The LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes
Claudia Rohr, Jürgen Prestel, Laurence Heidet, Hiltraud Hosser, Wilhelm Kriz, Randy L. Johnson, Corinne Antignac, Ralph Witzgall
Claudia Rohr, Jürgen Prestel, Laurence Heidet, Hiltraud Hosser, Wilhelm Kriz, Randy L. Johnson, Corinne Antignac, Ralph Witzgall
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Article Genetics

The LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes

Abstract

Patients with nail-patella syndrome often suffer from a nephropathy, which ultimately results in chronic renal failure. The finding that this disease is caused by mutations in the transcription factor LMX1B, which in the kidney is expressed exclusively in podocytes, offers the opportunity for a better understanding of the renal pathogenesis. In our analysis of the nephropathy in nail-patella syndrome, we have made use of the Lmx1b knockout mouse. Transmission electron micrographs showed that glomerular development in general and the differentiation of podocytes in particular were severely impaired. The glomerular capillary network was poorly elaborated, fenestrae in the endothelial cells were largely missing, and the glomerular basement membrane was split. In addition podocytes retained a cuboidal shape and did not form foot processes and slit diaphragms. Expression of the α4 chain of collagen IV and of podocin was also severely reduced. Using gel shift assays, we demonstrated that LMX1B bound to two AT-rich sequences in the promoter region of NPHS2, the gene encoding podocin. Our results demonstrate that Lmx1b regulates important steps in glomerular development and establish a link between three hereditary kidney diseases: nail-patella syndrome (Lmx1b), steroid-resistant nephrotic syndrome (podocin), and Alport syndrome (collagen IV α4).

Authors

Claudia Rohr, Jürgen Prestel, Laurence Heidet, Hiltraud Hosser, Wilhelm Kriz, Randy L. Johnson, Corinne Antignac, Ralph Witzgall

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Gel shift assays to demonstrate binding of LMX1B to FLAT-E and FLAT-F el...
Gel shift assays to demonstrate binding of LMX1B to FLAT-E and FLAT-F elements in the NPHS2 promoter region. The gel shift assays were carried out with an oligonucleotide from the first intron of the human COL4A4 gene COL4A4, whose recognition by LMX1B had been demonstrated before (38) and that therefore served as a positive control; an oligonucleotide containing a perfect FLAT-F element (–1087); an oligonucleotide containing two imperfect FLAT-E elements (–837); and a GC-rich oligonucleotide containing neither a FLAT-E nor a FLAT-F element (–287). (a) Two hundred fifty and 500 ng of bacterially expressed full-length LMX1B protein recognize not only the binding site in the COL4A4 intron, but also the perfect FLAT-F and imperfect FLAT-E elements, while the GC-rich oligonucleotide is not bound. np, oligonucleotide with no protein added. (b) Competition assays using 500 ng of bacterially expressed full-length LMX1B protein and a 25-fold, 100-fold, and 400-fold molar excess of the indicated unlabeled oligonucleotides demonstrate the specificity of the gel shift. Labeled oligonucleotides are indicated on the top, unlabeled oligonucleotides below. nc, no competing unlabeled oligonucleotide added. (c) Four microliters of the in vitro–translated LMX1B homeodomain were incubated with the oligonucleotides described above. It can be easily seen that the homeodomain (HD) recognizes the binding site in the COL4A4 intron, the perfect FLAT-F element, and the imperfect FLAT-E elements, but not the GC-rich oligonucleotide. Specific bands are indicated by arrows; the other bands can also be seen where no DNA was added to the in vitro transcription/translation reaction (H2O). (d) Nuclear extracts were prepared from stably transfected HeLa cells inducibly expressing a myc epitope–tagged full-length LMX1B protein, and 6 μg of nuclear proteins from noninduced (“off”) and induced (“on”) cells were used for a gel shift assay. Upon the induction of LMX1B, a clear shift can be recognized with the binding site in the COL4A4 intron and the perfect FLAT-F and imperfect FLAT-E elements (arrows). The specificity of the shift can be appreciated from the supershifted band appearing upon the addition of the anti–myc epitope antibody 9E10 (arrowhead in “on + Ab” lane).