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 alpha4 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 alpha4).

Figure 1

Development of the glomerular capillaries in Lmx1b^–/– mice. A comparison between newborn wild-type (a) and homozygous knockout (b) mice demonstrates the severely retarded outgrowth of glomerular capillaries when the Lmx1b gene is inactivated. Furthermore, a fibrillar material can be detected in Bowman’s space and adjoining proximal tubules of Lmx1b^–/– mice (asterisk in b). At a higher magnification, the reduction and sometimes even complete lack of fenestrae in the endothelium can be seen in the knockout mice (d) (arrows in c point to fenestrae in the glomerular endothelium of wild-type mice). Although by in situ hybridization VEGF mRNA can be detected in the podocytes of both wild-type (e) and Lmx1b^–/– (f) mice, an RNase protection assay with 20 μg of total kidney RNA (prepared from five Lmx1b^+/+ and four Lmx1b^–/– mice) demonstrates reduced levels of VEGF mRNA in kidneys from the knockout mice (tRNA served as a negative control). A protection assay with a probe directed against 18S rRNA shows that the RNA concentration was determined correctly (g). Bar: 20 μm (a, b, e, and f), 0.5 μm (c), 4 μm (d).

Figure 2

Podocyte differentiation in Lmx1b^–/– mice. Podocytes in newborn homozygous knockout animals do not spread out over the GBM (arrows) but retain a cuboidal shape (a). In many areas the GBM was split (b shows a transition between a one-layered GBM marked by arrows and a split GBM marked by arrowheads). In no glomerulus did we notice foot processes and slit diaphragms, but adjacent podocytes were connected by structures resembling adherens junctions (c; note the cytoplasmic plaques outlined by arrows on either side of the junction). In some glomeruli we also observed erythrocytes (E) and fibrillar material (arrows) in Bowman’s space (d), which upon higher magnification showed a striated pattern reminiscent of fibrin (e). Bar: 2 μm (a), 1 μm (b and d), 100 nm (c and e).

Figure 3

Expression of α3 integrin, podocalyxin, and nephrin in newborn Lmx1b^–/– mice. By immunohistochemistry, α3 integrin (a and b), podocalyxin (c and d), and nephrin (e and f) were all detected in the glomeruli of wild-type (a, c, and e) and homozygous knockout mice (b, d, and f). Immunohistochemical detection of the proteins in a and b was done with DAB, in c–f by fluorescence. Bar: 20 μm (a–f).

Figure 4

Expression of podocin in newborn Lmx1b^–/– mice. Podocin protein (a) and mRNA (c) were present only in podocytes of wild-type mice, not in those of homozygous knockout mice (b and d). The specificity of these results was corroborated by an RNase protection assay with podocin antisense RNA, where protected bands were observed only with RNA isolated from kidneys of wild-type mice (tRNA served as a negative control). A protection assay with a probe directed against 18S rRNA shows that the RNA concentration was determined correctly (e). Immunohistochemical detection of the proteins was done with DAB, and mRNA was detected by nonradioactive in situ hybridization (in order to demonstrate the negative glomerulus, the picture shown in d was taken using Nomarski optics). Bar: 20 μm (a and b), 40 μm (c and d).

Figure 5

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).

Figure 6

Expression of actin cytoskeleton–associated proteins and GBM components in newborn Lmx1b^–/– mice. The actin cytoskeleton–associated proteins synaptopodin (a and b) and CD2AP (c and d) are both expressed in kidneys of wild-type and homozygous knockout mice. An RNase protection assay with 20 μg of total kidney RNA demonstrates approximately equal levels of CD2AP mRNA (e) but reduced amounts of synaptopodin (Synpo) mRNA (f) in the knockout mice (tRNA served as a negative control). The protection assay with a probe directed against 18S rRNA is shown in Figure 1. The GBM components nidogen/entactin (g and h), fibronectin (i and j), and dystroglycan (k and l) can all be detected in kidneys of wild-type and homozygous knockout mice. Immunohistochemical detection of the proteins was done with DAB (a–d, k and l) and by fluorescence (g–j). Bars: 20 μm.

Figure 7

Expression of transcription factors in newborn Lmx1b^–/– mice. WT1 (a and b) and Foxc2 (c and d) are both expressed in kidneys of wild-type and homozygous knockout mice. WT1 protein was detected immunohistochemically with DAB, the Foxc2 mRNA with nonradioactive in situ hybridization. Bar: 20 μm.