JAK-STAT signaling is activated in the kidney and peripheral blood cells of patients with focal segmental glomerulosclerosis

Abstract

Focal segmental glomerular sclerosis (FSGS) is a devastating disease with limited treatment options and poor prognosis. Activated JAK-STAT signaling has been implicated in other kidney diseases. Since new technologies allow us to better evaluate changes in systemic and renal JAK-STAT activity as it relates to kidney function, we examined this in 106 patients with biopsy-proven FSGS compared to 47 healthy control individuals. Peripheral immune function was assessed in peripheral blood mononuclear cells by phosphoflow studies before and after cytokine stimulation. Kidney JAK-STAT activity was measured by immunofluorescence and by transcriptomics. A STAT1 activity score was calculated by evaluating message status of downstream targets of pSTAT 1. Peripheral blood mononuclear cells were found to be upregulated in terms of pSTAT production at baseline in FSGS and to have limited reserve to respond to various cytokines. Increased staining for components of the JAK-STAT system in FSGS by microscopy was found. Furthermore, we found transcriptomic evidence for activation of JAK-STAT that increased pSTAT 1 and pSTAT 3 in glomerular and tubulointerstitial sections of the kidney. Some of these changes were associated with the likelihood of remission of proteinuria and progression of disease. JAK-STAT signaling is altered in patients with FSGS as compared to healthy controls with activated peripheral immune cells, increased message in the kidney and increased activated proteins in the kidney. Thus, our findings support immune activation in this disease and point to the JAK-STAT pathway as a potential target for treatment of FSGS.

Keywords: cell signaling; focal segmental glomerulosclerosis; gene expression; lymphocytes.

Copyright © 2018. Published by Elsevier Inc.

Figures

Figure 1.

Work flow of FSGS JAK-STAT evaluation

Figure 2.

Unstimulated phosphoflow. Baseline of pSTAT1 (2A), pSTAT3 (2B), and pSTAT5 (2C) in different cell subsets of B lymphocytes, CD4+, CD4+CD45RA+, CD4+CD45RA-, CD8+, CD8+CD45RA+, CD8+CD45RA-, NK cells, and monocytes in FSGS were compared with controls. * compared with control, p

Figure 3.

Changes of pSTAT1, and pSTAT3 of B lymphocytes, CD4+, CD4+CD45RA+, CD4+CD45RA-, CD8+, CD8+CD45RA+, CD8+CD45RA-, NK cells, and monocytes stimulated by IFNα, IFNγ, IL-6, IL-7, IL-10, IL-2, and IL-21 were compared between FSGS and control. P values were determined by Wilcoxon rank sum test. P values lower than 0.05 were filtered and shown in a heatmap format (left panel). Direction of increased or decreased STAT phosphorylation in comparison to controls is represented by blue or red. Correlation analysis between 24 hour urine protein excretion, or serum creatinine with any fold change described above were conducted and the four significant correlations that overlapped with the significant change of pSTAT1 are shown in Figure 3A–D. Detailed raw data in change of pSTAT1, and pSTAT3 in any cell subset under any stimulant between control and FSGS were expressed as Mean±SEM in supplement file on request. P values from those comparisons are also provided in supplement file on request.

Figure 3.

Changes of pSTAT1, and pSTAT3 of B lymphocytes, CD4+, CD4+CD45RA+, CD4+CD45RA-, CD8+, CD8+CD45RA+, CD8+CD45RA-, NK cells, and monocytes stimulated by IFNα, IFNγ, IL-6, IL-7, IL-10, IL-2, and IL-21 were compared between FSGS and control. P values were determined by Wilcoxon rank sum test. P values lower than 0.05 were filtered and shown in a heatmap format (left panel). Direction of increased or decreased STAT phosphorylation in comparison to controls is represented by blue or red. Correlation analysis between 24 hour urine protein excretion, or serum creatinine with any fold change described above were conducted and the four significant correlations that overlapped with the significant change of pSTAT1 are shown in Figure 3A–D. Detailed raw data in change of pSTAT1, and pSTAT3 in any cell subset under any stimulant between control and FSGS were expressed as Mean±SEM in supplement file on request. P values from those comparisons are also provided in supplement file on request.

