Epigenetic regulation of renal development.

Developmental changes in cell fate are tightly regulated by cell-type specific transcription factors. Chromatin reorganization during organismal development ensures dynamic access of developmental regulators to their cognate DNA sequences. Thus, understanding the epigenomic states of promoters and enhancers is of key importance. Recent years have witnessed significant advances in our knowledge of the transcriptional mechanisms of kidney development. Emerging evidence suggests that histone deacetylation by class I HDACs and H3 methylation on lysines 4, 27 and 79 play important roles in regulation of early and late gene expression in the developing kidney. Equally exciting is the realization that nephrogenesis genes in mesenchymal nephron progenitors harbor bivalent chromatin domains which resolve upon differentiation implicating chromatin bivalency in developmental control of gene expression. Here, we review current knowledge of the epigenomic states of nephric cells and current techniques used to study the dynamic chromatin states. These technological advances will provide an unprecedented view of the enhancer landscape during cell fate commitment and help in defining the complex transcriptional networks governing kidney development and disease.

The polycomb proteins EZH1 and EZH2 co-regulate chromatin accessibility and nephron progenitor cell lifespan in mice.

SIX2 (SIX homeobox 2)-positive nephron progenitor cells (NPCs) give rise to all epithelial cell types of the nephron, the filtering unit of the kidney. NPCs have a limited lifespan and are depleted near the time of birth. Epigenetic factors are implicated in the maintenance of organ-restricted progenitors such as NPCs, but the chromatin-based mechanisms are incompletely understood. Here, using a combination of gene targeting, chromatin profiling, and single-cell RNA analysis, we examined the role of the murine histone 3 Lys-27 (H3K27) methyltransferases EZH1 (enhancer of zeste 1) and EZH2 in NPC maintenance. We found that EZH2 expression correlates with NPC growth potential and that EZH2 is the dominant H3K27 methyltransferase in NPCs and epithelial descendants. Surprisingly, NPCs lacking H3K27 trimethylation maintained their progenitor state but cycled slowly, leading to a smaller NPC pool and formation of fewer nephrons. Unlike Ezh2 loss of function, dual inactivation of Ezh1 and Ezh2 triggered overexpression of the transcriptional repressor Hes-related family BHLH transcription factor with YRPW motif 1 (Hey1), down-regulation of Six2, and unscheduled activation of Wnt4-driven differentiation, resulting in early termination of nephrogenesis and severe renal dysgenesis. Double-mutant NPCs also overexpressed the SIX family member Six1 However, in this context, SIX1 failed to maintain NPC stemness. At the chromatin level, EZH1 and EZH2 restricted accessibility to AP-1-binding motifs, and their absence promoted a regulatory landscape akin to differentiated and nonlineage cells. We conclude that EZH2 is required for NPC renewal potential and that tempering of the differentiation program requires cooperation of both EZH1 and EZH2.

Angiotensin II biphasically regulates cell differentiation in human iPSC-derived kidney organoids.

Human kidney organoid technology holds promise for novel kidney disease treatment strategies and utility in pharmacological and basic science. Given the crucial roles of the intrarenal renin-angiotensin system (RAS) and angiotensin II (ANG II) in the progression of kidney development and injury, we investigated the expression of RAS components and effects of ANG II on cell differentiation in human kidney organoids. Human induced pluripotent stem cell-derived kidney organoids were induced using a modified 18-day Takasato protocol. Gene expression analysis by digital PCR and immunostaining demonstrated the formation of renal compartments and expression of RAS components. The ANG II type 1 receptor (AT1R) was strongly expressed in the early phase of organoid development (around day 0), whereas ANG II type 2 receptor (AT2R) expression levels peaked on day 5. Thus, the organoids were treated with 100 nM ANG II in the early phase on days 0-5 (ANG II-E) or during the middle phase on days 5-10 (ANG II-M). ANG II-E was observed to decrease levels of marker genes for renal tubules and proximal tubules, and the downregulation of renal tubules was inhibited by an AT1R antagonist. In contrast, ANG II-M increased levels of markers for podocytes, the ureteric tip, and the nephrogenic mesenchyme, and an AT2R blocker attenuated the ANG II-M-induced augmentation of podocyte formation. These findings demonstrate RAS expression and ANG II exertion of biphasic effects on cell differentiation through distinct mediatory roles of AT1R and AT2R, providing a novel strategy to establish and further characterize the developmental potential of human induced pluripotent stem cell-derived kidney organoids.NEW & NOTEWORTHY This study demonstrates angiotensin II exertion of biphasic effects on cell differentiation through distinct mediatory roles of angiotensin II type 1 receptor and type 2 receptor in human induced pluripotent stem cell-derived kidney organoids, providing a novel strategy to establish and further characterize the developmental potential of the human kidney organoids.

