (blue). Figure subparts with prime indicate higher magnification. Bars = 50 mm. (e) Associated quantification of the P62+ area expressed as the percentage of the P62/PODXL positive area. Values are presented as individual plots and mean±SEM.