Primary cilia disappear in rat podocytes during glomerular development

Abstract

Most tubular epithelial cell types express primary cilia, and mutations of primary-cilium-associated proteins are well known to cause several kinds of cystic renal disease. However, until now, it has been unclear whether mammalian podocytes express primary cilia in vivo. In this study, we determined whether primary cilia are present in the podocytes of rat immature and mature glomeruli by means of transmission electron microscopy of serial ultrathin sections. In immature glomeruli of fetal rats, podocytes express the primary cilia with high percentages at the S-shaped body (88 +/- 5%, n = 3), capillary loop (95 +/- 4%, n = 4), and maturing glomerulus (76 +/- 13%, n = 5) stages. The percentage of ciliated podocytes was significantly lower at the maturing glomerulus stage than at the former two stages. In mature glomeruli of adult rats, ciliated podocytes were not found at all (0 +/- 0%, n = 11). These findings indicate that the primary cilia gradually disappear in rat podocytes during glomerular development. Since glomerular filtration rate increases during development, the primary cilia on the podocytes are subjected to a stronger bending force. Thus, the disappearance of the primary cilia presumably prevents the entry of excessive calcium-ions via the cilium-associated polycystin complexes and the disturbance of intracellular signaling cascades in mature podocytes.

Fig. 1

Immunofluorescence labeling for acetylated α-tubulin (Ac-tub, red) in adult rat kidney. a–a″ Strong immunofluorescence signal for Ac-tub is found in the glomerulus (G). Such strong signal is not found in the proximal tubules (PT) and distal tubules (DT). Actin filaments are visualized with FL-phallacidin (green) for identification of glomerulus, tubules, and vasculature. b–b″ In the proximal and distal tubules, the primary cilia (arrowheads) are detected with the anti-Ac-tub antibody at the luminal surface. c–c″ In the glomerulus, immunofluorescence signal for Ac-tub (red) is largely colocalized with that for podocalyxin (green), which is predominantly localized at the apical surface membrane of podocytes. It is difficult to determine whether or not the mature podocytes of adult rats possess the primary cilia in this staining. b–b″,c–c″ Z-stacked images of 5 µm thickness. Bars 20 µm

Fig. 2

Immunofluorescence labeling for Ac-tub (red) in fetal rat kidney. Unlike adult kidneys, immunofluorescence intensity of Ac-tub signals is as low in the glomeruli as in the tubules. However, prominent Ac-tub signals like little pieces of thread (arrowheads) are frequently recognized between or on the immature columnar podocytes, whose apical membrane is labeled with anti-podocalyxin antibody (green). Similar signals of Ac-tub are also found in the lumen of immature tubules (arrows). These thread-like signals for Ac-tub presumably represent the primary cilia. a–a″ Late S-shaped body stage. b–b″ Capillary loop stage. Z-stacked images of 5 µm thickness. Bars 20 µm

Fig. 3

Serial transmission electron micrographs of a mature podocyte in adult rat (a–g). These sections include both the mother (solid arrows) and daughter (open arrows) centrioles, which are located near the nucleus. The mother centriole is not in touch with the surface plasma membrane, and no primary cilium was projected from the mother centriole. Bar 1 µm. h The podocyte shown by the serial sections is located in the rectangle

Fig. 4

Transmission electron micrographs of centrioles and associated structures in the mature podocytes of adult rats. Axial (a–c) and longitudinal (d,e) sections of mother centrioles show transitional fibers (arrowheads) and basal feet (arrows). Even in the case where the mother centriole is close to the surface plasma membrane, the transitional fibers are not in contact with the membrane as indicated by arrowheads in (e). a–c Axial serial sections. f,g Striated rootlets (arrows) are occasionally found near the centrioles. G Golgi apparatus, N nucleus. Bars 500 nm

Fig. 5

Serial transmission electron micrographs of a ciliated immature podocyte in fetal rat (a–i). These sections include both basal body/mother centriole (solid arrows) and daughter centriole (open arrows). A primary cilia protruded from the bottom of shallow recess, and the basal body/mother centriole is located beneath the bottom membrane of the recess. Bar 1 µm. j The podocyte shown by the serial sections is located in the rectangle

Fig. 6

Serial transmission electron micrographs of a ciliated immature podocyte in fetal rat (a–k). These sections include both basal body/mother centriole (solid arrows) and daughter centriole (open arrows). In this case, the recess is very deep and forms a sheath-like invagination. Most part of the primary cilium is located in the sheath-like invagination (arrowheads). Bar 1 µm. l The podocyte shown by the serial sections is located in the rectangle

Fig. 7

Percentage of the ciliated podocytes in fetal and adult rat glomeruli. In fetal rat kidneys, the immature podocytes expressed primary cilia with high percentages at the S-shaped body (88 ± 5%, n = 3) and capillary loop stages (95 ± 4%, n = 4). The percentage significantly decreased at the maturing glomerulus stage (76 ± 13%, n = 5), in comparison with the former two stages. At the mature stage in adult kidneys, the podocytes express no primary cilia (0 ± 0%, n = 11). The numbers of glomeruli and podocytes examined are shown in Tables 1 and 2. p < 0.05 was considered significant

Fig. 8

Transmission electron micrographs of centrioles and associated structures in the immature podocytes of fetal rats. a In most cases, a primary cilium protruded from the bottom of the membrane recess. b A longitudinal section of a basal body/mother centriole shows transitional fibers (arrowheads) and a basal foot (arrow). In ciliated podocytes, the basal body/mother centriole is located beneath the bottom of the membrane recess, and the transitional fibers are in contact with the inner side of the bottom membrane. c,d The striated rootlets (arrows) are frequently found near centrioles. G Golgi apparatus, N nucleus. Bar 1 µm (a, c), 200 nm (b,d)

Fig. 9

Serial transmission electron micrographs of a non-ciliated immature podocyte in fetal rat (a–j). These sections include both mother (solid arrows) and daughter (open arrows) centrioles. The mother centriole is not in contact with the surface plasma membrane, and no primary cilium was projected from the mother centriole. Bar 1 µm. k The podocyte shown by the serial sections is located in the rectangle

Fig. 10

Diagram showing that the primary cilia disappear in rat podocytes during glomerular development. In immature glomeruli of fetal rats, the podocytes express the primary cilia with high percentages. However, in mature glomeruli of adult rats, the podocytes express no primary cilia