Histone deacetylases 1 and 2 regulate the transcriptional programs of nephron progenitors and renal vesicles.

Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a, as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles.

Human Cytomegalovirus Alters Host Cell Mitochondrial Function during Acute Infection.

Human cytomegalovirus (HCMV) is a large DNA herpesvirus that is highly prevalent in the human population. HCMV can result in severe direct and indirect pathologies under immunosuppressed conditions and is the leading cause of birth defects related to infectious disease. Currently, the effect of HCMV infection on host cell metabolism as an increase in glycolysis during infection has been defined. We have observed that oxidative phosphorylation is also increased. We have identified morphological and functional changes to host mitochondria during HCMV infection. The mitochondrial network undergoes fission events after HCMV infection. Interestingly, the network does not undergo fusion. At the same time, mitochondrial mass and membrane potential increase. The electron transport chain (ETC) functions at an elevated rate, resulting in the release of increased reactive oxygen species. Surprisingly, despite the stress applied to the host mitochondria, the network is capable of responding to and meeting the increased bioenergetic and biosynthetic demands placed on it. When mitochondrial DNA is depleted from the cells, we observed severe impairment of viral replication. Mitochondrial DNA encodes many of the ETC components. These findings suggest that the host cell ETC is essential to HCMV replication. Our studies suggest the host cell mitochondria may be a therapeutic target.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus present in up to 85% of some populations. Like all herpesviruses, HCMV infection is for life. No vaccine is currently available, neutralizing antibody therapies are ineffective, and current antivirals have limited long-term efficacy due to side effects and potential for viral mutation and resistance. The significance of this research is in understanding how HCMV manipulates the host mitochondria to support bioenergetic and biosynthetic requirements for replication. Despite a large genome, HCMV relies exclusively on host cells for metabolic functions. By understanding the dependency of HCMV on the mitochondria, we could exploit these requirements and develop novel antivirals.

Metabolic programming of nephron progenitor cell fate.

Metabolic pathways are one of the first responses at the cellular level to maternal/fetal interface stressors. Studies have revealed the previously unrecognized contributions of intermediary metabolism to developmental programs. Here, we provide an overview of cellular metabolic pathways and the cues that modulate metabolic states. We discuss the developmental and physiological implications of metabolic reprogramming and the key role of metabolites in epigenetic and epiproteomic modifications during embryonic development and with respect to kidney development and nephrogenesis.

Von Hippel-Lindau Acts as a Metabolic Switch Controlling Nephron Progenitor Differentiation.

