1. Volume 88, Issue 2, Pages 276-285 (August 2015)
Gluten exacerbates IgA nephropathy in humanized mice through gliadin–CD89 interaction 
Christina Papista, Sebastian Lechner, Sanae Ben Mkaddem, Marie-Bénédicte LeStang, Lilia Abbad, Julie Bex-Coudrat, Evangéline Pillebout, Jonathan M. Chemouny, Mathieu Jablonski, Martin Flamant, Eric Daugas, François Vrtovsnik, Minas Yiangou, Laureline Berthelot, Renato C. Monteiro 
Kidney International 
Volume 88, Issue 2, Pages (August 2015)
DOI: /ki
Copyright © 2015 International Society of Nephrology Terms and Conditions

2. Figure 1
Gluten induces mesangial deposition of IgA1 in α1KI-CD89Tg mice. From top to bottom: detection of human IgA1+, mouse TGase2+, and mouse TfR1+ cells by immunohistochemistry in frozen kidney sections from α1KI-CD89Tg versus α1KI-CD89Tg G- and α1KI-CD89Tg G+ mice. Right: number of positive cells per glomerulus counted in 20 randomly chosen fields for each mouse at magnification of × 200.n=3–4 mice per group. Bars represent the mean. **P<0.01 using the nonparametric Mann–Whitney’sU-test. Bars = 5 μm. IgA, immunoglobulin A.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

3. Figure 2
Gluten is required for complement deposition, glomerular inflammation, and hematuria. (a) From top to bottom: immunostaining for mouse C3+, CD11b+, and CD3+ cells in frozen kidney sections from α1KI-CD89Tg versus α1KI-CD89Tg G- and α1KI-CD89Tg G+ mice. Right: numbers of positive cells per glomerulus counted in 20 randomly chosen fields for each mouse at magnification of × 200.n=3–4 mice per group. Bars represent the mean. Bars = 5 μm. (b) mRNA levels of IL-6 normalized to GAPDH mRNA levels in kidney cortex of α1KI-CD89Tg, α1KI-CD89Tg G-, and α1KI-CD89Tg G+ mice, as detected by quantitative PCR. (c) Hematuria ( × 10,000 red blood cells/ml) measured in the urine samples of α1KI-CD89Tg, α1KI-CD89Tg G-, and α1KI-CD89Tg G+ mice.n=6–15 mice per group. *P<0.05, **P<0.01 and ***P<0.001 by Mann–Whitney’sU-test. IL-6, interleukin 6.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

4. Figure 3
Gluten is involved in IgA1–sCD89 complex formation. (a) Total IgA1 detected by ELISA in serum HPLC fractions of one representative α1KI-CD89Tg, one α1KI-CD89Tg fed during three generations in gluten-free diet (G-), and one α1KI-CD89Tg challenged with gluten (G+) mouse (n=4–5 mice per group). Detection of IgA1–sCD89 complexes in these fractions by ELISA showed that they corresponded to high-molecular-weight IgA1 fractions in α1KI-CD89Tg and α1KI-CD89Tg G+ mice. (b) Areas indicate high-molecular-mass complexes based on HPLC profiles, as measured by ImageJ. (c) IgA1 levels in the serum of α1KI-CD89Tg, α1KI-CD89Tg G-, and α1KI-CD89Tg G+ mice, as measured by ELISA.n=5–34 mice per group. (d) Surface staining of CD89 on blood monocytes from α1KI-CD89Tg compared with G- mice. The graphs show the mean fluorescent intensity of CD89 on CD11b+ cells.n=7–20 mice per group. (e) Detection of IgA1–sCD89 complexes in kidney eluates from α1KI-CD89Tg G- and α1KI-CD89Tg G+ mice by ELISA using a monoclonal anti-CD89 (A3) and a polyclonal anti-human IgA antibody.n=7 mice per group. Data represent the±S.E.M. *P<0.05 (Mann–Whitney’sU-test). ELISA, enzyme-linked immunosorbent assay; HPLC, high-performance liquid chromatography; IgA, immunoglobulin A.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

