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. 2013 Feb;24(2):253-67.
doi: 10.1681/ASN.2012060582. Epub 2013 Jan 18.

aPKCλ/ι and aPKCζ Contribute to Podocyte Differentiation and Glomerular Maturation

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Free PMC article

aPKCλ/ι and aPKCζ Contribute to Podocyte Differentiation and Glomerular Maturation

Björn Hartleben et al. J Am Soc Nephrol. .
Free PMC article

Abstract

Precise positioning of the highly complex interdigitating podocyte foot processes is critical to form the normal glomerular filtration barrier, but the molecular programs driving this process are unknown. The protein atypical protein kinase C (aPKC)--a component of the Par complex, which localizes to tight junctions and interacts with slit diaphragm proteins--may play a role. Here, we found that the combined deletion of the aPKCλ/ι and aPKCζ isoforms in podocytes associated with incorrectly positioned centrosomes and Golgi apparatus and mislocalized molecules of the slit diaphragm. Furthermore, aPKC-deficient podocytes failed to form the normal network of foot processes, leading to defective glomerular maturation with incomplete capillary formation and mesangiolysis. Our results suggest that aPKC isoforms orchestrate the formation of the podocyte processes essential for normal glomerular development and kidney function. Defective aPKC signaling results in a dramatically simplified glomerular architecture, causing severe proteinuria and perinatal death.

