Figure 4. Expression of the M-Type Phospholipase A₂ Receptor (PLA₂R) in Normal Kidney Tissue and Glomeruli.

Serial cryosections of the cortex of normal human kidneys were immunostained with anti-PLA₂R antibody (1:150 dilution), followed by Cy3-conjugated anti–guinea pig IgG antibody (1:500 dilution). Panel A shows two positively stained glomeruli (arrowheads) and scattered tubular staining (arrow). Panel B shows an adjacent section in which the anti-PLA₂R antiserum was preabsorbed with a recombinant fragment of PLA₂R containing C-type lectinlike domains (CTLDs) 4, 5, and 6 (Panel H). The glomerular PLA₂R staining is completely blocked by the recombinant fragment (arrowheads), whereas the scattered tubular staining (arrow) is not blocked and is therefore nonspecific. Panel C shows a human kidney-tissue specimen treated with nonimmune guinea pig serum (1:200 dilution), followed by Cy3-conjugated anti–guinea pig IgG antibody, and stained with Hoechst 33342 (a nuclear stain); the inset shows a glomerulus exposed instead to anti-PLA₂R antibody (1:200 dilution) but otherwise assessed under identical conditions. Next, cryosections of normal human kidney cortex were costained with antiagrin antibody, followed by an Alexa Fluor 488–conjugated antirabbit secondary antibody, to label the glomerular basement membrane (Panels D and E); in addition, PLA₂R staining with guinea pig anti-PLA₂R antibody was either left unblocked (Panel D) or was blocked (Panel E), as described above. The PLA₂R signal is clearly present outside the glomerular basement membrane and localizes to both the cell body and the processes of the podocyte. The PLA₂R staining is markedly reduced when the antibody is preabsorbed with the recombinant PLA₂R fragment (Panel E). Panel F shows an enlargement of the left lower portion of the glomerulus shown in Panel D, with demarcation of the capillary lumina (asterisks) and urinary space (US) to highlight the location of the PLA₂R-stained podocytes. Hoechst-stained nuclei of parietal epithelial cells in the Bowman’s capsule are indicated by the arrow. Panel G shows a portion of a human glomerulus stained with anti-PLA₂R antibody (as described above), anti–Wilms’ tumor 1 antibody (WT1) (1:100 dilution), and Alexa Fluor 488–conjugated anti–rabbit IgG antibody (1:500 dilution) to label podocyte nuclei. The inset shows the image of the entire glomerulus from which the enlarged view was taken. Panel H illustrates the domain structure of PLA₂R, which is composed of an N-terminal cysteine-rich domain (Cys-R), a fibronectin type II domain (FNII), eight CTLDs, a transmembrane domain (TM), and a short intracellular C-terminal tail (IC). The blocking fragment used in the experiments described above consists of CTLDs 4, 5, and 6 of a recombinant rabbit PLA₂R.