5. Figure 4
Gliadin directly interacts with sCD89. (a) Binding of sCD89 to gliadin, compared with blank and human serum albumin used as negative controls, as detected by ELISA using biotinylated sCD89. (b) Dose-dependent binding of sCD89 to gliadin.n=3 experiments. ELISA, enzyme-linked immunosorbent assay.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

6. Figure 5
Gluten is implicated in oral tolerance breakdown in IgAN. (a andb) Immunostaining for the detection of IgA1+ (a), F4-80+ (b, top), and CD3+ (b, bottom) cells in the epithelium, lamina propria, and crypts in frozen intestine sections of α1KI-CD89Tg G- and α1KI-CD89Tg G+ versus α1KI-CD89Tg mice. The histograms show the corresponding number of positive cells per field counted in 10 fields per mouse at magnification of × 200 (n=3 mice per group). Bars = 20 μm. (c) Hematoxylin and eosin (HE) staining of the jejunal region of α1KI-CD89Tg, α1KI-CD89Tg G-, and α1KI-CD89Tg G+ mice (original magnification × 200;n=3 mice per group). (d) Morphometric evaluation of villus height:crypt depth (V:C) ratio at HE-stained longitudinal sections of the small intestine of α1KI-CD89Tg, α1KI-CD89Tg G-, and α1KI-CD89Tg G+ mice (original magnification × 100,n=3 mice per group). The graphs represent the mean±s.e.m. *P<0.05 and ***P<0.001 using the nonparametric Mann–Whitney’sU-test. IEL, intraepithelial lymphocytes; IgAN, Immunoglobulin A nephropathy; LPL, lamina propria lymphocytes.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

7. Figure 6
Gliadin induces circulating IgA anti-gliadin antibodies (AGA), which correlate with proteinuria. (a) Detection of IgA1 AGA by ELISA in the sera of α1KI compared with α1KI-CD89Tg mice. For the detection of these antibodies, gliadin was used for coating and monoclonal anti-human IgA as a secondary reagent.n=7–12 mice per group. Bars represent the mean of optical density (OD). (b) Correlation between IgA1 AGA in the serum and proteinuria in α1KI-CD89Tg mice.n=11 mice. (c) The presence of IgA AGA in the sera of IgAN patients (n=26) compared with healthy individuals (n=28), as shown by ELISA. For the detection of these antibodies, gliadin was used for coating and monoclonal anti-human IgA as a secondary reagent. Bars represent the mean of OD. (d) Correlation between circulating IgA AGA and proteinuria in IgAN patients.n=26. **P<0.01 by Mann–Whitney’sU-test. ELISA, enzyme-linked immunosorbent assay; IgA, immunoglobulin A nephropathy.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

8. Figure 7
Early-stage treatment of α1KI-CD89Tg mice with gluten-free diet abolishes IgAN development. (a) Experimental design of treatment with gluten-free diet: α1KI-CD89Tg mice fed with standard gluten-containing diet were treated or not for 2, 6, or 9 weeks with a gluten-free diet and killed at 12 weeks of age. (b) Hematuria ( × 10,000 red blood cells/ml) measured in the urines of α1KI-CD89Tg mice treated or not with gluten-free diet.n=6–15 mice per group. *P<0.05, **P<0.01 and ***P<0.001 by Mann–Whitney’sU-test. (c) Immunostaining for human IgA1 in frozen kidney sections from α1KI-CD89Tg versus α1KI-CD89Tg mice bred in a gluten-free diet for 2, 6, or 9 weeks or three generations (magnification × 200).n=3-5 mice per group. Bars = 5 μm. The graph represents the the mean±S.E.M. ratio of the marked area/glomerular area as measured by ImageJ. *P<0.05, **P<0.01, and ***P<0.001 using the nonparametric Mann–Whitney’sU-test. IgAN, Immunoglobulin A nephropathy.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions