Autoimmunity in CD73/Ecto-5'-nucleotidase deficient mice induces renal injury

Abstract

Extracellular adenosine formed by 5'-ectonucleotidase (CD73) is involved in tubulo-glomerular feedback in the kidney but is also known to be an important immune modulator. Since CD73(-/-)mutant mice exhibit a vascular proinflammatory phenotype, we asked whether long term lack of CD73 causes inflammation related kidney pathologies. CD73(-/-)mice (13 weeks old) showed significantly increased low molecule proteinuria compared to C57BL6 wild type controls (4.8 ≥ 0.52 vs. 2.9 ± 0.54 mg/24 h, p<0.03). Total proteinuria increased to 5.97 ± 0.78 vs. 2.55 ± 0.35 mg/24 h at 30 weeks (p<0.01) whereas creatinine clearance decreased (0.161 ± 0.02 vs. 0.224 ± 0.02 ml/min). We observed autoimmune inflammation in CD73(-/-)mice with glomerulitis and peritubular capillaritis, showing glomerular deposition of IgG and C3 and enhanced presence of CD11b, CD8, CD25 as well as GR-1-positive cells in the interstitium. Vascular inflammation was associated with enhanced serum levels of the cytokines IL-18 and TNF-α as well as VEGF and the chemokine MIP-2 (CXCL-2) in CD73(-/-)mice, whereas chemokines and cytokines in the kidney tissue were unaltered or reduced. In CD73(-/-)mice glomeruli, we found a reduced number of podocytes and endothelial fenestrations, increased capillaries per glomeruli, endotheliosis and enhanced tubular fibrosis. Our results show that adult CD73(-/-)mice exhibit spontaneous proteinuria and renal functional deterioration even without exogenous stress factors. We have identified an autoimmune inflammatory phenotype comprising the glomerular endothelium, leading to glomeruli inflammation and injury and to a cellular infiltrate of the renal interstitium. Thus, long term lack of CD73 reduced renal function and is associated with autoimmune inflammation.

Figure 1. Increase of total proteinuria and decrease of renal function in mice lacking CD73.

(A) Using immunohistochemistry with a CD73-specific antibody and a rhodamin red λ conjugated secondary antibody, confocal microscopy at a magnitude of 10× revealed staining of glomerular cells in the mesangium as well as peritubular cells in renal tissue of WT mice which is absent in the mutant. Images are representative of 3 of 5 animals with more than 2 sections per kidney. (B) Renal function estimated by the measurement of serum creatinine, auto normalized, in WT and CD73^−/−mice. 8 to19 mice per group were used. Data are presented as means ± SEM, level of significance as indicated according to Student t-test. (C) Total proteinuria was elevated and increased with age in CD73^−/−mice as compared to the wild type controls. We detected proteinuria/creatinuria in [g/g], left panel, and total urine protein in [mg/24h], right panel. 8 to 19 mice per group were used. Data are presented as means ± SEM, level of significance as indicated according to Student t-test.

Figure 2. Total proteinuria in CD73^−/−mice is a low molecule proteinuria.

(A) Profiling the urinary protein content, 10 µg of total urinary protein from WT and CD73^−/−mice were subjected per lane to a 12.5% nonreducing SDS-PAGE (silver staining). Mice groups are shown as indicated, Alb  =  bovine serum albumin, M  =  rainbowmolecule weight marker with characteristic proteins as indicated (CA  =  carbonic anhydrase, GD  =  glutamatic dehydrogenase, Myo  =  myoglobin red). Proteins bands identified by size are indicated: α1-M  =  α 1-microglobulin, RBP  =  retinol binding protein. (B) Urinary albumin excretion was not different as compared to WT mice. 9 to 24 mice per group were used. Data are presented as means SEM.

Figure 3. CD73^−/−mice show an increased volume of podocytes, glomerular endothelial cells and capillaries.

