Disruption of Smad4 expression in T cells leads to IgA nephropathy-like manifestations

Abstract

The link between glomerular IgA nephropathy (IgAN) and T helper 2 (Th2) response has been implicated, however, the mechanisms are poorly defined because of the lack of an appropriate model. Here we report a novel murine model characterized by lineage-restricted deletion of the gene encoding MAD homologue 4 (Smad4) in T cells (Smad4(co/co;Lck-cre) ). Loss of Smad4 expression in T cells results in overproduction of Th2 cytokines and high serum IgA levels. We found that Smad4(co/co;Lck-cre) mice exhibited massive glomerular IgA deposition, increased albumin creatinine ratio, aberrant glycosylated IgA, IgA complexed with IgG1 and IgG2a, and polymeric IgA, all known features of IgAN in humans. Furthermore, we examined the β1, 4-galactosyltransferases (β4GalT) enzyme which is involved in the synthesis of glycosylated murine IgA, and we found reduced β4GalT2 and β4GalT4 mRNA levels in B cells. These findings indicate that Smad4(co/co;Lck-cre) mice could be a useful model for studying the mechanisms between IgAN and Th2 response, and further, disruption of Smad4-dependent signaling in T cells may play an important role in the pathogenesis of human IgAN and contributing to a Th2 T cell phenotype.

Figure 1. Loss of Smad4 signaling in T cells induces increased Th2 cytokine production by T cells.

The levels of (A) IL-4 and (C) IL-13 in T cell supernatant were significantly increased in Smad4^{co/co;Lck-cre} mice (n = 10) compared with WT mice (n = 10). Smad4^{co/co;Lck-cre} mice tended to have higher (B) IL-5 levels than WT mice (P = 0.066). Data are expressed as the mean±SEM; P values were calculated with Student's t test.*P<0.05

Figure 2. Levels of Th2 cytokines in serum of Smad4^{co/co;Lck-cre} mice.

The levels of IL-4, IL-5, and IL-13 in the serum from Smad4^{co/co;Lck-cre} (n = 7) and WT (n = 7) mice are were determined by ELISA. No significant differences were observed between the two groups. Data are expressed as the mean±SEM; P values were calculated with Student's t test.

Figure 3. Loss of Smad4 signaling in T cells induces high serum levels of IgA.

The serum levels of IgA, IgM, IgG1, and IgG2a were determined using ELISA. (A) Markedly increased serum levels of IgA in Smad4^{co/co;Lck-cre} mice (n = 8) compared with WT mice (n = 8) (1773±294.3 vs 545.2±71.39). The results from serum levels of (B) IgM, (C) IgG1, and (D) IgG2a showed no significant differences between the two groups. Data are expressed as the mean±SEM; P values were calculated with Student's t test. ***P< 0.001

Figure 4. Deletion of Smad4 in T cell induced predominant IgA deposition in glomeruli and proteinuria.

(A) Immunofluorecence studies showed glomerular deposition of predominantly IgA, with lesser amounts of IgG and IgM deposition in Smad4^{co/co;Lck-cre} mice compared with WT mice at 3 months of age. C3 deposition was occasionally observed in both mesangial areas and Bowman's capsule in Smad4^{co/co;Lck-cre} mice, but was restricted to Bowman's capsule in WT mice. PAS staining showed no significant difference between WT and Smad4^{co/co;Lck-cre} mice. Original magnification; ×400 (B) Electron microscopic overview (left panels) and detail of podocytic foot processes (right panels) for Smad4^{co/co;Lck-cre} (bottom panels) and WT (top panels) mice. Podocyte foot process effacement was partially observed in glomeruli from Smad4^{co/co;Lck-cre} mice compared to WT mice. Scale bars: 5 µm (left panels); 0.5 µm (right panels). (C) Increased albumin creatinine ratio (ACR) was observed in urines of Smad4^{co/co;Lck-cre} mice (n = 9) compared with WT mice (n = 9) (0.043±0.005 vs 0.083±0.007). (D) There is no significant difference in the urinary hemoglobin between WT (n = 10) and Smad4^{co/co;Lck-cre} mice (n = 10) (14.7± 1.94 vs 21.2±2.36). Data are expressed as the mean±SEM; P values were calculated with Student's t test. ***P< 0.001

Figure 5. Characteristics of circulating IgA in mice lacking Smad4 in T-cells are similar to those in patients with IgA nephropathy.

(A and B) Aberrant glycosylation of circulating IgA from Smad4^{co/co;Lck-cre} mice (n = 6, black square) and WT mice (n = 6, black triangle) was examined by lectin ELISA. Both (A) sialic acid and (B) galactose on circulating IgA from the mutants were lower than those from controls. Data are expressed as the ratios of specific lectin-binding IgA to total IgA. (C and D) Smad4^{co/co;Lck-cre} mice (n = 6, black square) exhibit an increased in both (C) IgG1-IgA complexes and (D) IgG2a-IgA complexes in the circulation, compared with WT mice (n = 6, black triangle). Data are expressed as mean±SEM; P values were calculated with t test or Bonferroni post test. ***P<0.001, **P<0.01, and *P<0.05 (E) The predominance of polymeric IgA was observed in serum from Smad4^{co/co;Lck-cre} mice, but not in WT mice, as determined by Western blot.

Figure 6. RT-PCR analysis of β4GalT mRNAs in Smad4^{co/co;Lck-cre} mice.

mRNA was isolated from purified B-cells (>95% CD19+), and RT-PCR was performed using primers specific for β4GalT1 to 7, and GAPDH. β4GalT 2 and 4 mRNA expression in Smad4^{co/co;Lck-cre} mice (n = 4, black column) were significantly reduced relative to controls (n = 4, white column). All samples were normalized to GAPDH levels. Data are expressed as mean±SEM; P values were calculated with t test. *P<0.05