Eishin Yaoita

Up-Regulation of Connexin43 in Glomerular Podocytes in Response to Injury

Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of focal segmental glomerulosclerosis. However, it is poorly understood how podocytes respond to injury. In this study, glomerular expression of connexin43 (Cx43) gap junction protein was examined at both protein and transcript levels in an experimental model of podocyte injury, puromycin aminonucleoside (PAN) nephrosis. A striking increase in the number of immunoreactive dots with anti-Cx43 antibody was demonstrated along the glomerular capillary wall in the early to nephrotic stage of PAN nephrosis. The conspicuous change was not detected in the other areas including the mesangium and Bowmans capsule. Immunoelectron microscopy showed that the immunogold particles for Cx43 along the capillary wall were localized predominantly at the cell-cell contact sites of podocytes. Consistently, Western blotting and ribonuclease protection assay revealed a distinct increase of Cx43 protein, phosphorylation, and transcript in glomeruli during PAN nephrosis. The changes were detected by 6 hours after PAN injection. These findings indicate that the increase of Cx43 expression is one of the earliest responses that have ever been reported in podocyte injury. To show the presence of functional gap junctional intercellular communication (GJIC) in podocytes, GJIC was assessed in podocytes in the primary culture by transfer of fluorescent dye, Lucifer yellow, after a single-cell microinjection. Diffusion of the dye into adjacent cells was observed frequently in the cultured podocytes, but scarcely in cultured parietal epithelial cells of Bowmans capsule, which was compatible with their Cx43 staining. Thus, it is concluded that Cx43-mediated GJIC is present between podocytes, suggesting that podocytes may respond to injury as an integrated epithelium on a glomerulus rather than individually as a separate cell.

Phenotypic modulation of parietal epithelial cells of Bowman's capsule in culture

Abstract. The origin of cobblestone-like polygonal cells (the most numerous in renal glomerular cell culture) remains controversial; they could be either dedifferentiated podocytes or parietal epithelial cells (PECs) of Bowmans capsule. Poor cellular outgrowth from glomeruli devoid of Bowmans capsule (decapsulated glomeruli) hinders podocytes being obtained without contamination of PECs in culture. Since podocytes are easily damaged during the isolation of glomeruli by the conventional sieving method, we devised a gentle isolation method without forced sieving, resulting in substantial numbers of arborized cells growing out from decapsulated glomeruli. The cells were distinctly different from cobblestone-like polygonal cells in their irregular and often arborized shape and extended long cytoplasmic processes that often crossed over adjacent cells. The arborized cells from decapsulated glomeruli showed intense staining for a podocyte-specific marker, podocalyxin, but no staining for markers specific to PECs (pan cadherin), mesangial cells (Thy-1) or endothelial cells (von Willebrand factor, RECA-1), indicating their podocyte origin. Polygonal cells growing out from encapsulated glomeruli were negative for podocalyxin and positive for pan cadherin at the peripheral cell-cell contact. Thus, the cell population from decapsulated glomeruli is distinctly different from that from encapsulated glomeruli, supporting the idea that polygonal cells originate from PECs, although immunocytochemical markers specific to podocytes in vivo such as WT1, synaptopodin, HSP27 and P-31 antigen were expressed significantly in the polygonal cells. Occasionally, large irregular-shaped cells appeared at the periphery of the outgrowths from encapsulated glomeruli. They were similar in shape to the arborized cells from decapsulated glomeruli but were identical in antigenic properties to cobblestone-like polygonal cells and thus may be named pseudo-arborized cells. We conclude that PECs in culture modulate their phenotype to resemble podocytes.

Coxsackievirus and Adenovirus Receptor, a Tight Junction Membrane Protein, Is Expressed in Glomerular Podocytes in the Kidney

In nephrosis, filtration slits of podocytes are greatly narrowed, and slit diaphragms are displaced by junctions with close contact. Freeze-fracture studies have shown that the newly formed junctions consist of tight junctions and gap junctions. Several tight-junction proteins are known as integral membrane components, including occludin and claudins; but none of them have been found in podocytes. Coxsackievirus and adenovirus receptor (CAR) has recently been identified as a virus receptor that is a 46-kDa integral membrane protein with two Ig-like domains in the extracellular region. In polarized epithelial cells, CAR is expressed at the tight junction, where it associates with ZO-1 and plays a role in the barrier to the movement of macromolecules and ions. In the present study, we investigated the expression and localization of CAR in rat kidneys treated with puromycin aminonucleoside (PAN) and in rat kidneys perfused for 15 minutes with protamine sulfate (PS). Both the experimental models have been used to induce tight junctions in podocytes. Ribonuclease protection assay and Western blot analysis revealed a distinct increase of CAR transcript and protein in glomeruli during PAN nephrosis but no increase in glomeruli by PS perfusion. Immunohistochemistry revealed a significant increase in CAR staining intensity along the glomerular capillary wall in PAN nephrosis and after PS perfusion. Immunoelectron microscopy demonstrated in both the models that the immunogold particles for CAR along the capillary wall were found predominantly at close cell-cell contact sites of podocytes but were rarely found at slit diaphragms. In cultured podocytes, CAR was localized at cell-cell contact sites. CAR distribution was identical to that of ZO-1 and different from that of a gap junction protein, connexin43. These findings indicate that CAR is an integral membrane component of tight junction in podocytes and that CAR expression in podocytes is regulated at the transcriptional level and in the redistribution of protein.

Involvement of Endothelial Cell Adhesion Molecules in the Development of Anti-Thy-1 Nephritis

To study an involvement of glomerular endothelial cells in the development of anti-Thy-1 nephritis, we examined the expression of endothelial cell adhesion molecules during the course of this model. Ribonuclease protection assay elucidated that expression of mRNA for intercellular adhesion molecule-1 (ICAM-1) was markedly enhanced in the glomeruli with a peak at 2 h (6.5-fold, p < 0.05) after the anti-Thy-1 antibody injection when mesangial cell lysis was recognized and IL-1β mRNA expression was induced in the glomeruli. The glomerular ICAM-1 was predominantly localized in the endothelial cells and was intensely immunostained at day 1 in the glomerular endothelial cells. In contrast, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin mRNA expression increased gradually with a peak at day 6 (2.6-fold (p < 0.05) and 4.2-fold (p < 0.05), respectively) in the glomeruli with mesangial proliferative lesion. PECAM-1 was also immunolocalized in the glomerular endothelial cells and the immunoreactivity was greatly enhanced at day 6. Glomerular expression of vascular cell adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 (E-selectin) was unchanged at a low level during the course of anti-Thy-1 nephritis. Blocking of ICAM-1 by administration of anti-ICAM-1 antibody showed significant decrease in the number of polymorphonuclear leukocytes accumulating in the glomeruli by 45.7% (9.4 ± 0.2 vs. 5.1 ± 0.1 per glomerular cross section, p < 0.01) at 2 h. These results suggest a significant involvement of glomerular endothelial cells in the development and repair of anti-Thy-1 nephritis via direct or indirect intercellular interactions between mesangial cells and glomerular endothelial cells.