Caspase-1, but Not Caspase-3, Promotes Diabetic Nephropathy

Abstract

Glomerular apoptosis may contribute to diabetic nephropathy (dNP), but the pathophysiologic relevance of this process remains obscure. Here, we administered two partially disjunct polycaspase inhibitors in 8-week-old diabetic (db/db) mice: M-920 (inhibiting caspase-1, -3, -4, -5, -6, -7, and -8) and CIX (inhibiting caspase-3, -6, -7, -8, and -10). Notably, despite reduction in glomerular cell death and caspase-3 activity by both inhibitors, only M-920 ameliorated dNP. Nephroprotection by M-920 was associated with reduced renal caspase-1 and inflammasome activity. Accordingly, analysis of gene expression data in the Nephromine database revealed persistently elevated glomerular expression of inflammasome markers (NLRP3, CASP1, PYCARD, IL-18, IL-1β), but not of apoptosis markers (CASP3, CASP7, PARP1), in patients with and murine models of dNP. In vitro, increased levels of markers of inflammasome activation (Nlrp3, caspase-1 cleavage) preceded those of markers of apoptosis activation (caspase-3 and -7, PARP1 cleavage) in glucose-stressed podocytes. Finally, caspase-3 deficiency did not protect mice from dNP, whereas both homozygous and hemizygous caspase-1 deficiency did. Hence, these results suggest caspase-3-dependent cell death has a negligible effect, whereas caspase-1-dependent inflammasome activation has a crucial function in the establishment of dNP. Furthermore, small molecules targeting caspase-1 or inflammasome activation may be a feasible therapeutic approach in dNP.

Keywords: apoptosis; diabetic nephropathy; immunology; renal protection.

Figure 1.

Differential effect of caspase inhibitors M-920 and CIX on diabetic nephropathy in db/db mice. (A) Albuminuria and extracellular matrix accumulation, as reflected by the fractional mesangial area (FMA), are decreased in M-920-treated db/db mice (+M-920; targeting caspases 1, 3, 4, 5, 6, 7, and 8) as compared with PBS-treated db/db mice (db/db). Treatment with CIX (+CIX; targeting caspases 3, 6, 7, 8, and 10) has no effect on these markers in db/db mice. Treatment was initiated at age 8 weeks and continued for 12 weeks. Mean value±SEM. At least six mice in each group. (B) FMA; periodic acid–Schiff–stained glomeruli (size bar: 20 µm). (C) FMA; mean value±SEM. *P<0.05. alb, albumin; crea, creatinine; db/m, nondiabetic control; ns, not significant.

Figure 2.

M-920 prevents inflammasome activation in addition to glomerular cell death. (A, B) Both caspase inhibitors (M-920 and CIX) decrease glomerular cell death in db/db mice as detected by the TUNEL assay. (A) Representative TUNEL-stained glomeruli (size bar: 20 µm Higher magnification (top) of glomeruli indicated by black dotted lines and TUNEL-positive cells indicated by black arrowheads. (C, D) Both M-920 and CIX decrease caspase-3/7 activity, as detected by the FLICA assay. However, only M-920, but not CIX, also reduces caspase-1 activity (C, E). (F) In db/db mice treated with M-920, but not in those treated with CIX, the cleaved forms of IL-1β and IL-18 and expression of Nlrp3 are reduced in renal cortex extracts. (G) Renal inflammasome inhibition in M-920-treated db/db mice is associated with lower plasma IL-1β levels. (B, D–G) Mean value±SEM of at least six mice in each group; (C) representative images of frozen sections incubated with FLICA probes – overviews (size bar: 20 µm) and higher (4×) magnification of glomeruli (glomeruli indicated by white dotted circles); (F) representative immunoblots; *P<0.05. cl-Casp1, cleaved caspase-1; cl-Casp3/7, cleaved caspase-3/7; cl-IL-1β, cleaved IL-1β; cl-IL-18, cleaved IL-18; DAPI, 4′,6-diamidino-2-phenylindole; db/m, nondiabetic control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPF, high-power field; ns, not significant; P-IL-1β, plasma-IL-1β.

Figure 3.

Inflammasome and apoptosis markers are differentially regulated in dNP and glucose-treated podocytes in vitro. We screened the Woroniecka cohort in the Nephromine database (human dNP) for glomerular expression of inflammasome- and apoptosis-related genes. (A) Only the inflammasome-related regulators, caspase-1 (CASP1), IL-18, ASC (PYCARD), and Nlrp3 (NLRP3), but not the apoptosis-related genes, caspase-3 (CASP3), caspase-7 (CASP7), and PARP-1 (PARP1) are significantly induced in microdissected glomeruli of patients with diabetes with dNP (h-glomerular) as compared with nondiabetic controls. Likewise, significant induction of inflammasome-related genes (CASP1, IL-18, and IL-1β for db/db C57BLKS mice, and CASP1 and IL-18 for eNOS^−/− db/db mice), but not of apoptosis-related genes (CASP3, CASP7, and PARP1), is observed within the Nephromine Hodgin dataset as compared with nondiabetic db/m mice or eNOS^+/+ m/m mice. The fold changes of the log2 median centered intensity from the Nephromine database (Life Technologies, Ann Arbor, MI) are used for graphical representation. High glucose (25 mM; [B, C]), but not mannitol (25 mM; [B]), induces the inflammasome markers Nlrp3 and cleaved caspase-1 within 3 hours in murine podocytes in vitro, preceding the induction of cleaved PARP1 (12 hours), cleaved caspase-3 (24 hours), and cleaved caspase-7 (48 hours; [C]). (B) Representative immunoblots of at least three repeat experiments; (C, E) mean value±SEM; *P<0.05. cl-Casp1, cleaved caspase-1; cl-Casp3, cleaved caspase-3; cl-Casp7, cleaved caspase-7; cl-PARP1, cleaved PARP1.

Figure 4.

Caspase-3 deficiency fails to protect against diabetic nephropathy. (A) Compared with nondiabetic wild-type control mice (WT C), STZ induced diabetic mice (WT DM) show markedly increased albuminuria, and (B, C) increased fractional mesangial area (FMA). (A–C) Caspase-3 deficiency (Casp3^−/−) fails to reduce albuminuria and the FMA in diabetic mice, whereas homozygous (Casp1^−/−) or hemizygous (Casp1^+/−) caspase-1 deficiency is protective. (D) IL-1β and (E) IL-18 plasma levels and (F) renal IL-1β/IL-18 expression are unaffected in caspase-3^−/− mice, but are reduced in caspase-1^+/−, and reduced to a larger extent in caspase-1^−/− mice. (A, C–F) Mean value±SEM; at least six mice in each group; (B) representative periodic acid–Schiff–stained glomeruli (size bar: 20 µm); (F) representative agarose-gels are shown; *P<0.05. alb, albumin; C, control; crea, creatinine; ns, not significant.