BACKGROUND: Nephron progenitors, the cell population that give rise to the functional unit of the kidney, are metabolically active and self-renew under glycolytic conditions. A switch from glycolysis to mitochondrial respiration drives these cells toward differentiation, but the mechanisms that control this switch are poorly defined. Studies have demonstrated that kidney formation is highly dependent on oxygen concentration, which is largely regulated by von Hippel-Lindau (VHL; a protein component of a ubiquitin ligase complex) and hypoxia-inducible factors (a family of transcription factors activated by hypoxia). METHODS: To explore VHL as a regulator defining nephron progenitor self-renewal versus differentiation, we bred Six2-TGCtg mice with VHLlox/lox mice to generate mice with a conditional deletion of VHL from Six2+ nephron progenitors. We used histologic, immunofluorescence, RNA sequencing, and metabolic assays to characterize kidneys from these mice and controls during development and up to postnatal day 21. RESULTS: By embryonic day 15.5, kidneys of nephron progenitor cell-specific VHL knockout mice begin to exhibit reduced maturation of nephron progenitors. Compared with controls, VHL knockout kidneys are smaller and developmentally delayed by postnatal day 1, and have about half the number of glomeruli at postnatal day 21. VHL knockout nephron progenitors also exhibit persistent Six2 and Wt1 expression, as well as decreased mitochondrial respiration and prolonged reliance on glycolysis. CONCLUSIONS: Our findings identify a novel role for VHL in mediating nephron progenitor differentiation through metabolic regulation, and suggest that VHL is required for normal kidney development.

Histone deacetylase 1 and 2 regulate Wnt and p53 pathways in the ureteric bud epithelium.

Histone deacetylases (HDACs) regulate a broad range of biological processes through removal of acetyl groups from histones as well as non-histone proteins. Our previous studies showed that Hdac1 and Hdac2 are bound to promoters of key renal developmental regulators and that HDAC activity is required for embryonic kidney gene expression. However, the existence of many HDAC isoforms in embryonic kidneys raises questions concerning the possible specificity or redundancy of their functions. We report here that targeted deletion of both the Hdac1 and Hdac2 genes from the ureteric bud (UB) cell lineage of mice causes bilateral renal hypodysplasia. One copy of either Hdac1 or Hdac2 is sufficient to sustain normal renal development. In addition to defective cell proliferation and survival, genome-wide transcriptional profiling revealed that the canonical Wnt signaling pathway is specifically impaired in UB(Hdac1,2-/-) kidneys. Our results also demonstrate that loss of Hdac1 and Hdac2 in the UB epithelium leads to marked hyperacetylation of the tumor suppressor protein p53 on lysine 370, 379 and 383; these post-translational modifications are known to boost p53 stability and transcriptional activity. Genetic deletion of p53 partially rescues the development of UB(Hdac1,2-/-) kidneys. Together, these data indicate that Hdac1 and Hdac2 are crucial for kidney development. They perform redundant, yet essential, cell lineage-autonomous functions via p53-dependent and -independent pathways.

p53 Enables metabolic fitness and self-renewal of nephron progenitor cells.

Contrary to its classic role in restraining cell proliferation, we demonstrate here a divergent function of p53 in the maintenance of self-renewal of the nephron progenitor pool in the embryonic mouse kidney. Nephron endowment is regulated by progenitor availability and differentiation potential. Conditional deletion of p53 in nephron progenitor cells (Six2Cre(+);p53(fl/fl)) induces progressive depletion of Cited1(+)/Six2(+) self-renewing progenitors and loss of cap mesenchyme (CM) integrity. The Six2(p53-null) CM is disorganized, with interspersed stromal cells and an absence of a distinct CM-epithelia and CM-stroma interface. Impaired cell adhesion and epithelialization are indicated by decreased E-cadherin and NCAM expression and by ineffective differentiation in response to Wnt induction. The Six2Cre(+);p53(fl/fl) cap has 30% fewer Six2(GFP(+)) cells. Apoptotic index is unchanged, whereas proliferation index is significantly reduced in accordance with cell cycle analysis showing disproportionately fewer Six2Cre(+);p53(fl/fl) cells in the S and G2/M phases compared with Six2Cre(+);p53(+/+) cells. Mutant kidneys are hypoplastic with fewer generations of nascent nephrons. A significant increase in mean arterial pressure is observed in early adulthood in both germline and conditional Six2(p53-null) mice, linking p53-mediated defects in kidney development to hypertension. RNA-Seq analyses of FACS-isolated wild-type and Six2(GFP(+)) CM cells revealed that the top downregulated genes in Six2Cre(+);p53(fl/fl) CM belong to glucose metabolism and adhesion and/or migration pathways. Mutant cells exhibit a ∼ 50% decrease in ATP levels and a 30% decrease in levels of reactive oxygen species, indicating energy metabolism dysfunction. In summary, our data indicate a novel role for p53 in enabling the metabolic fitness and self-renewal of nephron progenitors.

