Involvement of the NLRC4-Inflammasome in Diabetic Nephropathy

Abstract

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease worldwide but current treatments remain suboptimal. The role of inflammation in DN has only recently been recognized. It has been shown that the NLRP3-inflammasome contributes to DN development by inducing interleukin (IL)-1β processing and secretion. In an effort to understand other IL-1β activating mechanism during DN development, we examined the role of the NLRC4-inflammasome in DN and found that NLRC4 is a parallel mechanism, in addition to the NLRP3-inflammasome, to induce pro-IL-1β processing and activation. We found that the expression of NLRC4 is elevated in DN kidneys. NLRC4-deficiency results in diminished DN disease progression, as manifested by a decrease in blood glucose and albumin excretion, as well as preserved renal histology. We further found that DN kidneys have increased F4/80+ macrophages, increased IL-1β production, and other signaling pathways related to kidney pathology such as activation of NF-κB and MAP kinase pathways, all of which were rescued by NLRC4-deficiency. This study demonstrates NLRC4-driven IL-1β production as critical for the progression of DN, which underscores the importance to target this pathway to alleviate this devastating disease.

Fig 1. Increased expression of NLRC4 and macrophage infiltration in renal tissues of DN patients.

(A): PAS staining showing GBM thickening and mesangial expansion in renal biopsies in DN patients (Aa vs. Ab, Scale Bar: 200 μm). IHC studies revealed increased expression of NLRC4 and CD68 in DN patients (Ac-Af, Scale Bar: 100 μm). (B): Averaged relative intensity for the staining of NLRC4 (top panel) and CD68 (bottom panel) in kidney biopsies of control versus DN patients. (C and D): Blood glucose levels and serum creatinine (SCR) in control and DN patients. Values are means ± s.e.m. Con: control patient; DN: diabetic patient. *P<0.05.

Fig 2. NLRC4-deficiency ameliorates hyperglycemia and renal morphological changes in mice.

A-C. Mice were fed an HFD for 4 weeks then injected with STZ and monitored for 8 weeks. Blood glucose levels (A), body weight changes (B), and urinary albumin to creatinine ratio (ACR) (C) at different time pointes after STZ treatment were measured from normal WT, diabetic WT and diabetic Nlrc4^-/- mice. D. Mice were killed 8 weeks after STZ treatment and the kidneys were harvested. Kidney sections were stained with hematoxylin and Eosin staining (H&E) and PAS staining (magnification×400). *, P<0.05 vs. control WT; #, P <0.05 vs. diabetic WT. n = 6 for all these experiments. Scale bar: 100 μm.

Fig 3. NLRC4-inflammasome activation in the renal tissues of DN mice.

(A) ELISA analysis of IL-1β levels in kidney tissues from different groups including normal WT, diabetic WT and diabetic Nlrc4^-/- mice. Values are means ± s.e.m. n = 3. (B) The mRNA levels of Nlrc4 and Casp-1 in renal tissues from different groups by real time PCR analysis, using Gapdh as a reference gene. Values are mean fold change compared to control mice ± s.e.m. *, P<0.05 vs. control WT; #, P <0.05 vs. diabetic WT. n = 3 each group. (C) Western blot analyses of cleaved Casp-1 p10 and p20 subunits in renal lysates from different groups. n = 3 each group. (D) Protein densitometry analysis of C to show the processing and activation of pro-casp1 into p20 and p10 subunits. Data represents mean protein densitometry in pixels and normalized to the corresponding Tubulin, quantitated by Image J ± s.e.m. n = 3.

Fig 4. NLRC4-deficiency reduces kidney macrophage accumulation in the DN mice.

(A-C). Flow cytometric analysis of CD45⁺ leukocytes (A) and CD11b⁺F4/80⁺ macrophages (B-C) in the renal tissues from different groups. (B). Representative images for flow cytometry for CD11b⁺F4/80⁺ macrophages from different groups. (C). Statistics for total numbers (left panel) and percentages (right panel) of macrophages from B. Values are means ± s.e.m. *, P<0.05 vs. control WT; #, P <0.05 vs. diabetic WT. (D). Immunofluorescent staining showed for F4/80⁺ macrophages in the renal interstitial space from the indicated mice. n = 6 for all these experiments.

Fig 5. NLRC4-deficiency leads to decreased signaling pathways related to DN pathology.

(A). The mRNA fold change of Mcp1, Ccl3, Ccl4, and Cxcl1 in the kidney tissues was determined by real-time PCR, using Gapdh as a reference gene, from the indicated groups. Data is presented as the average fold change compared to control ± s.e.m. n = 3–4. (B). Western blot showing altered activation of different signaling pathways including NF-κB and JNK pathways in the renal tissues from different groups. Images represent two independent experiments from a total of 3 kidneys within each indicated groups. (C). The mRNA fold change of Tnfα, Tgfβ, and Ctgf in the renal tissues was determined by real-time PCR, using Gapdh as a reference gene. Data is presented as the average fold change compared to controls ± s.e.m. n = 3. *, P<0.05 vs. control WT; #, P <0.05 vs. diabetic WT. n = 3.