Podocyte Activation of NLRP3 Inflammasomes Contributes to the Development of Proteinuria in Lupus Nephritis

Abstract

Objective: Development of proteinuria in lupus nephritis (LN) is associated with podocyte dysfunction. The NLRP3 inflammasome has been implicated in the pathogenesis of LN. The purpose of this study was to investigate whether NLRP3 inflammasome activation is involved in the development of podocyte injury in LN.

Methods: A fluorescence-labeled caspase 1 inhibitor probe was used to detect the activation of NLRP3 inflammasomes in podocytes derived from lupus-prone NZM2328 mice and from renal biopsy tissues obtained from patients with LN. MCC950, a selective inhibitor of NLRP3, was used to treat NZM2328 mice. Proteinuria, podocyte ultrastructure, and renal pathology were evaluated. In vitro, sera from diseased NZM2328 mice were used to stimulate a podocyte cell line, and the cells were analyzed by flow cytometry.

Results: NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from patients with LN. Inhibition of NLRP3 with MCC950 ameliorated proteinuria, renal histologic lesions, and podocyte foot process effacement in lupus-prone mice. In vitro, sera from diseased NZM2328 mice activated NLRP3 inflammasomes in the podocyte cell line through the production of reactive oxygen species.

Conclusion: NLRP3 inflammasomes were activated in podocytes from lupus-prone mice and from LN patients. Activation of NLRP3 is involved in the pathogenesis of podocyte injuries and the development of proteinuria in LN.

Figure 1. Activation of NLRP3 inflammasome in podocytes of diseased NZM2328 mice

A, Representative Western blot bands (left) and relative expression levels in each group (right) showed the expression of NLRP3 and caspase-1 p20, normalized to the values of GAPDH and caspase-1 in the kidneys of 36-week-old NZM2328 mice versus 12-week-old NZM2328 mice. B, Levels of IL-1β in renal extracts of NZM2328 mice. Glomeruli were isolated from NZM2328 mice and single cell suspensions were prepared. Flow cytometry analysis was used to assess the active caspase-1 levels in podocytes (C) and endothelial cells (D). Results are mean ± SD. *p<0.05, **p<0.01, NS = no significance, n=3 per group.

Figure 2. Activation of NLRP3 inflammasome in podocytes of human LN biopsies

Human kidney biopsies were collected and prepared for immunofluorescence staining. Anti- synaptopodin Ab (red), FAM-FLICA Caspase-1 assay kit (green) and mAb anti-human IL-1β (green) were used to detect expression of active caspase-1 and IL-1β in podocytes in the human kidneys. (A) Normal kidney isolated from non-disease area of nephrectomy specimen from a patient with renal cell cancer. (B) Class IV LN biopsy and (C) Class V LN biopsy. Original magnification ×400.

Figure 3. Activated NLRP3 inflammasome in urine podocytes

Fresh urine was collected and cells were spun onto a slide by a Cytospin. They were fixed and stained with anti- synaptopodin Ab (red), FAM-FLICA Caspase-1 assay kit (green) and mAb anti-human IL-1β (green) to detect expression of active caspase-1 and IL-1β in urine podocytes. DAPI (blue) was used to stain nuclei. (A) Expression of caspase 1 in the urinary podoycyte in a patient with LN but not in that of a normal individual. (B) Detection of IL-1β in the urine podocytes in a patient with LN but not from a normal control. Original magnification ×630.

Figure 4. MCC950 inhibited activation of NLRP3 inflammasome in NZM2328 mice and reduced IL-1β production

Following 6 weeks’ treatment of MCC950, mouse kidneys were collected for the following analysis. A–C, the kidneys were subjected to Western blot (representative bands shown) for expression of caspase 1 and its active form caspase-1 p20. D, levels of IL-1β in kidney extract determined by ELISA. E, anti-synaptopodin Ab (red) and FAM-FLICA Caspase-1 assay kit were used to detect caspase-1 expression (green) in glomerular podocytes, cell nuclei were marked by DAPI (blue). Original magnification ×400. After 2 weeks’ treatment, flow cytometry analysis was used to assess the active caspase-1 levels in podocytes (F) and endothelial cells (G). Results are mean ± SD. *P<0.05, **P<0.01, n=8 per group.

Figure 5. MCC950 treatment reduced renal lesions and proteinuria in NZM2328 mice

A, Severity of proteinuria (recorded weekly). B, Transmission electron microscopy was used to assess podocytes foot process. Original magnification ×5800 (left) and ×26500 (right). C, Renal sections were stained with PAS to assess features of glomerulonephritis. Original magnification ×400. D, Histopathological scores were recorded. *P<0.05, **P<0.01, n=8 per group.

Figure 6. Serum from diseased NZM2328 mice activate NLRP3 inflammasome in podocytes

The NZM2328 podocyte cell line was stimulated with a control serum or a serum from diseased NZM2328 mice or serum from diseased NZM2328 mice depleted of IgG, with or without MCC950 or Mito TEMPO for 24 hours. A, Representative Western blot (left) and relative expression levels (right) in each group showed the expression of caspase-1 p20, normalized to the values of caspase-1. B, Levels of IL-1β in supernatant following different stimulation. Flow cytometry analysis was used to assess active caspase-1 levels (C), production of ROS (D) and mitochondrial membrane potential (E). F, Expression of P2X7R detected by Western blot. G, Caspase-1 p20 expression analysis followed stimulation with diseased serum or diseased serum depleted of IgG. Results were mean ± SD. *p<0.05, **p<0.01, NS = no significance.