Pathogenic Role of a Proliferation-Inducing Ligand (APRIL) in Murine IgA Nephropathy

Abstract

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) superfamily. Despite advances in clinical and genetic studies, the details of the pathological roles of APRIL in IgA nephropathy (IgAN) remain to be fully defined. The present study aimed to further assess the pathological role of APRIL using a mouse model of IgAN. Mice with IgAN designated "grouped ddY" (gddY) were intraperitoneally administered an anti-APRIL monoclonal antibody (anti-APRIL Ab) or control IgG (Control Ab) twice each week for 2 weeks starting during the early stage of IgAN (6-7 weeks of age). Urinary albumin, serum IgA, and glomerular IgA deposition were evaluated. We further assessed the inflammatory responses during treatment by measuring the levels of the chemokine fractalkine (FKN) and its receptor CX3CR1 as well as the level of peripheral blood monocytosis. Anti-APRIL Ab treatment significantly decreased albuminuria and tissue damage combined with decreases in serum IgA levels and deposition of glomerular IgA. In contrast, the abundance of IgA+/B220+ or CD138+/B220+ B cells in the spleen and bone marrow, respectively, was unchanged. Treating gddY mice with anti-April Ab reduced the overexpression of FKN/CX3CR1 in the kidney and the increase in the population of circulating Gr1-/CD115+ monocytes. The size of the population of Gr1-/CD115+ monocytes correlated with renal FKN and urinary albumin levels. Moreover, mice treated with anti-APRIL Ab exhibited reduced progression of IgAN, serum IgA levels, and glomerular IgA deposition as well as an attenuated inflammatory process mediated by FKN-associated activation of monocytes. To the best of our knowledge, this is the first study to implicate the APRIL signal transduction pathway in the pathogenesis of nephrogenic IgA production. Moreover, our findings identify APRIL as a potential target of therapy.

Fig 1. Effects of APRIL blockade on albuminuria, serum IgA levels, and B cell populations in spleen and BM.

(A) The urine ACR of the anti-ARPIL Ab group differed significantly from that of the Control Ab group after 2 weeks (D14) of treatment (*p < 0.05 vs. Control Ab group). (B) The change in serum IgA levels was different from that for controls on days 7 and 14 (D7 and D14) (*p < 0.01 for D7 and p < 0.001 for D14 vs. Control Ab group). (C, D) The populations of (C) PB (CD138⁺/B220⁺) and PC (CD138⁺/B220⁻) and (D) IgA-secreting PBs (IgA⁺/B220⁺) and IgA-secreting PCs (IgA⁺/B220⁻) did not differ between the spleen and BM of each group.

Fig 2. Anti-APRIL Ab ameliorates histopathological alterations and glomerular IgA deposition.

(A) The pathological images of the Control Ab group (left) and anti-APRIL Ab group (right) were obtained after PAS staining (original magnification, ×200). (B) Histological analysis revealed significant attenuation of glomerular and tubulointerstitial changes in the anti-APRIL Ab group compared with the Control Ab group. (C) Representative immunofluorescence images show glomerular IgA and IgG deposition in the Control Ab group (left) and the anti-APRIL Ab group (right) (original magnification, ×400). (D) Densitometric analyses show significantly less deposition of IgA, but not IgG, in the anti-APRIL Ab group compared with the Control Ab group *p < 0.05.

Fig 3. APRIL blockade reduces glomerular monocyte/macrophage infiltration.

(A) Immunofluorescence analysis of F4/80 expression in the glomeruli of the Control Ab group (left) and the anti-APRIL Ab group (right) (original magnification, ×400). (B) The average number of infiltrated monocytes/macrophages per glomerulus was calculated from more than 30 glomeruli. The number of infiltrated glomerular monocytes/macrophages was decreased in the anti-APRIL Ab group. *p < 0.05.

Fig 4. APRIL blockade is associated with attenuated renal FKN and CX3CR1 expression.

(A) Real-time PCR analysis shows that renal FKN/CX3CR1 mRNA levels in 6-week-old untreated type BB gddY mice were higher than those in age-matched HIGA and Balb/c mice. (*vs. HIGA, ^†vs. Balb/c; p < 0.01 for kidney FKN and p < 0.05 for kidney CX3CR1). In contrast, spleen CX3CR1 mRNA expression was similar in gddY, HIGA, and Balb/c mice (mRNAs levels were normalized to those of GAPDH). (B) Real-time PCR analysis of kidney samples shows that after 2 weeks of treatment, FKN and CX3CR1 mRNAs levels in the anti-APRIL Ab group were lower than those in the controls (*p < 0.05 vs. Control Ab group).

Fig 5. APRIL blockade affects the abundance of Gr1⁻/CD115⁺ cells among PBMCs.

(A) The numbers of total monocytes, Gr1⁺/CD115⁺ monocytes, and Gr1⁻/CD115⁺ monocytes in peripheral blood were measured using flow cytometry. The percentages of Gr1⁺ and Gr1⁻ cells among PBMCs from 3 different age-matched strains of untreated mice are compared. The percentage of Gr1⁻/CD115⁺ monocytes was increased in type BB gddY mice compared with HIGA and Balb/c mice (*p < 0.001 vs. HIGA; ^†p < 0.001 vs. Balb/c). HIGA mice had a higher percentage of Gr1⁻/CD115⁺ monocytes than Balb/c mice (^‡p < 0.01 vs. Balb/c). The size of the population of Gr1⁺/CD115⁺ monocytes was similar between type BB gddY and Balb/c mice but was significantly lower in HIGA mice (*p < 0.001 vs. gddY, ^‡p < 0.01 vs. Balb/c). (B) After 2 weeks (D14) of APRIL Ab treatment, the difference (D14/D0) in monocyte numbers was decreased significantly only in the Gr1⁻/CD115⁺ monocyte population (*p < 0.05 vs. Control Ab group).

Fig 6. Percentage of Gr1⁻/CD115⁺ monocytes correlates with renal FKN levels and degree of albuminuria.

The percentage of Gr1⁻/CD115⁺ monocytes in all mice (anti-APRIL Ab group + Control Ab group) correlated positively with (A) renal FKN mRNA expression and (B) degree of albuminuria according to Spearman rank correlation.