Identification d'un nouveau médiateur du développement rénal (Mitf-A)

Le développement du rein dépend d interactions réciproques entre le bourgeon urétéral et le mésenchyme métanéphrique. Etroitement régulée dans le temps et l espace, cette interaction est essentielle à la croissance et à la division du bourgeon urétéral. Au cours de ma thèse, nous avons identifié un nouveau facteur de transcription, Mitf-A, impliqué dans la régulation de ce processus. En établissant une nouvelle lignée de souris transgéniques surexprimant Mitf-A spécifiquement dans le rein, nous avons montré Mitf-A joue un rôle clé dans l arborisation du bourgeon urétéral et dans la détermination du capital néphronique. En effet, les souris transgéniques surexprimant Mitf-A présentent une augmentation du nombre de branchements du bourgeon urétéral qui conduit à un nombre final de néphrons plus important. Au contraire, les souris FVB/N portant un variant hypomorphe du gène Mitf-A présentent, de façon intéressante, d une diminution de l arborisation du bourgeon urétéral et du capital néphronique. Des études in vivo et in silico nous ont permis d élucider, en partie, les mécanismes moléculaires et cellulaires mis en jeu. Ainsi, nous avons montré que Mitf-A participerait à l arborisation du bourgeon urétéral en contrôlant la balance prolifération/ apoptose cellulaire via la régulation de l expression de gènes clés du développement rénal tels que Ret, Wnt11, Sprouty1, et Bcl2. En conclusion, nous avons découvert un nouveau médiateur du développement rénal : Mitf-A. Cette découverte ouvre de nouvelles perspectives quant à la compréhension des réseaux transcriptionnels impliqués dans le contrôle du développement rénalPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

TGF-α Mediates Genetic Susceptibility to Chronic Kidney Disease

The mechanisms of progression of chronic kidney disease (CKD) are poorly understood. Epidemiologic studies suggest a strong genetic component, but the genes that contribute to the onset and progression of CKD are largely unknown. Here, we applied an experimental model of CKD (75% excision of total renal mass) to six different strains of mice and found that only the FVB/N strain developed renal lesions. We performed a genome-scan analysis in mice generated by back-crossing resistant and sensitive strains; we identified a major susceptibility locus (Ckdp1) on chromosome 6, which corresponds to regions on human chromosome 2 and 3 that link with CKD progression. In silico analysis revealed that the locus includes the gene encoding the EGF receptor (EGFR) ligand TGF-α. TGF-α protein levels markedly increased after nephron reduction exclusively in FVB/N mice, and this increase preceded the development of renal lesions. Furthermore, pharmacologic inhibition of EGFR prevented the development of renal lesions in the sensitive FVB/N strain. These data suggest that variable TGF-α expression may explain, in part, the genetic susceptibility to CKD progression. EGFR inhibition may be a therapeutic strategy to counteract the genetic predisposition to CKD

MITF - A controls branching morphogenesis and nephron endowment.

Congenital nephron number varies widely in the human population and individuals with low nephron number are at risk of developing hypertension and chronic kidney disease. The development of the kidney occurs via an orchestrated morphogenetic process where metanephric mesenchyme and ureteric bud reciprocally interact to induce nephron formation. The genetic networks that modulate the extent of this process and set the final nephron number are mostly unknown. Here, we identified a specific isoform of MITF (MITF-A), a bHLH-Zip transcription factor, as a novel regulator of the final nephron number. We showed that overexpression of MITF-A leads to a substantial increase of nephron number and bigger kidneys, whereas Mitfa deficiency results in reduced nephron number. Furthermore, we demonstrated that MITF-A triggers ureteric bud branching, a phenotype that is associated with increased ureteric bud cell proliferation. Molecular studies associated with an in silico analyses revealed that amongst the putative MITF-A targets, Ret was significantly modulated by MITF-A. Consistent with the key role of this network in kidney morphogenesis, Ret heterozygosis prevented the increase of nephron number in mice overexpressing MITF-A. Collectively, these results uncover a novel transcriptional network that controls branching morphogenesis during kidney development and identifies one of the first modifier genes of nephron endowment

Generation of MITF-A transgenic mice.

<p><b>A)</b> Schematic representation of the Ksp-cadherin-FLAG-MITF-A transgene. <b>B)</b> <i>Mitf-A</i> mRNA expression evaluated by quantitative RT-PCR in kidneys from wild-type (WT), heterozygous (HE) and homozygous (HO) MITF-A transgenic mice (line 42) 2 months after birth. Data are means ± SEM; n = 4–6 per each genotype. ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: ** P < 0.01, *** P < 0.001. <b>C)</b> MITF-A protein expression evaluated by western blot on kidney nuclear protein extracts from WT, HE and HO MITF-A transgenic mice 2 months after birth. This is a representative image of three experiments. Nuclear protein extracts from <i>Mitfa</i>^-/- kidneys were used as a negative control; crude extracts from renal cells transfected with either FLAG-MITF-A plasmid (lane 1) or MITF-A plasmid (lane 2) were used as a positive control. Lamin A/C was used as control of nuclear protein amount. IB = immunoblot.</p

<i>Mitfa</i> inactivation results in reduced glomeruli number.

<p><b>A)</b> Schematic representation of the targeting strategy used to inactivate <i>Mitfa</i>. <b>B-C)</b> <i>Mitf-A</i> <b>(B)</b> and total <i>Mitf</i> mRNA <b>(C)</b> expression evaluated by quantitative RT-PCR in kidneys from 2 months-old <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> mice. <b>D)</b> Glomerular number in kidneys from 2 months-old <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> mice. Data are means ± SEM, n = 8–10 per each genotype. Mann-Whitney test; <i>Mitfa</i>^<i>-/-</i> <i>versus Mitfa</i>^<i>+/+</i>: *** P < 0.001.</p

Expression pattern of MITF-A during kidney development.

<p><b>A-B)</b><i>In situ</i> hybridization of <i>Mitf-A</i> of E13.5 kidneys from wild-type (WT) and homozygous (HO) MITF-A transgenic embryos using an antisense RNA probe directed against a sequence encompassing exon 1A, specific for <i>Mitf-A</i>, and exon 1B common to <i>Mitf-A</i>, <i>Mitf-H</i>, <i>Mitf-C</i>, <i>Mitf-J</i> and <i>Mitf-Mc</i> isoforms. The inset shows the staining of E13.5 kidneys using the sense RNA probe. Magnifications are X100 (left panels), X200 (middle panels) and X400 (right panels). In WT kidneys <b>(A)</b> a weak staining is observed in branches of UB (black arrow), in S-shaped body (blue arrow) and in metanephric mesenchyme (asterisk). Consistent with the use of the Ksp-cadherin promoter, the signal in MITF transgenic kidneys <b>(B)</b> was strongly increased in UB and tips (black arrow), in ureteric tip (black arrow) and to a lesser extent in S-shaped body (blue arrow). <b>C)</b> <i>In situ</i> hybridization of <i>Mitf-A</i> in transgenic HO kidneys after laminin immunohistochemistry (red). Note <i>Mitf</i> expression in ureteric bud and tip (black arrow), in and S-shaped body (blue arrow). Magnification X400. Sections are representative images of 4 kidneys per genotype. <b>D</b>) Immunostaining of MITF-A in WT and HO MITF-A transgenic metanephroi at E13.5. Note the increase of MITF-A expression in UB stalks, tips and S-bodies. Magnification X400.</p

Impact of MITF-A overexpression on cell survival.

<p><b>A-B)</b> Cell proliferation in E13.5 kidneys from wild-type (WT) and homozygous (HO) MITF-A transgenic embryos. Proliferating cells were identified using an anti-phospho-histone H3 (pH3) <b>(A)</b> and an anti-PCNA antibody <b>(B)</b>. Magnifications are X400 and X600, respectively. Left panels: representative images of 5 kidneys; right panels: quantification of the number of pH3-positive and PCNA-positive cells per UB structure. <b>C)</b> Apoptosis was evaluated by TUNEL assay in E13.5 kidneys from WT and HO MITF-A transgenic embryos. Left panels: representative images of 5 kidneys (magnification X400); right panels: quantification of the number of TUNEL-positive cells per microscopic field. Data are means ± SEM. Quantifications were performed on three sections for each kidney (n = 5 mice per genotype). Mann-Whitney test; transgenic <i>versus</i> wild-type mice: *** <i>P</i> < 0.001.</p

Expression of candidate MITF-A targets in E13.5 kidneys.

<p><b>A)</b><i>In situ</i> hybridization of <i>Bmp7</i>, <i>Pax2</i> and <i>Wnt9b</i> in wild-type (WT) and homozygous (HO) MITF-A transgenic kidneys at E13.5 (magnification X200, n = 5–6 per genotype). <b>B)</b> Quantitative RT-PCR analysis of <i>Bmp7</i>, <i>Pax2</i> and <i>Wnt9b</i> mRNA expression in E13.5 kidneys of WT, heterozygous (HE) and HO MITF-A transgenic embryos (n = 6–9 per genotype). <b>C)</b> <i>In situ</i> hybridization of <i>Re</i>t, <i>Wnt11</i> and <i>Spry1</i> in WT and HO MITF-A transgenic kidneys at E13.5 (magnification X200, n = 5–6 per genotype). Note the increased staining of <i>Re</i>t mRNA in transgenic kidneys at E13.5. <b>D)</b> Quantitative RT-PCR analysis of <i>Re</i>t, <i>Wnt11</i> and <i>Spry1</i> mRNA expression in E13.5 kidneys of WT, HE and HO MITF-A transgenic embryos (n = 6–9 per genotype). Data are means ± SEM. ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: * P < 0.05, ** P < 0.01.</p

MITF-A modulates kidney branching morphogenesis.

<p><b>A)</b> Whole mount E13.5 metanephroi in wild-type (WT), heterozygous (HE) and homozygous (HO) MITF-A transgenic embryos (line 42) after staining with anti-Calbindin antibody. These are representative images of at least 6 embryos for each genotype. Bar = 100 μm. <b>B)</b> Morphology of kidneys in WT, HE and HO MITF-A transgenic embryos at E13.5. These are representative images of at least 6 embryos for each genotype. <b>C-D)</b> Ureteric bud (UB) branching, as assayed by counting the number of UB tips in (<b>C</b>) WT (n = 17), HE (n = 14) and HO (n = 25) MITF-A transgenic embryos and (<b>D</b>) <i>Mitfa</i>^<i>+/+</i> (n = 15) and <i>Mitfa</i>^<i>-/-</i> (n = 20) embryos at E13.5. <b>E-F)</b> <i>Mitf-A</i> mRNA expression evaluated by quantitative RT-PCR in kidneys from (<b>E</b>) WT, HE and HO MITF-A transgenic embryos (n = 7–8 per each genotype) and (<b>F</b>) <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> embryos (n = 7 and 3 per genotype, respectively) at E 13.5. Data are means ± SEM. For transgenic MITF-A mice: ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: *** P < 0.001, HE v<i>ersus</i> HO MITF-A transgenic mice: ## P < 0.01, ### P < 0.01. For <i>Mitfa</i> knockout mice: Mann-Whitney test; <i>Mitfa</i>^<i>-/-</i> versus: <i>Mitfa</i>^<i>+/+</i>: * P < 0.05, *** P < 0.001.</p