Although the pattern of IgG accumulation in FcRn-deficient mice was predominantly mesangial, our results suggest that the podocyte is mainly responsible for clearing protein that is trapped at the slit diaphragms. pathway in disease, saturating the clearance mechanism potentiated the pathogenicity of nephrotoxic sera. These studies support the idea that podocytes play an active role in removing proteins from the GBM and suggest that genetic or acquired impairment of the clearance machinery is likely to be a common mechanism promoting glomerular diseases. 0.0002 by two-tailed Student’s test. Results are the combined results from two independent experiments using age- and sex-matched mice with similar results. FcRn Deficiency Results in Increased Glomerular IgG Retention. To test the long-term consequences of FcRn deficiency on the clearance of IgG from the GBM, we harvested kidneys from wild-type and FcRn-deficient mice up to 6 months of age and stained for mouse IgG. We consistently noted increased IgG staining in FcRn-deficient animals compared with wild-type animals as young as 2.5 months of age (data not shown). The difference, however, was most apparent in 6-month-old animals (Fig. 3). The pattern of glomerular IgG localization was mostly mesangial, but capillary loop staining also was readily observed. This increased IgG accumulation in FcRn-deficient mice was observed GSK-3326595 (EPZ015938) despite the fact that, in the absence of FcRn, the serum IgG concentration in these mice is only 10C20% of the normal level (17). Thus, the absence of FcRn leads to the glomerular accumulation of IgG with age. Open in a separate window Fig. 3. FcRn-deficient mice accumulate glomerular IgG with age. Kidneys from FcRn wild-type and knockout mice were stained for mouse IgG retention at 6 months of age. The results are representative of two different experiments with six to eight age-matched mice per group. (for injection protocol). The protein content of urine for all mice in HSA alone and NTS alone groups was 30 mg/dl. Six of seven mice in the HAS + NTS group had proteinuria of 3,000 mg/dl, and one mouse had proteinuria of 300 mg/dl. The four remaining mice in this group had 30 mg/dl of protein in their urine. Values are the combined results from two independent experiments with similar results and were significant ( 0.002) by Fisher’s exact test. Discussion Current models propose that the glomerular filtration barrier consists of a fenestrated endothelium, the GBM, and finally the slit diaphragm of the podocytes. The charge selectivity of the glomerular filtration barrier resides at the level of the charged glycocalyx of the endothelium and the negatively charged matrix of the GBM (27, 28). Although this charge barrier may repel a significant amount of serum protein, proteins that do enter the GBM are directed toward the podocyte slit diaphragm by fluid flow and GSK-3326595 (EPZ015938) diffusion (8). Assuming that the slit diaphragm is not freely permeable to protein, large serum proteins that enter the GBM should accumulate behind this final component of the filter. Why, then, doesn’t the kidney filter routinely clog with abundant endogenous serum proteins such as albumin and IgG? Even assuming very low permeability coefficients for albumin and IgG into the GBM, given the high blood flow to the kidney, podocytes would still be expected to encounter significant quantities of these proteins each day. Consequently, internalization and degradation of this protein would be maladaptive. Rather, receptor-mediated transcytosis of protein into the urinary space, followed by reabsorption in the proximal tubule, would solve two problems. First, transcytosis of protein accumulated at the filtration slit would serve to keep the basement membrane and slit diaphragms clear of protein. Second, reabsorption of proteins by the proximal tubule would prevent loss of transcytosed protein into the urine. We therefore hypothesized that podocytes use an active clearance mechanism to keep the GBM and the slit diaphragms clear of protein. In RNA microarray studies, we found only one transcytotic receptor that satisfied these criteria, the epithelial transport receptor, FcRn. We confirmed that FcRn was expressed in podocytes by RT-PCR and immunoblotting. Because FcRn is expressed mainly in intracellular vesicles, how does it encounter IgG? It is conceivable that podocytes use the classical FcRs and megalin to internalize protein. Consistent with this idea, a recent report describes the receptor-mediated uptake of IgG into intracellular vesicles of cultured murine podocytes (29). Once internalized, FcRn can bind IgG in an acidic intracellular endosome and transport it to the urinary space. Previous work has shown that primary rat podocytes return internalized IgG intact COL4A3 back to the cell surface rather than degrading it (30). Thus, the expression of FcRn in podocytes provides a mechanism to clear IgG from the GBM and deliver it intact into the urinary space. Consistent with this mechanism, FcRn-deficient mice showed reduced clearance of radioactive IgG from their kidneys. Also, GSK-3326595 (EPZ015938) as they aged, FcRn-deficient mice had increased IgG deposition in their glomeruli. This.