Five sections per animal were analyzed using the point-counting method and a 100-point-eyepiece (integration plate II, Zeiss Co.) at a magnitude of 100× (oil immersion). (A) Volume of glomerular capillaries in 10³ µm³ in CD73^−/−mice as compared to WT (n = 5−6, means ± SD, p<0.02 according to Student t-test.). (B) Volume of glomerular cells in [µm³] CD73^−/−mice as compared to WT (n = 5−6, means ± SD, p<0.01 according to Student t-test.).

Figure 4. Reduced number of podcytes in CD73^−/−mice.

(A) The expression of the podocyte markers WT1, synaptopodin and nephrin was immunohistochemically detected on 6 µM cryosections of the renal cortex of CD73^−/−mice vs. WT-mice. Images are representative of 6–7 animals per group. (B) Statistical analysis showed a decreased number of WT1-stained podocytes per glomerulus (counted in 30 glomeruli per section and 7 mice per group), means ± SEM, p<0.0001 according to Student t-test (left panel). Histomorphometrical analysis (integration plate II, Zeiss Co., magnitude of 100×, oil immersion) confirmed reduced podocytes per glomerulus as compared to other glomerular cells (n = 5−6 mice per group), means ± SD, p<0.01 according to Student t-test (right panel).

Figure 5. Glomerular endotheliosis and injury in CD73^−/−mice.

Electrone microscopic analysis was performed n = 7 CD73^−/−mice vs. WT, original magnification x 10000 or 5000. (A) Ultrastructural analysis of the glomerular filtration barrier from a 3 months old WT mouse with regular appearance of foot processes and slit membranes and fenestration of the endothelium. (B) Analysis of the glomerular filtration barrier from a 3 months old CD73^−/− mouse shows cytoplasmic swelling apparent in endothelial cells (cap. Lumen  =  capillary lumen, ery  =  erythrocyte), endothelial fenestrations and single foot effacements were reduced (arrow). No subepithelial depositis were detectable. (C) Overview of a glomerular segment with regular appearance and without glomerulitis (3 months old WT-mouse). (D) Overview of a glomerular segment in a 3 months old CD73^−/−mouse (mes  =  mesangial cell, ly  =  lymphocytes) shows lymphocytic glomerulitis.

Figure 6. Collagen deposition in the renal cortex, peritubular and surrounding the glomeruli in CD73^−/−mice.

(A) Representative images of fibrillar collagen deposition shown by Sirius Red staining (in CD73^−/−as compared to WT controlc mice, ×200 magnification). (B) Image analysis demonstrates that CD73^−/−mice exhibit increased levels of fibrillary collagen deposition within the cortex compared with WT controls (significance as indicated, p<0.002 CD73^−/− vs WT mice, Sirius red staining, 3–5 images per animal, n = 7 animals/group, Student t-test).

Figure 7. Humoral inflammation in glomeruli and cellular infiltrates in the interstitium of CD73^−/−mice.

The renal cortex of CD73^−/−mice showed glomerular deposits of IgG and C3 as well as increased presence of CD 11b- and CD8- positive cells as well as CD25- and GR-1–positive cells in the interstitium of CD73^−/− mutants which were absent in WT-mice. (A) IgG staining (green, FITC conjugated secondary antibodies) and C3 staining (red, rhodamin red λ conjugated secondary antibodies) was carried out in cryo-conserved, 7-µm sections of the kidneys of WT and CD73^−/−mice. Images are representative of >3 independent experiments. (B) CD11b and CD 8 staining (green, FITC conjugated secondary antibodies) and CD25 and GR-1 staining (red, Cy 3) was performed in cryo-conserved, 6–7-µm sections of the mice kidneys. Images are representative of 5 to 7 animals with more than 2 sections per kidney.

Figure 8. The levels of IL-18, VEGF, MIP-2 and TNFα is increased in the serum but not in the kidney tissue of CD73^−/−mice.

The levels of IL-18, VEGF, MIP-2 and TNFα were analyzed in serum and kidney tissue of CD73^−/− and WT mice using BioPlex analysis. 7 animals from each mice group were analyzed. Data are presented as mean l ± SEM. Level of significance as indicated according to according to Mann-Whitney-U-test (p<0.005).