Anti-PR3 antibodies cause kidney disease.

<p>(A–C) PAS stained images of glomeruli from chimera mice 6 days after injection with anti-PR3 (n = 18, A, 400×; C, 600×) or control IgG (n = 8, B, 600×). Note extra-capillary proliferation and peri-glomerular inflammation (arrowhead) (A), and mesangiolysis (C, arrow) in anti-PR3 treated mice. (D–F) H & E stained sections of kidney from chimera mice treated with anti-PR3 (D, 40×) or disease control (E, 40×) IgG. There are regions of tubulointerstitial injury, with red cell cast formation (arrow). (F) Demonstrates intense peri-glomerular inflammation in an animal treated with anti-PR3 IgG (arrowhead, 400×). By comparison mice treated with disease control IgG showed minimal glomerular or tubulointerstitial changes. (G) Fractions of glomeruli affected in anti-PR3 (n = 18) and control IgG (n = 8) treated animals (Error bars depict SEM; ***<i>p</i> = 0.001) (H). Degree of tubulointerstitial disease in mice treated with anti-PR3 antibodies and control antibodies (*<i>P</i><0.05, median ± IQ ± max/min values). (Bars = 50 µm).</p

Characterization of chimerism in NOD-<i>scid</i>-<i>IL2Rγ^−/−</i> mice.

<p>(A–D) Flow cytometric analysis of leukocytes from tail bleeds six weeks after administration of HSCs (n = 26 mice). (A) Plots showing mouse leukocytes labelled with anti-mouse CD45 antibodies. Compared with control wild-type mouse blood, chimeras have populations of mCD45 negative leukocytes that show SSC characteristics of granulocytes (High), monocytes (Int) and lymphocytes (low). (B) Chimera blood leukocytes express human CD45 and many of these express CD11b. hCD45+,CD11b+ leukocytes predominantly express hCD15 and hCD66b compared with hCD45+,CD11b− leukocytes shown in histograms. (C) A proportion of hCD45+ leukocytes express CD19. (D) Some hCD45 leukocytes are CD14^high and some are CD16+,CD14^low. (E) In chimera bone marrow there are CD11b+ leukocytes which do not express mCD45 and among hCD45+ leukocytes a proportion express CD14 and a proportion express CD66b. (F) In chimera spleen there are CD11b+ leukocytes which express hCD45 and among hCD45+ leukocytes many express both CD14 and CD16. (G) Bone marrow spreads from wild type or chimera mice, labelled with anti-hMPO or anti-hPR3 IgG antibodies (red) purified from patients with vasculitis. Note that chimera bone marrow demonstrates anti-hMPO or anti-hPR3 antibody positive leukocytes with characteristic human neutrophil nuclear morphology. Wild type mouse bone marrow shows no cells positive for these antigens indicating that the anti-human antibodies do not cross react with mouse neutrophils.</p

Anti-PR3 antibodies induce infiltration of kidneys with leukocytes of murine and human origin.

<p>Kidney sections were incubated with anti-mCD45 (red) and anti-hCD45 (green) antibodies and images were captured by fluorescence microscopy (T = tubule). Occasional (<5%) glomeruli of anti-PR3 treated mice displayed intense extracapillary leukocyte infiltration (A) in the shape of crescents (arrows). Most glomeruli in animals treated with anti-PR3 antibodies (n = 18) had evidence of intraglomerular (B,G) and peri-glomerular (C,G) leukocyte infiltration. These were comprised mostly of mCD45+ cells, although some hCD45 leukocytes were also present (arrowheads). In addition, there was a significant increase in peri-vascular leukocyte (mCD45+ and hCD45+) infiltration in anti-PR3 treated mice (D,G [per arteriolar section (art.sec.)]). Sections were also stained for deposition of IgG [red] (E,G) and C3 [green] (F,G). IgG was detectable within periglomerular cells, but there was minimal deposition within the glomeruli. Mouse C3 was weakly deposited in glomeruli but was no different between control group (n = 8) and anti-PR3 group (n = 18). Note mouse C3 can be detected normally binding avidly to tubular basement membranes. (Marker = 10 µm) (*<i>P</i><0.05, **<i>P</i><0.01. median ± IQ ± max/min values). (H) Kidney sections from anti-PR3 and control treated animals were incubated with anti-PR3 positive ANCA IgG. In the peritubular capillaries of chimera mice that received anti-PR3 hIgG occasional leukocytes detected by anti-hPR3 hIgG could be detected. No positively stained human neutrophils were seen in glomeruli.</p

Characterization of the chimeric immune system.