Figures

Figure 1.
Figure 1.
The double aPKCλ/ι and aPKCζ knockout in podocytes results in severe proteinuria and perinatal death. (A and B) In situ hybridization studies of kidneys of newborn mice reveal high expression of both aPKC isoforms, aPKCλ/ι and aPKCζ, in glomerular podocytes (arrows). (C and D) Western blot analyses confirm significant reduction of aPKCλ/ι and loss of aPKCζ in glomerulus lysates of the accordant knockout mice at P14. (E) Follow-up analysis of urinary albumin/creatinine ratio (n=7–48 each group). * P<0.05, ** P<0.01, *** P<0.001, aPKC double mutant compared with aPKCλ/ι PcKO; + P<0.05, ++ P<0.01, +++ P<0.001, aPKC double mutant or aPKCλ/ι PcKO respectively compared with aPKCζ KO; # P<0.05, ## P<0.01, ### P<0.001, aPKC double mutant, aPKCλ/ι PcKO or aPKCζ KO respectively compared with control. (F) Serum creatinine (n=7–12 each group). (G) Body weight (male mice, n=4–24 each group). (H) Survival (median survival aPKC double mutant = 2.5 days, aPKCλ/ι PcKO = 16 days. Log-rank (Mantel–Cox) test, P<0.001. Scale bars, 50 µm.
Figure 2.
Figure 2.
Severely impaired foot process network formation in aPKC double mutant mice. (A) Whereas scanning electron micrographs of kidneys of control and aPKCζ KO mice show developing foot processes that enwrap the capillaries, podocyte processes of aPKCλ/ι PcKO mice display an irregular pattern. In aPKC double mutant mice, podocyte cell bodies are covered with microvilli and blebs. Podocyte processes cannot be detected. (B) Podocytes of newborn aPKCλ/ι PcKO mice exhibit partially broadened processes and long projections to the glomerular basement membrane. aPKC double knockout podocytes display no elaborated process network (arrows). Furthermore, capillary dilations are observed (asterisk). Scale bars, 10 µm in upper panel in A; 2.5 µm in lower panel in A; 2 µm in upper and middle panels in B; 1 µm in lower panel in B.
Figure 3.
Figure 3.
Glomerular development arrests at late capillary loop stage in aPKC double mutant mice. Frozen kidney sections of newborn mice of the indicated genotypes (P0) were stained using antibodies against the polarity protein Par3 and the basement membrane marker nidogen and were subjected to confocal laser microscopy. Because glomerular development is asynchronous, kidneys of newborn mice display all glomerular developmental stages (from left to right) as follows: comma-shaped body (I), s-shaped body (II), early and late capillary loop stage (III and IV), and glomerulus (V). (A and B) Par3 is already expressed during the comma-shaped body stage (I) and localizes at the apical sited cell-cell junctions (arrows). During the s-shaped body stage (II), Par3 and the cell-cell junctions translocate to basal (arrows), where during capillary loop stage (IV) process development starts (arrows). In the following, glomerular surface infolding occurs (V), when Par3-positive stained protrusions enwrap the developing capillaries (arrows). (C) Whereas the glomerular development displays no obvious abnormality in aPKCζ KO mice, (D) Par3 positive protrusions display reduced branching in aPKCλ/ι PcKO mice. (E) The infolding process and enwrapping of the capillaries is almost completely absent in aPKC double mutant mice. Arrows mark localization of Par3. Scale bars, 5 µm.
Figure 4.
Figure 4.
aPKC double mutant mice display severely disturbed glomerular maturation. (A) Periodic acid–Schiff staining of P4 kidney sections of mice of the genotypes as indicated. Arrows mark mesangiolysis and mesangial hemorrhagia in aPKC double mutant mice. Asterisks mark tubular dilations with protein casts. (B) Ultrastructural analysis of P4 kidney sections reveals a regular foot process network in control and aPKCζ KO mice. In aPKCλ/ι PcKO mice, broadened foot processes with narrowed filtration slits (arrows) can be observed. In aPKC double mutant mice, podocytes (arrowheads) exhibit an undifferentiated appearance, sitting directly on the glomerular basement membrane. Asterisks indicate dilated capillaries, dotted line indicates the glomerular basement membrane, and white arrowhead indicates mesangiolysis. Scale bars, 20 µm in A; 2 µm in B.
Figure 5.
Figure 5.
aPKC double mutant mice develop capillary aneurysms during glomerular maturation. (A) Frozen kidney sections of mice of the indicated genotypes at P4 are stained using antibodies against the mesangial marker desmin and the podocyte marker podocin and are subjected to confocal laser microscopy. Arrows indicate podocyte extensions to the mesangium, which regress during glomerular maturation in aPKC double mutant mice. (B) Frozen kidney sections of mice at P4 were stained using antibodies against the endothelial marker CD31 and the podocyte marker podocin. Arrows mark CD31-positive stained capillaries. In aPKC double mutant mice, massive aneurysms can be observed (asterisks). Scale bars, 5 µm.
Figure 6.
Figure 6.
Impaired expression of slit diaphragm proteins in aPKC double mutant mice. (A–C) Staining of frozen kidney sections at P4 for the slit diaphragm proteins Par3, nephrin, podocin, and the basement membrane marker nidogen. Arrows mark expression of the indicated proteins, asterisks mark aneurysms, and arrowheads label mesangiolysis. (D) Staining of frozen kidney sections at P4 for the podocyte primary process marker detyrosinated tubulin and the slit diaphragm protein nephrin. Arrows mark expression of detyrosinated tubulin. Scale bars, 5 µm.
Figure 7.
Figure 7.
Translocation of the centrosome and Golgi apparatus during podocyte differentiation. (A) Frozen kidney sections of newborn wild-type rat are stained using antibodies against the centrosome marker protein pericentrin (arrows) and the junction marker ZO-1 and are subjected to confocal laser microscopy (top, comma-shaped body stage; middle, late s-shaped body stage; bottom, immature glomerulus). (B) Schematic illustration of the pericentrin signal during podocyte differentiation. (C) Frozen kidney sections of newborn wild-type rat are stained using antibodies against the Golgi apparatus marker giantin (arrows) and ZO-1. Arrows mark translocation of the Golgi apparatus from apical to basal during podocyte differentiation (top, comma-shaped body stage; middle, late s-shaped body stage/early capillary loop stage; bottom, immature glomerulus). (D) Schematic illustration of the giantin signal during podocyte differentiation. Scale bars, 5 µm.
Figure 8.
Figure 8.
Aberrant centrosome and Golgi apparatus localization in aPKC double knockout podocytes. (A and B) Frozen kidney sections of control and aPKC double mutant mice at P4 are stained for the podocyte marker nephrin, the centrosome marker pericentrin, or the Golgi apparatus marker giantin, respectively. Whereas the centrosome (arrows) and the Golgi apparatus (arrows) are restricted to the basal pole of the cytoplasm in control mice, aberrant localization (arrows) can be detected in aPKC double knockout podocytes. (C) Electron micrographs of P4 control and aPKC double knockout podocytes. C, centrosome; G, Golgi apparatus; GBM, glomerular basement membrane. Scale bars, 5 µm in A and B; 1 µm in C.
Figure 9.
Figure 9.
aPKC is required for glomerular maintenance. Time-dependant induction of aPKCλ/ι knockout in podocytes of aPKCλ/ιflox/flox;NPHS2.rtTA;tetO.Cre mice between 5 and 7 weeks of age results in the following: (A) loss of glomerular aPKCλ/ι expression, (B) proteinuria, and (C) glomerulosclerotic changes with synechia, crescent formation (arrows), and proteinaceous casts in dilated tubules (asterisks) 3 weeks after start of induction. Scale bars, 5 µm in A; 20 µm in C.
Figure 10.
Figure 10.
The podocyte foot process network is required for glomerular maturation. (A) Under physiologic conditions, podocytes establish a foot process network during glomerular development and maturation. Surface infolding is formed by podocyte foot processes, glomerular basement membrane, and mesangial cells, separating the capillaries from each other. (B) In aPKC double mutant mice, failure to establish a process network results in an arrest of glomerular maturation.

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