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The role of the transcription factor GATA3 in kidney function and disease

Final Report Summary - GATA3 IN KIDNEY (The role of the transcription factor GATA3 in kidney function and disease)

GATA-binding protein 3 (GATA3) is a dual-zinc finger transcription factor that regulates developmental gene expression in a variety of tissues. In the kidney, GATA3 is known to be essential for ureteric bud branching and its deficiency in mice causes renal agenesis. In man, autosomal dominant GATA3 mutations can cause renal aplasia as part of the hypoparathyroidism, renal dysplasia, deafness (HDR) syndrome that also includes mesangioproliferative glomerulonephritis. This suggests that GATA3 might have a previously unrecognized role in glomerular development, homeostasis or in response to injury and the overall purpose of this project was to determine whether this was the case. To define the role of GATA3 in kidney function both in health and in individuals with progressive glomerular disease and their rodent analogues, four objectives have been addressed and experimental data has been generated. The specific objectives were to:
1) Assess the expression pattern of GATA3 in developing and mature kidneys and in kidneys with glomerulonephritis;
2) Determine whether heterozygous (+/-) Gata3 knockout mice have glomerular abnormalities;
3) Investigate the role of GATA3 in the Wnt/beta-catenin pathway and other cellar pathways;
4) Identify the target genes of GATA3 in normal adult kidneys.

After optimising immunohistological techniques to detect cell-specific expression of GATA3 in human and rodent kidneys by light, confocal and electron microscopy, I was able to identify the precise cell types where GATA3 is expressed in the mature kidneys from humans, mice and rats, as well as their progenitor cells in the developing kidneys. These techniques were then used to investigate rodent models of glomerulonephritis to assess alterations in GATA3 expression in during glomerular injury. The immunohistological studies were complemented by in vitro studies using appropriate renal cell lines and primary cell cultures to identify changes in GATA3 expression under various growth conditions, and cellular changes which result when GATA3 is over- or under-expressed. Chromatin immunoprecipitation experiments and luciferase assays were optimised and have been used to identify target genes of GATA3 in the renin and mesangial cells lines. I have used the Gata3 knockout mice (in collaboration with Professor Meinrad Busslinger, IMP, Vienna) where GFP is expressed in all endogenous Gata3 expression domains, to optimise a strategy for isolating renal Gata3-expressing cells by fluorescence-activated cell sorting (FACS) for studies which will allow me to assess the molecular changes which occur during glomerular injury and how these are affected by haploinsufficiency of Gata3 in the Gata3 heterozygote mice.

Results of experiments during the course this 2-year project provide the first systematic analysis of GATA3 expression in the developing and mature kidneys. They show that GATA3 is strongly expressed in a subset of stromal progenitor cells during early stages of kidney development, as well as in the descendent cells originating from them in the adult mouse and human kidneys. Thus, GATA3 is expressed in: (i) intra- and extra-glomerular mesangial cells identified by co-expression of the platelet-derived growth factor receptor-beta (Pdgfr-beta); (ii) vascular smooth muscle cells of the renal arterioles that co-express alpha-smooth muscle actin (alpha-sma); (iii) renin-producing cells of the juxtaglomerular apparatus (JGA) that synthesise the aspartyl-protease renin; and iv) a subset of peritubular fibroblasts. This suggests GATA3 may influence specification of undifferentiated progenitor cells to their differentiated descendant cell types. Subsequently, I investigated this hypothesis by focusing on mesangial cells and their response to glomerular injury, and on the JGA cells in which GATA3 co-localises with renin. The main results achieved so far include:

1. The identification of strong GATA3 expression by mesangial cells, but not in other intrinsic glomerular cells including podocytes, endothelial or parietal epithelial cells; and that Gata3+/- mice have a reduced number of mesangial cells, identifying for the first time, the importance of GATA3 for normal glomerular development.

2. The demonstration that during development, GATA3 is expressed in mouse kidneys from embryonic day (E) 13.5 in a subset of stromal cells migrating into the vascular cleft of the developing glomeruli, co-localising with Pdgfr-beta and alpha-sma, thus identifying GATA3 as a novel early marker for mesangial cell precursors.

3. The discovery that GATA3 makes a non-redundant contribution to mesangial development because embryonic kidneys from Gata3+/- mice have significantly fewer GATA3-positive cells within the developing glomeruli than wild-type mice.

4. The demonstration that nuclear GATA3 expression is a much more robust marker for mesangial cells in health and disease that those currently available, and it can be used as a clinical biomarker to assess the severity of proliferative glomerulonephritis.

5. The discovery that GATA3 is also expressed in the renin producing cells of the juxtaglomerular apparatus (JGA) and is located in a cytoplasmic compartment that partially colocalises with renin, suggesting a novel function beyond its role as a transcription factor.

6. The demonstration that GATA3 is expressed as two isoforms in the kidney which are likely to have functionally different roles.

There is an urgent need for more detailed understanding of renal stromal progenitors and their differentiation into daughter cells, as these stromal cells have recently been recognised to play a crucial role in the overall development of the kidney, adult kidney repair, and in the pathobiology of renal fibrosis. An immediate benefit of the data arising from this project is that GATA3 was identified to be a good marker for stromal cells and their descendent cell types. Stromal-specific inactivation of Gata3 in mice will form the basis of future studies.
The phenotypic observation that Gata3+/- mice have fewer mesangial cells in glomeruli suggests that the Gata3+/- mice may provide a model to study the kidney abnormalities observed in HDR patients that are haploinsufficient for GATA3 and that have been reported to develop mesangioproliferative nephritis.
I have demonstrated that nuclear GATA3 expression is a robust marker for mesangial cells in health and disease that can be used as a biomarker to assess proliferative glomerulonephritis and may have clinical implications.