<p>Blood, bone marrow and splenic digests were analysed by flow cytometry to define the leukocyte populations (n = 26 mice). Treatment groups were matched by degree of peripheral blood chimerism prior to IgG injection. hMono = human monocytes. There was no significant difference in degree of chimerism or human granulocyte reconstitution between the experimental groups.</p

The GAGs effectively reduced heparanase-1 expression in the glomeruli of treated DM rats.

<p>From A–D, immunofluorescence photomicrographs of heparanase-1–stained renal glomerular sections with an evident increase in expression in the DM (B) animals that was prevented by FCS (C) and ENX (D) administration, as confirmed by a semiquantitative scoring system (E). The data represent the means ± SEMs. *<i>p<</i>0.0001 vs. groups; #<i>p<</i>0.01 vs. ENX. Bar = 25 µm.</p

Gene expression of proteoglycan core proteins in DM and GAGs treated groups related to control.

<p>Gene expression of: agrin, the major GBM proteoglycan; perlecan and collagen XVIII, predominantly mesangial proteoglycans; decorin, a small rich-leucine proteoglycan and glypican-1, a cell suface associated proteoglycan. Gray columns, DM; dark gray, FCS and black, ENX. *<i>p<</i>0.01 vs. control; #<i>p<</i>0.05 vs. DM.</p

Functional parameters at the end of the experiment.

<p>The results are presented as the means ± SEMs. BWt, body weight; RWt, renal weight; SBP, systolic blood pressure; SCr, serum creatinine; eGFR, estimated glomerular filtration rate; DM, diabetic group; FCS, diabetic group treated with fucosylated chondroitin sulfate; ENX, diabetic group treated with enoxaparin;</p><p>*all of the diabetic groups vs. control;</p>#<p>DM vs. FCS and ENX groups;</p><p>**<i>p<</i>0.01 DM vs. control.</p><p>Functional parameters at the end of the experiment.</p

Gene expression analysis for glycosaminoglycan modifying/degrading enzymes in DM and GAGs treated groups related to control.

<p>Gray columns, DM; dark gray, FCS and black, ENX. *<i>p<</i>0.05 vs. control. HPSE, heparanase-1; CS, chondroitin sulfate; NDST-1, N-deacetylase/N-sulfotransferase; HS-3-ST-1, heparan sulfate-3-O-sulfotransferase 1.</p

Morphological aspects of the mesangial axis and tubulointerstitial area in the experimental animals at the end of the study.

<p>A–D show PAS-stained glomerular photomicrographs with a significant increase in the mesangial area in the DM group (B) compared to the control group (A) and no increase in the DM groups treated with FCS (C) or ENX (D). In E, a semi-quantitative analysis demonstrated a 1.4-fold increase in the mesangial area in the DM group compared to the other groups. In F–I, PAS-stained renal sections show expansion of the interstitial area with tubular dilation in the DM group (G), compared to the control animals (F) and GAG-treated groups (H and I). In J, the semiquantitative analysis shows a discrete but significant expansion in the DM group. In L–N, the Sirius Red staining area shows a trend toward increased deposition of collagen fibers in the interstitial area in the DM animals (M) compared to the other groups but without statistical significance, as shown in (O). In N–P, Masson’s trichrome staining depicts the expansion of the interstitial area in the DM rats due to edema and tubular dilation compared to the treated groups. The data represent the means ± SEMs. *<i>p<</i>0.001, **<i>p<</i>0.0001. A–N, bar = 25 µm; N–P, bar = 50 µm.</p