Renal involvement in PMM2-CDG, a mini-review.

Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common N-linked glycosylation disorder. The majority of patients present with a multisystem phenotype, including central nervous system involvement, hepatopathy, gastrointestinal and cardiac symptoms, endocrine dysfunction and abnormal coagulation. Renal abnormalities including congenital malformations and altered renal function are part of the multisystem manifestations of congenital disorders of glycosylation. We reviewed the literature on 933 patients with molecularly and/or enzymatically confirmed PMM2 deficiency to evaluate the incidence of renal involvement in PMM2-CDG. Renal abnormalities were reported in 56 patients. Congenital abnormalities were present in 41 out of these 55. Cystic kidney and mild proteinuria were the most common findings. One of the most severe renal manifestations, congenital nephrotic syndrome, was detected in 6 children. Renal manifestations were not associated with the presence of specific PMM2 alleles. This review summarizes the reported renal abnormalities in PMM2-CDG and draws attention to the pathophysiological impact of abnormal glycosylation on kidney structure and function.

Regulation of Nephron Progenitor Cell Self-Renewal by Intermediary Metabolism.

Nephron progenitor cells (NPCs) show an age-dependent capacity to balance self-renewal with differentiation. Older NPCs (postnatal day 0) exit the progenitor niche at a higher rate than younger (embryonic day 13.5) NPCs do. This behavior is reflected in the transcript profiles of young and old NPCs. Bioenergetic pathways have emerged as important regulators of stem cell fate. Here, we investigated the mechanisms underlying this regulation in murine NPCs. Upon isolation and culture in NPC renewal medium, younger NPCs displayed a higher glycolysis rate than older NPCs. Inhibition of glycolysis enhanced nephrogenesis in cultured embryonic kidneys, without increasing ureteric tree branching, and promoted mesenchymal-to-epithelial transition in cultured isolated metanephric mesenchyme. Cotreatment with a canonical Wnt signaling inhibitor attenuated but did not entirely block the increase in nephrogenesis observed after glycolysis inhibition. Furthermore, inhibition of the phosphatidylinositol 3-kinase/Akt self-renewal signaling pathway or stimulation of differentiation pathways in the NPC decreased glycolytic flux. Our findings suggest that glycolysis is a pivotal, cell-intrinsic determinant of NPC fate, with a high glycolytic flux supporting self-renewal and inhibition of glycolysis stimulating differentiation.

Cardiac-restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis, and Dysfunction.

TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates IκB kinase (IKK)/NF-κB, JNK/AP-1, and c/EBPß and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of IKK/NF-κB, JNK/AP-1, c/EBPß, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction.

Hypoxia-inducible factor prolyl-4-hydroxylation in FOXD1 lineage cells is essential for normal kidney development.

Hypoxia in the embryo is a frequent cause of intra-uterine growth retardation, low birth weight, and multiple organ defects. In the kidney, this can lead to low nephron endowment, predisposing to chronic kidney disease and arterial hypertension. A key component in cellular adaptation to hypoxia is the hypoxia-inducible factor pathway, which is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3. In the adult kidney, PHD oxygen sensors are differentially expressed in a cell type-dependent manner and control the production of erythropoietin in interstitial cells. However, the role of interstitial cell PHDs in renal development has not been examined. Here we used a genetic approach in mice to interrogate PHD function in FOXD1-expressing stroma during nephrogenesis. We demonstrate that PHD2 and PHD3 are essential for normal kidney development as the combined inactivation of stromal PHD2 and PHD3 resulted in renal failure that was associated with reduced kidney size, decreased numbers of glomeruli, and abnormal postnatal nephron formation. In contrast, nephrogenesis was normal in animals with individual PHD inactivation. We furthermore demonstrate that the defect in nephron formation in PHD2/PHD3 double mutants required intact hypoxia-inducible factor-2 signaling and was dependent on the extent of stromal hypoxia-inducible factor activation. Thus, hypoxia-inducible factor prolyl-4-hydroxylation in renal interstitial cells is critical for normal nephron formation.

