Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases.

The progression of proteinuric kidney diseases is associated with podocyte loss, but the mechanisms underlying this process remain unclear. Podocytes reenter the cell cycle to repair double-stranded DNA breaks. However, unsuccessful repair can result in podocytes crossing the G1/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity - its tissue-specific loss in mouse podocytes resulted in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle reentry was identified using FUCCI2aR mice, and we observed altered expression of cell-cycle associated proteins, such as p21, p53, cyclin B1, and cyclin D1. Podocyte-specific translating ribosome affinity purification and RNA-Seq revealed the downregulation of ribosomal RNA-processing 8 (Rrp8). Overexpression of Rrp8 in Pfn1-KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilin1 is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss.

Cell Cycle and Senescence Regulation by Podocyte Histone Deacetylase 1 and 2.

SIGNIFICANCE STATEMENT: The loss of integrity of the glomerular filtration barrier results in proteinuria that is often attributed to podocyte loss. Yet how damaged podocytes are lost remains unknown. Germline loss of murine podocyte-associated Hdac1 and Hdac2 ( Hdac1/2 ) results in proteinuria and collapsing glomerulopathy due to sustained double-stranded DNA damage. Hdac1/2 deletion induces loss of podocyte quiescence, cell cycle entry, arrest in G1, and podocyte senescence, observed both in vivo and in vitro . Through the senescence secretory associated phenotype, podocytes secrete proteins that contribute to their detachment. These results solidify the role of HDACs in cell cycle regulation and senescence, providing important clues in our understanding of how podocytes are lost following injury. BACKGROUND: Intact expression of podocyte histone deacetylases (HDAC) during development is essential for maintaining a normal glomerular filtration barrier because of its role in modulating DNA damage and preventing premature senescence. METHODS: Germline podocyte-specific Hdac1 and 2 ( Hdac1 / 2 ) double-knockout mice were generated to examine the importance of these enzymes during development. RESULTS: Podocyte-specific loss of Hdac1 / 2 in mice resulted in severe proteinuria, kidney failure, and collapsing glomerulopathy. Hdac1 / 2 -deprived podocytes exhibited classic characteristics of senescence, such as senescence-associated ß-galactosidase activity and lipofuscin aggregates. In addition, DNA damage, likely caused by epigenetic alterations such as open chromatin conformation, not only resulted in podocyte cell-cycle entry as shown in vivo by Ki67 expression and by FUCCI-2aR mice, but also in p21-mediated cell-cycle arrest. Through the senescence secretory associated phenotype, the damaged podocytes secreted proinflammatory cytokines, growth factors, and matrix metalloproteinases, resulting in subsequent podocyte detachment and loss, evidenced by senescent podocytes in urine. CONCLUSIONS: Hdac1 / 2 plays an essential role during development. Loss of these genes in double knockout mice leads to sustained DNA damage and podocyte senescence and loss.

Podocyte Endocytosis in Regulating the Glomerular Filtration Barrier.

Endocytosis is a mechanism that internalizes and recycles plasma membrane components and transmembrane receptors via vesicle formation, which is mediated by clathrin-dependent and clathrin-independent signaling pathways. Podocytes are specialized, terminally differentiated epithelial cells in the kidney, located on the outermost layer of the glomerulus. These cells play an important role in maintaining the integrity of the glomerular filtration barrier in conjunction with the adjacent basement membrane and endothelial cell layers within the glomerulus. An intact podocyte endocytic machinery appears to be necessary for maintaining podocyte function. De novo pathologic human genetic mutations and loss-of-function studies of critical podocyte endocytosis genes in genetically engineered mouse models suggest that this pathway contributes to the pathophysiology of development and progression of proteinuria in chronic kidney disease. Here, we review the mechanism of cellular endocytosis and its regulation in podocyte injury in the context of glomerular diseases. A thorough understanding of podocyte endocytosis may shed novel insights into its biological function in maintaining a functioning filter and offer potential targeted therapeutic strategies for proteinuric glomerular diseases.

A UVB-responsive common variant at chromosome band 7p21.1 confers tanning response and melanoma risk via regulation of the aryl hydrocarbon receptor, AHR.

Genome-wide association studies (GWASs) have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). Because ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWASs identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. Because AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 introduced via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression and altering melanocyte growth phenotypes upon exposure.

Hypothalamic estrogen receptor alpha establishes a sexually dimorphic regulatory node of energy expenditure.

Estrogen receptor a (ERa) signaling in the ventromedial hypothalamus (VMH) contributes to energy homeostasis by modulating physical activity and thermogenesis. However, the precise neuronal populations involved remain undefined. Here, we describe six neuronal populations in the mouse VMH by using single-cell RNA transcriptomics and in situ hybridization. ERa is enriched in populations showing sex biased expression of reprimo (Rprm), tachykinin 1 (Tac1), and prodynorphin (Pdyn). Female biased expression of Tac1 and Rprm is patterned by ERa-dependent repression during male development, whereas male biased expression of Pdyn is maintained by circulating testicular hormone in adulthood. Chemogenetic activation of ERa positive VMH neurons stimulates heat generation and movement in both sexes. However, silencing Rprm gene function increases core temperature selectively in females and ectopic Rprm expression in males is associated with reduced core temperature. Together these findings reveal a role for Rprm in temperature regulation and ERa in the masculinization of neuron populations that underlie energy expenditure.

Partial Rescue of Ocular Pigment Cells and Structure by Inducible Ectopic Expression of Mitf-M in MITF-Deficient Mice.

Purpose: Complete deficiency of microphthalmia transcription factor (MITF) in Mitfmi-vga9/mi-vga9 mice is associated with microphthalmia, retinal dysplasia, and albinism. We investigated the ability of dopachrome tautomerase (DCT) promoter-mediated inducible ectopic expression of Mitf-M to rescue these phenotypic abnormalities. Methods: A new mouse line was created with doxycycline-inducible ectopic Mitf-M expression on an Mitf-deficient Mitfmi-vga9 background (DMV mouse). Adult DMV mice were phenotypically characterized and tissues were collected for histology, immunohistochemistry, and evaluation of Mitf, pigmentary genes, and retinal pigment epithelium (RPE) gene expression. Results: Ectopic Mitf-M expression was specifically induced in the eyes, but was not detected in the skin of DMV mice. Inducible expression of Mitf-M partially rescued the microphthalmia, RPE structure, and pigmentation as well as a subset of the choroidal and iris melanocytes but not cutaneous melanocytes. RPE function and vision were not restored in the DMV mice. Conclusions: Ectopic expression of Mitf-M during development of Mitf-deficient mice is capable of partially rescuing ocular and retinal structures and uveal melanocytes. These findings provide novel information about the roles of Mitf isoforms in the development of mouse eyes.