Nephrotic Syndrome and Subepithelial Deposits in a Mouse Model of Immune-Mediated Anti-Podocyte Glomerulonephritis

Abstract

Subepithelial immune complex deposition in glomerular disease causes local inflammation and proteinuria by podocyte disruption. A rat model of membranous nephropathy, the passive Heymann nephritis, suggests that Abs against specific podocyte Ags cause subepithelial deposit formation and podocyte foot process disruption. In this study, we present a mouse model in which a polyclonal sheep anti-mouse podocyte Ab caused subepithelial immune complex formation. Mice developed a nephrotic syndrome with severe edema, proteinuria, hypoalbuminemia, and elevated cholesterol and triglycerides. Development of proteinuria was biphasic: an initial protein loss was followed by a second massive increase of protein loss beginning at approximately day 10. By histology, podocytes were swollen. Electron microscopy revealed 60–80% podocyte foot process effacement and subepithelial deposits, but no disruption of the glomerular basement membrane. Nephrin and synaptopodin staining was severely disrupted, and podocyte number was reduced in anti-podocyte serum-treated mice, indicating severe podocyte damage. Immunohistochemistry detected the injected anti-podocyte Ab exclusively along the glomerular filtration barrier. Immunoelectron microscopy localized the Ab to podocyte foot processes and the glomerular basement membrane. Similarly, immunohistochemistry localized mouse IgG to the subepithelial space. The third complement component (C3) was detected in a linear staining pattern along the glomerular basement membrane and in the mesangial hinge region. However, C3-deficient mice were not protected from podocyte damage, indicating a complement-independent mechanism. Twenty proteins were identified as possible Ags to the sheep anti-podocyte serum by mass spectrometry. Together, these data establish a reproducible model of immune-mediated podocyte injury in mice with subepithelial immune complex formation.

Rho-kinase inhibition prevents proteinuria in immune-complex-mediated antipodocyte nephritis

“…Podocytes were stained with WT-1 and counted per glomerular section in all four groups. Total podocyte number significantly decreased in antipodocyte serum-treated mice compared with preimmune serum-treated mice as shown previously (25,26). Interestingly podocyte number was significantly less reduced in Rho-kinaseinhibited mice receiving antipodocyte serum, indicating that podocytes were protected from detachment by Rho-kinase inhibition.…”

“…We therefore hypothesized that increased activation of Rho-kinases leads to cytoskeletal rearrangement in podocytes in the course of antibody-mediated podocyte injury, culminating in foot process retraction, proteinuria, and podocyte loss into the urine and that this could be prevented by Rho-kinase inhibition. To test this hypothesis, we recently developed a mouse model of immune-mediated podocyte injury, in which a polyclonal antipodocyte antiserum was used to induce podocyte injury and proteinuria in mice (25,26). Here, we show for the first time, that Rho-kinase inhibition prevented proteinuria and podocyte disruption in vivo in a model of immune complex-mediated glomerulonephritis.…”

“…Podocytes are the primary target of the injected antipodocyte serum and were found to be severely injured in antipodocyte nephritis (26). Antipodocyte serum causes podocyte and mesangial swelling by deposition of subepithelial immune complexes (Fig.…”

MicroRNA-193a Regulates the Transdifferentiation of Human Parietal Epithelial Cells toward a Podocyte Phenotype

“…Albuminuria was measured by a commercial ELISA kit (Bethyl), and values were normalized to creatinine values assessed by the Department of Clinical Chemistry (University Clinic HamburgEppendorf, Hamburg, Germany) as described before. 27 Periodic acidSchiff staining to detect polysaccharides/glycoproteins and to evaluate the glomerulus and Sour Fuchsin Orange G staining to visualize immune deposits were performed according to routine protocols. Crescent formation was measured in 40 glomeruli per mouse and five mice per condition.…”

Impairment of podocyte function by diphtheria toxin—a new reversible proteinuria model in mice

“…Conditionally immortalized podocytes were incubated with various amounts of DTx, LPS or a sheep serum reacting against murine podocytes. 22 Figure 7a and Supplementary Figure 4A show that despite high concentration of DTx the podocytes do not lose viability. When this experiment was repeated with isolated podocytes from murine kidneys we found the same result.…”