Conditional knockout of collecting duct bradykinin B2 receptors exacerbates angiotensin II-induced hypertension during high salt intake.

We elucidated the role of collecting duct kinin B2 receptor (B2R) in the development of salt-sensitivity and angiotensin II (ANG II)-induced hypertension. To this end, we used a Cre-Lox recombination strategy to generate mice lacking Bdkrb2 gene for B2R in the collecting duct (Hoxb7-Cre(tg/+):Bdkrb2(flox/flox)). In 3 groups of control (Bdkrb2(flox/flox)) and 3 groups of UB(Bdkrb2-/-) mice, systolic blood pressure (SBP) responses to high salt intake (4 or 8% NaCl; HS) were monitored by radiotelemetry in comparison with standard salt diet (0.4% NaCl) prior to and during subcutaneous ANG II infusion (1000 ng/min/kg) via osmotic minipumps. High salt intakes alone for 2 weeks did not alter SBP in either strain. ANG II significantly increased SBP equally in control (121 ± 2 to 156 ± 3 mmHg) and UB(Bdkrb2-/-) mice (120 ± 2 to 153 ± 2 mmHg). The development of ANG II-induced hypertension was exacerbated by 4%HS in both control (125 ± 3 to 164 ± 5 mmHg) and UB(Bdkrb2-/-) mice (124 ± 2 to 162 ± 3 mmHg) during 2 weeks. Interestingly, 8%HS caused a more profound and earlier ANG II-induced hypertension in UB(Bdkrb2-/-) (129 ± 2 to 166 ± 3 mmHg) as compared to control (128 ± 2 to 158 ± 2 mmHg) and it was accompanied by body weight loss and increased mortality. In conclusion, targeted inactivation of B2R in the renal collecting duct does not cause salt-sensitivity; however, collecting duct B2R attenuates the hypertensive actions of ANG II under conditions of very high salt intake.

Angiotensin II regulates brain (pro)renin receptor expression through activation of cAMP response element-binding protein.

We reported that brain (pro)renin receptor (PRR) expression levels are elevated in DOCA-salt-induced hypertension; however, the underlying mechanism remained unknown. To address whether ANG II type 1 receptor (AT1R) signaling is involved in this regulation, we implanted a DOCA pellet and supplied 0.9% saline as the drinking solution to C57BL/6J mice. Sham pellet-implanted mice that were provided regular drinking water served as controls. Concurrently, mice were intracerebroventricularly infused with the AT1R blocker losartan, angiotensin-converting-enzyme inhibitor captopril, or artificial cerebrospinal fluid for 3 wk. Intracerebroventricular infusion of losartan or captopril attenuated DOCA-salt-induced PRR mRNA elevation in the paraventricular nucleus of the hypothalamus, suggesting a role for ANG II/AT1R signaling in regulating PRR expression during DOCA-salt hypertension. To test which ANG II/AT1R downstream transcription factors were involved in PRR regulation, we treated Neuro-2A cells with ANG II with or without CREB (cAMP response element-binding protein) or AP-1 (activator protein-1) inhibitors, or CREB siRNA. CREB and AP-1 inhibitors, as well as CREB knockdown abolished ANG II-induced increases in PRR levels. ANG II also induced PRR upregulation in primary cultured neurons. Chromatin immunoprecipitation assays revealed that ANG II treatment increased CREB binding to the endogenous PRR promoter in both cultured neurons and hypothalamic tissues of DOCA-salt hypertensive mice. This increase in CREB activity was reversed by AT1R blockade. Collectively, these findings indicate that ANG II acts via AT1R to upregulate PRR expression both in cultured cells and in DOCA-salt hypertensive mice by increasing CREB binding to the PRR promoter.