Figure 4.

Representative images of IHC stain of JAK1, JAK2, pSTAT1, STAT3 and pSTAT3 in control kidney and FSGS patient. A: Control JAK1, B: Control JAK2, C: Control pSTAT1, D: Control STAT3, E: Control pSTAT3, F: FSGS JAK1, G: FSGS JAK2, H: FSGS pSTAT1, I: FSGS STAT3, J: FSGS pSTAT3. (200×) K: Control JAK1, L: Control JAK2, M: Control pSTAT1, N: Control STAT3, O: Control pSTAT3, P: FSGS JAK1, Q: FSGS JAK2, R: FSGS pSTAT1, S: FSGS STAT3, T: FSGS pSTAT3. (400×)

Figure 5.

Semi-quantification of the JAK2, pSTAT1, STAT3, and pSTAT3 immunohistochemistry staining in FSGS patients (n=9). Healthy non-neoplastic kidney dissected from tumor nephrectomy was used as control. For JAK2 (A), the scoring was used as 0: none; +1 75% cells positive with a strong intensity.

Figure 6.

Differentially expressed transcripts in FSGS were enriched in the JAK-STAT pathway in the tubulointerstitium. (A) Signaling network nodes highlighted in purple indicate those genes that were differentially expressed in FSGS relative to living donor controls, while shading of the nodes indicates up-regulation (red) or down-regulation (green) in FSGS relative to living donor controls. (B) Upstream regulator analysis identified a predicated activation of IFNG and STAT1 in the tubulointerstitium with evidence supporting activation of STAT1 indicated (C).

Figure 7.

Differentially expressed transcripts in FSGS were enriched in the JAK-STAT pathway in the glomeruli. (A) Signaling network nodes highlighted in purple indicate those genes that were differentially expressed in FSGS relative to living donor controls, while shading of the nodes indicates up-regulation (red) or down-regulation (green) in FSGS relative to living donor controls. (B) Predicated activation of STAT related signaling nodes in the glomeruli with evidence supporting activation of STAT1 indicated (C). Network supporting STAT1 activation in the glomeruli. Orange and blue connections indicate that STAT1 can induce or inhibit expression of downstream genes. These findings are consistent with STAT1 activation. Yellow connections indicate findings that are inconsistent with STAT1 activation, while grey lines are findings that neither support nor reject the predicated activation of STAT1.

Figure 8.

Dual IHC stain of pSTAT1 (red) and CD3 (grey) in 12 FSGS patient from NEPTUNE cohort according to isolated glomerular genetic analysis results for JAK1, JAK2 or STAT1expression. A and G: two FSGS patients with low glomerular expression of JAK1; B and H: two FSGS patients with high glomerular expression of JAK1; C and I: two FSGS patients with low glomerular expression of JAK2; D and J: two FSGS patients with high glomerular expression of JAK2; E and K: two FSGS patients with low glomerular expression of STAT1; F and L: two FSGS patients with high glomerular expression of STAT1 (200×). A1 to L1 are corresponding images for the cases labeled as A to L (200×) under higher magnification (400×).

Figure 9.

Correlation of JAK-STAT1 activation score from glomerular compartment with baseline eGFR (panel A) and baseline UPCR (panel B) and JAK-STAT activation score from tubular compartment with baseline eGFR (panel C) and baseline UPCR (panel D) in NEPTUNE patients.

Figure 10.

Adjusted Kaplan Meier survival curve for complete remission by tertile of JAK-STAT1 Activation z-score (Glomerular Compartment, panel A, p-value 0.14 and Tubular Compartment, panel B, p-value 0.08). Adjusted Kaplan Meier survival curve for Composite of 40% loss in eGFR and ESRD by tertile of JAK-STAT1 Activation z-score (Glomerular Compartment, panel C, p-value 0.41 and Tubular Compartment, panel D, p-value 0.02).