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. 2002 Apr;109(8):1065-72.
doi: 10.1172/JCI13954.

Transcriptional induction of slit diaphragm genes by Lmx1b is required in podocyte differentiation

Jeffrey H Miner  1 , Roy Morello, Kaya L Andrews, Cong Li, Corinne Antignac, Andrey S Shaw, Brendan Lee
Affiliations

Transcriptional induction of slit diaphragm genes by Lmx1b is required in podocyte differentiation

Jeffrey H Miner et al. J Clin Invest. 2002 Apr.

Abstract

LMX1B encodes a LIM-homeodomain transcription factor. Mutations in LMX1B cause nail-patella syndrome (NPS), an autosomal dominant disease with skeletal abnormalities, nail hypoplasia, and nephropathy. Expression of glomerular basement membrane (GBM) collagens is reduced in Lmx1b(-/-) mice, suggesting one basis for NPS nephropathy. Here, we show that Lmx1b(-/-) podocytes have reduced numbers of foot processes, are dysplastic, and lack typical slit diaphragms, indicating an arrest in development. Using antibodies to podocyte proteins important for podocyte function, we found that Lmx1b(-/-) podocytes express near-normal levels of nephrin, synaptopodin, ZO-1, alpha3 integrin, and GBM laminins. However, mRNA and protein levels for CD2AP and podocin were greatly reduced, suggesting a cooperative role for these molecules in foot process and slit diaphragm formation. We identified several LMX1B binding sites in the putative regulatory regions of both CD2AP and NPHS2 (podocin) and demonstrated that LMX1B binds to these sequences in vitro and can activate transcription through them in cotransfection assays. Thus, LMX1B regulates the expression of multiple podocyte genes critical for podocyte differentiation and function. Our results indicate that reduced levels of proteins associated with foot processes and the glomerular slit diaphragm likely contribute, along with reduced levels of GBM collagens, to the nephropathy associated with NPS.

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Figures

Figure 1
Figure 1
Ultrastructural analysis of podocytes from control and Lmx1b–/– newborn mice. Low- and high-magnification micrographs from control (a and b) and mutant (d and e) show reduced foot process formation in the mutant. Those processes that do form have aberrant cell-cell junctions (f, arrows). Normal and aberrant processes and junctions have been schematized for clarity (insets in b and f). Mutant podocytes resemble those observed in immature control glomeruli (c), suggesting that mutant podocytes are developmentally arrested. P, podocyte; rbc, red blood cell; E, endothelial cell; arrowheads, foot processes. Bar, 2.3 μm for a, c, and d; 0.7 μm for b, e, and f.
Figure 2
Figure 2
Immunohistochemical analysis of podocyte gene expression in Lmx1b+/+ and Lmx1b–/– glomeruli from newborn mice. No differences in levels of nephrin (a and b), laminin (Lam) α5 (c and d), or integrin α3 (e and f) were detected, suggesting that Lmx1b does not normally regulate their expression. G, glomerulus. Bar, 50 μm.
Figure 3
Figure 3
Immunohistochemical analysis of podocyte gene expression in Lmx1b+/+ and Lmx1b–/– glomeruli from newborn mice. Levels of CD2AP (a and b) and podocin (e and f) were greatly reduced in the mutant, suggesting that Lmx1b regulates their expression. Sections in a and b were doubly labeled with an antibody to laminin γ1 chain (c and d, respectively), and sections in e and f were doubly labeled with an antibody to ZO-1 (g and h, respectively). Laminin γ1 was not reduced, and ZO-1 was only slightly reduced. G, glomerulus. Bar, 50 μm.
Figure 4
Figure 4
In situ hybridization analysis of CD2AP and podocin mRNA in Lmx1b+/+ and Lmx1b–/– glomeruli from newborn mice. (a) In podocytes (arrows) of capillary loop stage glomeruli (outlined in black), CD2AP transcripts were detected in control (Lmx1b+/+), but levels were greatly reduced in mutant (Lmx1b–/–). Transcripts were easily detected in the branched mutant collecting duct (arrowheads). (b) Podocin transcripts were detected in control but not mutant podocytes. These data suggest that Lmx1b regulates podocyte transcription of Cd2ap and Nphs2 (podocin). Bar in a, 50 μm; in b, 500 μm.
Figure 5
Figure 5
LMX1B interacts specifically with putative regulatory elements in CD2AP and NPHS2. (a) Schematic diagram of CD2AP and NPHS2 5′ genomic structures. The putative LMX1B binding sites (FLAT sites) are numbered relative to the ATG translation start codon. Ovals represent FLAT-E sites (consensus TAATTA) and rectangles represent FLAT-F sites (consensus TTAAKAM). (b) EMSA of full-length LMX1B (lanes 2, 6, 10, 14), LMX1B homeodomain (HD) (lanes 3, 7, 11, 15, 17), mutant LMX1B HD (N246K) (lanes 4, 8, 12, 16), and empty vector (lanes 1, 5, 9, 13) using the following probes: CD2AP –1170, CD2AP –1817, CD2AP –2855, NPHS2 –825, and the COL4A3-COL4A4 enhancer element (FLAT-E) as positive control. Arrows indicate the shifted bands containing full-length LMX1B and LMX1B HD protein/DNA complexes. (c) Binding of full-length LMX1B (lanes 1, 3, 5, 7, 9, 11, 13, 15) and LMX1B HD (lanes 2, 4, 6, 8, 10, 12, 14, 16) to the CD2AP –1817 probe (lanes 1–10) was successfully competed with 30-, 100-, and 300-fold excess of cold CD2AP –1817 but not with excess unrelated cold probe. Binding to FLAT-E (lanes 11–16) was successfully competed with 100- and 300-fold excess of cold CD2AP –1817 probe. Arrows indicate the full-length LMX1B and LMX1B HD DNA/polypeptide complexes.
Figure 6
Figure 6
LMX1B transactivates expression of a reporter driven by NPHS2 –825 FLAT sites. Luciferase reporter constructs containing four copies of the wild-type (WT) or mutant (mut) FLAT site and a minimal promoter were cotransfected with an empty vector (–) or with an LMX1B expression plasmid (+) into NIH 3T3 cells. LMX1B upregulated expression of luciferase directed by the wild-type but not by the mutant FLAT sites.
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References

    1. Smoyer WE, Mundel P. Regulation of podocyte structure during the development of nephrotic syndrome. J Mol Med. 1998;76:172–183. - PubMed
    1. Somlo S, Mundel P. Getting a foothold in nephrotic syndrome. Nat Genet. 2000;24:333–335. - PubMed
    1. Tryggvason K, Ruotsalainen V, Wartiovaara J. Discovery of the congenital nephrotic syndrome gene discloses the structure of the mysterious molecular sieve of the kidney. Int J Dev Biol. 1999;43:445–451. - PubMed
    1. Tryggvason K. Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm. J Am Soc Nephrol. 1999;10:2440–2445. - PubMed
    1. Kerjaschki D. Caught flat-footed: podocyte damage and the molecular bases of focal glomerulosclerosis. J Clin Invest. 2001;108:1583–1587. DOI:10.1172/JCI200114629. - PMC - PubMed

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