Simultaneous activation of innate and adaptive immunity participates in the development of renal injury in a model of heavy proteinuria

Abstract

Protein overload of proximal tubular cells (PTCs) can promote interstitial injury by unclear mechanisms that may involve activation of innate immunity. We investigated whether prolonged exposure of tubular cells to high protein concentrations stimulates innate immunity, triggering progressive interstitial inflammation and renal injury, and whether specific inhibition of innate or adaptive immunity would provide renoprotection in an established model of massive proteinuria, adriamycin nephropathy (ADR). Adult male Munich-Wistar rats received a single dose of ADR (5 mg/kg, iv), being followed for 2, 4, or 20 weeks. Massive albuminuria was associated with early activation of both the NF-κB and NLRP3 innate immunity pathways, whose intensity correlated strongly with the density of lymphocyte infiltration. In addition, ADR rats exhibited clear signs of renal oxidative stress. Twenty weeks after ADR administration, marked interstitial fibrosis, glomerulosclerosis, and renal functional loss were observed. Administration of mycophenolate mofetil (MMF), 10 mg/kg/day, prevented activation of both innate and adaptive immunity, as well as renal oxidative stress and renal fibrosis. Moreover, MMF treatment was associated with shifting of M from the M1 to the M2 phenotype. In cultivated NRK52-E cells, excess albumin increased the protein content of Toll-like receptor (TLR) 4 (TLR4), NLRP3, MCP-1, IL6, IL-1β, Caspase-1, α-actin, and collagen-1. Silencing of TLR4 and/or NLRP3 mRNA abrogated this proinflammatory/profibrotic behavior. Simultaneous activation of innate and adaptive immunity may be key to the development of renal injury in heavy proteinuric disease. Inhibition of specific components of innate and/or adaptive immunity may be the basis for future strategies to prevent chronic kidney disease (CKD) in this setting.

Keywords: NF-κB system; NLRP3 inflammasome; adaptive immunity; chronic kidney disease; proteinuria.

Figure 1. NLRP3 and NF-κB patwhays 2, 4 and 20 weeks after adriamycin injection

Renal protein content of TLR4 (A), nuclear p65 (B), IL-6 (C), interstitial NLRP3+ cells (D), caspase-1 (E), and IL-1β 2 (F) 2, 4, and 20 weeks after ADR injection; representative Western blot images appear at the bottom (G). C n=9, ADR_2wn=12, ADR_4wn=12, ADR_20wn=10. ANOVA ^aP<0.05 compared with C; ^bP<0.05 compared with ADR_2w; ^cp<0.05 compared with ADR_4w.

Figure 2. Linear correlation between albuminuria and renal NLRP3 or 1L-1β abundance

Linear correlation (Pearson’s correlation coefficient) between the intensity of albuminuria and the abundance of NLRP3 (A) and IL-1β (B). Additional correlations between albuminuria and parameters of inflammation and innate immunity activation are shown in Supplementary data.

Figure 3. Representative microphotographs showing detection by immunohistochemistry of TLR4, Caspase-1, NLRP3, and IL-1β, the predominant staining is located in the tubular and interstitium (renal tissue)

Figure 4. Markers of oxidative stress after adriamycin injection

Renal abundance of HO-1 (A) and SOD2 (B) 2, 4, and 20 weeks after ADR injection. Representative Western blot images appear at the bottom (C). C n=9, ADR_2wn=12, ADR_4wn=12, ADR_20wn=10. ANOVA ^aP<0.05 compared with C; ^bP<0.05 compared with ADR_2w; ^cP<0.05 compared with ADR_4w.

Figure 5. NLRP3, Casp-1 and IL-1β production by cultivated proximal tubular cells after exposure to BSA

Production of NLRP3 (A), Caspase-1 (B), and IL-1β (C) by cultivated PTCs exposed to BSA and transfected with either scramble or siRNA for NLRP3. Illustrative immunofluorescence microphotographs of NLRP3 (D) and IL-1β (E) in these cells are also shown, while representative Western blot images appear at the bottom. ANOVA ^aP<0.05 compared with C; ^bP<0.05 compared with BSA; ^cP<0.05 compared with scramble.

Figure 6. TLR4 and IL-6 production by cultivated proximal tubular cells after exposure to BSA

Production of TLR4 (A) and IL-6 (B) by cultivated PTCs exposed to BSA and transfected with either scramble or siRNA for TLR-4. Illustrative immunofluorescence microphotographs of TLR-4 (C) and IL-6 (D) in these cells are also shown. ANOVA ^aP<0.05 compared with C; ^bP<0.05 compared with BSA; ^cP<0.05 compared with scramble.

Figure 7. MCP-1, COLL-1 and α-SMA production by cultivated proximal tubular cells after exposure to BSA

Production of MCP-1 (A), collagen-1 (B), and α-SMA (D) by cultivated PTCs exposed to BSA and transfected with scramble, siRNA for NLRP3, or siRNA for TLR-4. Illustrative immunofluorescence microphotographs of α-SMA (E) in these cells are also shown, while representative Western blot images appear in (C). ANOVA ^aP<0.05 compared with C; ^bP<0.05 compared with BSA; ^cP<0.05 compared with scramble.

Figure 8. Effect of MMF treatment on renal injury and inflamation after adriamycin injection

Effect of MMF treatment on renal T-lymphocyte (CD3-positive) infiltration (A), albuminuria (B), percent glomerulosclerosis (C), cortical α-SMA (D), and collagen-1 deposition (E) 2, 4, and 20 weeks after ADR injection, n=10 per group. Student’s ‘t’ test; *P<0.05 compared with ADR.

Figure 9. Effect of MMF treatment on innate immunity after adriamycin injection

Effect of MMF treatment on interstitial abundance of NLRP3 (A), Caspase-1 (B), IL-1β (C), TLR4 (D), and nuclear NF-κB (E), with representative Western blot images (F). The renal content of HO-1 (G) and SOD2 (H), along with representative Western blot images (I) are also shown, n=10 per group. Student’s ‘t‘ test; *P<0.05 compared with ADR.

Figure 10. Linear correlation (Pearson’s correlation coefficient) between the intensity of T-lymphocyte (CD3-positive) infiltration and the percent cortical area staining for α-SMA (A) or collagen-1 (B)

Figure 11. Effect of MMF treatment on polarization machophages and content of IL-10 after adriamycin injection

Renal density of macrophages ED-1 (A), M2 macrophages (CD206+ cells) (B), percent M2 macrophages (C), and renal IL-10 abundance (D) 2, 4, and 20 weeks after ADR injection. Figure 11E shows representative microphotographs of M2 macrophage detection by immunohistochemistry in C and 20 weeks after ADR injection in untreated and MMF-treated rats, n=10 per group. Student’s ‘t’ test; *P<0.05 compared with ADR.