Inhibition of podocyte binding to laminin by anti α-dystroglycan monoclonal antibodies IIH6 and VIA 4.1.

<p>Podocytes were seeded on laminin (A) or collagen A coatings (B) after pre-incubation with either monoclonal antibody IIH6 using TEPC as an isotype control, or with VIA 4.1 using MOPC 21 as an isotype control. Monoclonal antibody IIH6 specifically inhibits podocyte binding to laminin, but not to collagen A.</p

Pulmonary tumor foci formation in syndencan-1 −/− versus wild type mice with and without treatment with LMWH.

<p>Syndecan-1 −/− and wild type mice were administered 2.0×10⁵ B16F10 melanoma cells into the lateral tail vein. One group of mice was treated with LMWH (15 mg/kg enoxaparin ) prior to the administration of B16F10 melanoma cells and LMWH treatment was repeated after 6, 12 and 24 h. Mice were sacrificed 14 days after cancer cell injection and the number of tumor foci at the surface of the lungs was determined. Error bars represent medians ± interquartile range (n = 8), * p<0.05; *** p<0.001.</p

pNS sera did not induce proteinuria in Balb/c^Thy-1.1 males.

Balb/cThy-1.1 males were injected intravenously, at 5 weeks ± 3 days old, with 2 mg serum protein from patients with recurrent FSGS, FSGS in the native kidneys, non-recurrent FSGS, or healthy controls. Individual animal data and group means of urinary albumin/creatinine (alb/creat, mg/mg) ratio are presented. No significant differences (p<0.05) compared to sera from healthy donors.</p

Ligation of α-dystroglycan results in a loss of pedicles in cultured mouse podocytes.

<p>Differentiated podocytes on collagen coatings were incubated with either monoclonal antibodies IIH6 or VIA 4.1, their respective isotype controls (TEPC or MOPC) or fibronectin. The podocyte pedicles (arrow) are probed with antibodies against Mena (green, which also localizes along stress fibers and in focal contacts at the tips of stress fibers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005979#pone.0005979-YanagidaAsanuma1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005979#pone.0005979-Asanuma1" target="_blank">[65]</a>). The pedicles were lost after 6 hours of incubation with the antibodies. As these pedicles may be the <i>in vitro</i> manifestation of foot processes, their retraction may imply foot process effacement. Percentage of podocytes with smooth surfaces were counted, which yielded for IIH6 60% <i>versus</i> TEPC 30% (χ² = 20.8, p<0.001), for VIA4.1 51% <i>versus</i> MOPC 28% (χ² = 9.01, p<0.01) and fibronectin 38% versus control 30% (χ² = 1.3, p = 0.25). (Red = phalloidin (actin); blue = DAPI (DNA)).</p

Calcium fluxes in cultured mouse podocytes after ligation of α-dystroglycan.

<p>Mouse podocytes were differentiated on collagen A coatings for 2 weeks. Intracellular calcium concentrations were monitored by the fura-2 method, with and without extracellular calcium. Both α-dystroglycan specific monoclonal antibodies induced calcium fluxes, most likely from intracellular stores. Isotype controls were negative (A, B). The laminin G modules-containing proteins agrin and laminin were unable to induce a calcium influx in podocytes (C, D). From the natural ligands of α-dystroglycan tested, fibronectin induced the most pronounced calcium influx. Note that blocking of potential integrin binding sites with a RGD peptide did not influence the fibronectin effect (E). The proteoglycan biglycan induced a modest calcium influx (F).</p

Different doses healthy plasma were injected in the Thy-1.1 mice with different backgrounds to determine maximum injection dose.

(A) Balb/cThy-1.1 males, (B) Balb/cThy-1.1 females, (C) C57BL/6Thy-1.1 males, (D) C57BL/6Thy-1.1 females were injected intravenously with various doses healthy plasma protein at 5 weeks ± 3 days old. (E) 129X1/SvThy-1.1 males, (F) 129X1/SvThy-1.1 females, (G) 129S2/SvPasThy-1.1 males, and (H) 129S2/SvPasThy-1.1 females received PBS vehicle (veh) injection only. Individual animal data and group means of urinary albumin/creatinine (alb/creat, mg/mg) ratio are presented. One-way ANOVA: * p≤0.05, ** p≤0.01, *** p≤0.001 versus vehicle.</p

Development of spontaneous proteinuria of Thy-1.1 transgenic mouse strains.

Urine was collected weekly and urinary albumin was analyzed of (A) Balb/cThy-1.1 males, (B) Balb/cThy-1.1 females, (C) C57BL/6Thy-1.1 males, (D) C57BL/6Thy-1.1 females, (E), 129X1/SvThy-1.1 males, (F) 129X1/SvThy-1.1 females, (G) 129S2/SvPasThy-1.1 males, (H) 129S2/SvPasThy-1.1 females, (I) FVB/NThy-1.1 males, and (J) FVB/NThy-1.1 females. (DOCX)</p

<i>In vitro</i> and <i>in vivo</i> responses to simultaneously collected serum, plasma, and PP effluent.

(A) Cellular granularity of cultured human podocytes (hPod) exposed to simultaneously collected serum, plasma, and PP effluent, as measured by flow cytometry, is represented by the median side scatter relative to healthy controls. Relative granularity of two replicate experiments and their mean are presented. ** pB) Albuminuria of Balb/cThy-1.1 males injected intravenously, at 5 weeks ± 3 days old, with 2 mg protein from serum, plasma, or PP effluent from three FSGS patients during active disease (rec7, rec8, nat3) and serum and plasma from one FSGS patient not treated with PP, during active disease and remission (nat4). Individual animal data and group means of urinary albumin/creatinine (alb/creat, mg/mg) ratio are presented. Data of healthy donor sera derived from Fig 3 and pooled healthy plasma derived from Fig 1 are shown for comparison with patient samples. Although none of the patient sera induced proteinuria, the ages of the patients in this study do not match the ages of the healthy donors, which may limit interpretation of these data.</p

FSGS plasma induced proteinuria mostly in Balb/c^Thy-1.1 males.

FSGS plasma induced proteinuria mostly in Balb/cThy-1.1 males.</p

Development of spontaneous proteinuria in Thy-1.1 mice with different backgrounds.

Development of spontaneous proteinuria in Thy-1.1 mice with different backgrounds.</p