Genome-wide analysis of gestational gene-environment interactions in the developing kidney.

The G protein-coupled bradykinin B2 receptor (Bdkrb2) plays an important role in regulation of blood pressure under conditions of excess salt intake. Our previous work has shown that Bdkrb2 also plays a developmental role since Bdkrb2(-/-) embryos, but not their wild-type or heterozygous littermates, are prone to renal dysgenesis in response to gestational high salt intake. Although impaired terminal differentiation and apoptosis are consistent findings in the Bdkrb2(-/-) mutant kidneys, the developmental pathways downstream of gene-environment interactions leading to the renal phenotype remain unknown. Here, we performed genome-wide transcriptional profiling on embryonic kidneys from salt-stressed Bdkrb2(+/+) and Bdkrb2(-/-) embryos. The results reveal significant alterations in key pathways regulating Wnt signaling, apoptosis, embryonic development, and cell-matrix interactions. In silico analysis reveal that nearly 12% of differentially regulated genes harbor one or more Pax2 DNA-binding sites in their promoter region. Further analysis shows that metanephric kidneys of salt-stressed Bdkrb2(-/-) have a significant downregulation of Pax2 gene expression. This was corroborated in Bdkrb2(-/-);Pax2(GFP+/tg) mice, demonstrating that Pax2 transcriptional activity is significantly repressed by gestational salt-Bdkrb2 interactions. We conclude that gestational gene (Bdkrb2) and environment (salt) interactions cooperate to impact gene expression programs in the developing kidney. Suppression of Pax2 likely contributes to the defects in epithelial survival, growth, and differentiation in salt-stressed BdkrB2(-/-) mice.

The MDM2-p53 pathway: multiple roles in kidney development.

The molecular basis of nephron progenitor cell renewal and differentiation into nascent epithelial nephrons is an area of intense investigation. Defects in these early stages of nephrogenesis lead to renal hypoplasia, and eventually hypertension and chronic kidney disease. Terminal nephron differentiation, the process by which renal epithelial precursor cells exit the cell cycle and acquire physiological functions is equally important. Failure of terminal epithelial cell differentiation results in renal dysplasia and cystogenesis. Thus, a better understanding of the transcriptional frameworks that regulate early and late renal cell differentiation is of great clinical significance. In this review, we will discuss evidence implicating the MDM2-p53 pathway in cell fate determination during development. The emerging central theme from loss- and gain-of-function studies is that tight regulation of p53 levels and transcriptional activity is absolutely required for nephrogenesis. We will also discuss how post-translational modifications of p53 (e.g., acetylation and phosphorylation) alter the spatiotemporal and functional properties of p53 and thus cell fate during kidney development. Mutations and polymorphisms in the MDM2-p53 pathway are present in more than 50 % of cancers in humans. This raises the question of whether sequence variants in the MDM2-p53 pathway increase the susceptibility to renal dysgenesis, hypertension or chronic kidney disease. With the advent of whole exome sequencing and other high throughput technologies, this hypothesis is testable in cohorts of children with renal dysgenesis.

Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney.

