Final Report Summary - RENAL EPIGENETICS (Epigenetic modifications in glomerular nephropathy and renal aging)
The way in which the renal cell epigenome is altered by environmental modifiers driving the onset and progression of renal diseases has extended our understanding of the pathophysiology of kidney disease progression. Until recently a thorough characterization of the transcriptome and proteome of endogenous podocytes, the highly specific epithelial cells of the kidney that form the filtration barrier, has been hampered by low cell yields during isolation. We established a double fluorescent reporter mouse model combined with an optimized bead perfusion protocol and efficient single cell dissociation allowing us global, unbiased downstream applications. Combining mRNA and miRNA transcriptional profiling with quantitative proteomic analyses revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport, and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. This approach let us to the discovery and integration of novel gene, protein, and organelle regulatory networks of renal aging and diabetic nephropathy. In our subsequent studies, we identified autophagic cell repair as a direct determinant of cellular aging for long-lived glomerular podocytes. However, the molecular mechanisms of autophagy/mTOR-signaling that maintain podocytes and consequently the integrity of the glomerular filtration barrier and kidney aging remained only very incompletely understood. We show that the mTOR-regulated class III phosphoinositide 3-kinase vacuolar protein sorting 34 (Vps34) is a key player in endocytotic and autophagic vesicle maturation, maintaining podocyte homeostasis.
Epigenetic mechanisms are fundamental key features of developing cells connecting developmental regulatory factors to chromatin modification, i.e. DNA methylation and histone modifications determine renal programming and disease progression. Changes in the environment during renal development can have long-lasting effects on the permanent tissue structure and the level of expression of important functional genes. These changes are believed to contribute to kidney disease occurrence and progression. Although the mechanisms of early patterning and cell fate have been well described for renal development, little was known about associated epigenetic modifications and their impact on how genes interact to specify the renal epithelial cells of nephrons and how this specification is relevant to maintaining normal renal function. Observational studies suggest that maternal nutritional and metabolic factors during gestation contribute to the high variability of nephron endowment. However, the underlying molecular mechanisms have been unclear.
Analyzing the differential role of MOF, the major H4K16 lysine acetyltransferase for embryonic development in Drosophila and mammals, revealed that MOF is absolutely critical for podocyte maintenance in response to injury although it seems to be dispensable under physiological conditions. Genome-wide expression analyses of podocytes uncovered several MOF-regulated pathways required for stress response. We found that MOF directly binds to genes encoding the lysosome, endocytosis and vacuole pathways, known regulators of podocyte maintenance. Thus, our work identified MOF as a key regulator of cellular stress response in glomerular podocytes. Since nephron number is a major determinant of long-term renal function and cardiovascular risk, we used mouse models, including DNA methyltransferase (Dnmt1, Dnmt3a, and Dnmt3b) knockout mice, optical projection tomography, three-dimensional reconstructions of the nephrogenic niche, and transcriptome and DNA methylation analyses to characterize the role of DNA methylation for kidney development. We demonstrate that DNA hypomethylation is a key feature of nutritional kidney growth restriction in vitro and in vivo, and that DNA methyltransferases Dnmt1 and Dnmt3a are highly enriched in the nephrogenic zone of the developing kidneys. Deletion of Dnmt1 in nephron progenitor cells mimics nutritional models of kidney growth restriction and results in a significant reduction of nephron number as well as renal hypoplasia at birth. In Dnmt1-deficient mice, optical projection tomography and three-dimensional reconstructions uncovered a significant reduction of stem cell niches and progenitor cells. RNA sequencing analysis revealed that global DNA hypomethylation interferes in the progenitor cell regulatory network, leading to downregulation of genes crucial for initiation of nephrogenesis, Wt1 and its target Wnt4. Derepression of germline genes, protocadherins, Rhox genes, and endogenous retroviral elements resulted in the upregulation of IFN targets and inhibitors of cell cycle progression. These findings establish DNA methylation as a key regulatory event of prenatal renal programming, which possibly represents a fundamental link between maternal nutritional factors during gestation and reduced nephron number. The analyses of epigenetic regulators using podocyte-specific knockout mice allowed us to comprehensively understand the role of epigenetic modifications in podocytes, to identify key mechanisms of the molecular programs that promote kidney aging and disease progression and to detect new therapeutic targets for the chronic loss of renal function.
Epigenetic mechanisms are fundamental key features of developing cells connecting developmental regulatory factors to chromatin modification, i.e. DNA methylation and histone modifications determine renal programming and disease progression. Changes in the environment during renal development can have long-lasting effects on the permanent tissue structure and the level of expression of important functional genes. These changes are believed to contribute to kidney disease occurrence and progression. Although the mechanisms of early patterning and cell fate have been well described for renal development, little was known about associated epigenetic modifications and their impact on how genes interact to specify the renal epithelial cells of nephrons and how this specification is relevant to maintaining normal renal function. Observational studies suggest that maternal nutritional and metabolic factors during gestation contribute to the high variability of nephron endowment. However, the underlying molecular mechanisms have been unclear.
Analyzing the differential role of MOF, the major H4K16 lysine acetyltransferase for embryonic development in Drosophila and mammals, revealed that MOF is absolutely critical for podocyte maintenance in response to injury although it seems to be dispensable under physiological conditions. Genome-wide expression analyses of podocytes uncovered several MOF-regulated pathways required for stress response. We found that MOF directly binds to genes encoding the lysosome, endocytosis and vacuole pathways, known regulators of podocyte maintenance. Thus, our work identified MOF as a key regulator of cellular stress response in glomerular podocytes. Since nephron number is a major determinant of long-term renal function and cardiovascular risk, we used mouse models, including DNA methyltransferase (Dnmt1, Dnmt3a, and Dnmt3b) knockout mice, optical projection tomography, three-dimensional reconstructions of the nephrogenic niche, and transcriptome and DNA methylation analyses to characterize the role of DNA methylation for kidney development. We demonstrate that DNA hypomethylation is a key feature of nutritional kidney growth restriction in vitro and in vivo, and that DNA methyltransferases Dnmt1 and Dnmt3a are highly enriched in the nephrogenic zone of the developing kidneys. Deletion of Dnmt1 in nephron progenitor cells mimics nutritional models of kidney growth restriction and results in a significant reduction of nephron number as well as renal hypoplasia at birth. In Dnmt1-deficient mice, optical projection tomography and three-dimensional reconstructions uncovered a significant reduction of stem cell niches and progenitor cells. RNA sequencing analysis revealed that global DNA hypomethylation interferes in the progenitor cell regulatory network, leading to downregulation of genes crucial for initiation of nephrogenesis, Wt1 and its target Wnt4. Derepression of germline genes, protocadherins, Rhox genes, and endogenous retroviral elements resulted in the upregulation of IFN targets and inhibitors of cell cycle progression. These findings establish DNA methylation as a key regulatory event of prenatal renal programming, which possibly represents a fundamental link between maternal nutritional factors during gestation and reduced nephron number. The analyses of epigenetic regulators using podocyte-specific knockout mice allowed us to comprehensively understand the role of epigenetic modifications in podocytes, to identify key mechanisms of the molecular programs that promote kidney aging and disease progression and to detect new therapeutic targets for the chronic loss of renal function.