Despite mounting evidence that p53 senses and responds to physiological cues in vivo, existing knowledge regarding p53 function and target genes is largely derived from studies in cancer or stressed cells. Herein we utilize p53 transcriptome and ChIP-Seq (chromatin immunoprecipitation-high throughput sequencing) analyses to identify p53 regulated pathways in the embryonic kidney, an organ that develops via mesenchymal-epithelial interactions. This integrated approach allowed identification of novel genes that are possible direct p53 targets during kidney development. We find the p53-regulated transcriptome in the embryonic kidney is largely composed of genes regulating developmental, morphogenesis, and metabolic pathways. Surprisingly, genes in cell cycle and apoptosis pathways account for <5% of differentially expressed transcripts. Of 7,893 p53-occupied genomic regions (peaks), the vast majority contain consensus p53 binding sites. Interestingly, 78% of p53 peaks in the developing kidney lie within proximal promoters of annotated genes compared with 7% in a representative cancer cell line; 25% of the differentially expressed p53-bound genes are present in nephron progenitors and nascent nephrons, including key transcriptional regulators, components of Fgf, Wnt, Bmp, and Notch pathways, and ciliogenesis genes. The results indicate widespread p53 binding to the genome in vivo and context-dependent differences in the p53 regulon between cancer, stress, and development. To our knowledge, this is the first comprehensive analysis of the p53 transcriptome and cistrome in a developing mammalian organ, substantiating the role of p53 as a bona fide developmental regulator. We conclude p53 targets transcriptional networks regulating nephrogenesis and cellular metabolism during kidney development.

Interactions between BdkrB2 and p53 genes in the developing kidney.

Gene-environment interactions are implicated in congenital disorders. Accordingly, there is a pressing need to develop animal models of human disease, which are the product of defined gene-environment interactions. Work from our laboratory demonstrates the presence of genetic interactions between the bradykinin B2 receptor (BdkrB2) and the tumor suppressor protein p53 in the developing kidney. Our studies have shown that the Bdkrb2(-/-) embryos exposed to gestational salt stress develop renal dysgenesis. The underlying mechanism is p53 stabilization and mediated apoptosis and repression of the terminal epithelial differentiation program. We also uncovered a novel functional cross-talk between p53 and BdkrB2. Thus, while BdkrB2 is a target for p53-mediated transcriptional activation, BdkrB2 inactivation results in the upregulation of checkpoint kinase 1 (Chk1) levels, thus potentiating phosphorylation of p53 on Ser23 by Chk1, an essential step in the pathway leading to renal dysgenesis in salt-stressed BdkrB2(-/-) mutant mice. Future studies will now focus on defining how this G-protein-coupled receptor is coupled to the activation of p53, a tumor suppressor gene that is mutated in more than 50% of all human cancers.

Endoplasmic reticulum stress is involved in cardiac damage and vascular endothelial dysfunction in hypertensive mice.

OBJECTIVE: Cardiac damage and vascular dysfunction are major causes of morbidity and mortality in hypertension. In the present study, we explored the beneficial therapeutic effect of endoplasmic reticulum (ER) stress inhibition on cardiac damage and vascular dysfunction in hypertension. METHODS AND RESULTS: Mice were infused with angiotensin II (400 ng/kg per minute) with or without ER stress inhibitors (taurine-conjugated ursodeoxycholic acid and 4-phenylbutyric acid) for 2 weeks. Mice infused with angiotensin II displayed an increase in blood pressure, cardiac hypertrophy and fibrosis associated with enhanced collagen I content, transforming growth factor-ß1 (TGF-ß1) activity, and ER stress markers, which were blunted after ER stress inhibition. Hypertension induced ER stress in aorta and mesenteric resistance arteries (MRA), enhanced TGF-ß1 activity in aorta but not in MRA, and reduced endothelial NO synthase phosphorylation and endothelium-dependent relaxation (EDR) in aorta and MRA. The inhibition of ER stress significantly reduced TGF-ß1 activity, enhanced endothelial NO synthase phosphorylation, and improved EDR. The inhibition of TGF-ß1 pathway improved EDR in aorta but not in MRA, whereas the reduction in reactive oxygen species levels ameliorated EDR in MRA only. Infusion of tunicamycin in control mice induced ER stress in aorta and MRA, and reduced EDR by a TGF-ß1-dependent mechanism in aorta and reactive oxygen species-dependent mechanism in MRA. CONCLUSIONS: ER stress inhibition reduces cardiac damage and improves vascular function in hypertension. Therefore, ER stress could be a potential target for cardiovascular diseases.