Constans, Megan M

Significance Statement The lack of rapidly progressive murine models reflecting the more severe end of the spectrum of autosomal recessive polycystic kidney disease (ARPKD) inhibits progress to understanding ARPKD pathogenesis. Defects in primary cilia have been implicated in polycystic kidney disease, but their potential role is poorly understood. The authors generated and characterized new mouse and rat models of ARPKD and autosomal dominant polycystic kidney disease (ADPKD) and investigated the interaction between causative genes for these two conditions. Their digenic models demonstrated a synergistic interaction that better reflects the early-onset disease characteristic of ARPKD. Analysis of mRNA expression in the models highlighted different disrupted pathways, but with a commonality of dysregulated mechanisms associated with primary cilia. These models may improve understanding of ARPKD and preclinical testing for this disease. Background Autosomal recessive polycystic kidney disease (ARPKD) and autosomal dominant polycystic kidney disease (ADPKD) are genetically distinct, with ADPKD usually caused by the genes PKD1 or PKD2 (encoding polycystin-1 and polycystin-2, respectively) and ARPKD caused by PKHD1 (encoding fibrocystin/polyductin [FPC]). Primary cilia have been considered central to PKD pathogenesis due to protein localization and common cystic phenotypes in syndromic ciliopathies, but their relevance is questioned in the simple PKDs. ARPKD’s mild phenotype in murine models versus in humans has hampered investigating its pathogenesis. Methods To study the interaction between Pkhd1 and Pkd1 , including dosage effects on the phenotype, we generated digenic mouse and rat models and characterized and compared digenic, monogenic, and wild-type phenotypes. Results The genetic interaction was synergistic in both species, with digenic animals exhibiting phenotypes of rapidly progressive PKD and early lethality resembling classic ARPKD. Genetic interaction between Pkhd1 and Pkd1 depended on dosage in the digenic murine models, with no significant enhancement of the monogenic phenotype until a threshold of reduced expression at the second locus was breached. Pkhd1 loss did not alter expression, maturation, or localization of the ADPKD polycystin proteins, with no interaction detected between the ARPKD FPC protein and polycystins. RNA-seq analysis in the digenic and monogenic mouse models highlighted the ciliary compartment as a common dysregulated target, with enhanced ciliary expression and length changes in the digenic models. Conclusions These data indicate that FPC and the polycystins work independently, with separate disease-causing thresholds; however, a combined protein threshold triggers the synergistic, cystogenic response because of enhanced dysregulation of primary cilia. These insights into pathogenesis highlight possible common therapeutic targets.

The failure of the polycystins (PCs) to function in primary cilia is thought to be responsible for autosomal dominant polycystic kidney disease (ADPKD). Primary cilia integrate multiple cellular signaling pathways, including calcium, cAMP, Wnt, and Hedgehog, which control cell proliferation and differentiation. It has been proposed that mutated PCs result in reduced intracellular calcium, which in turn upregulates cAMP, protein kinase A (PKA) signaling, and subsequently other proliferative signaling pathways. However, the role of PKA in ADPKD has not been directly ascertained in vivo, although the expression of the main regulatory subunit of PKA in cilia and other compartments (PKA-RIα, encoded by PRKAR1A) is increased in a mouse model orthologous to ADPKD. Therefore, we generated a kidney-specific knockout of Prkar1a to examine the consequences of constitutive upregulation of PKA on wild-type and Pkd1 hypomorphic ( Pkd1 RC ) backgrounds. Kidney-specific loss of Prkar1a induced renal cystic disease and markedly aggravated cystogenesis in the Pkd1 RC models. In both settings, it was accompanied by upregulation of Src, Ras, MAPK/ERK, mTOR, CREB, STAT3, Pax2 and Wnt signaling. On the other hand, Gli3 repressor activity was enhanced, possibly contributing to hydronephrosis and impaired glomerulogenesis in some animals. To assess the relevance of these observations in humans we looked for and found evidence for kidney and liver cystic phenotypes in the Carney complex, a tumoral syndrome caused by mutations in PRKAR1A. These observations expand our understanding of the pathogenesis of ADPKD and demonstrate the importance of PRKAR1A highlighting PKA as a therapeutic target in ADPKD.

Abstract To generate functional peripheral T cells, proper gene regulation during T cell development is critical. In this study, we found that histone deacetylase (HDAC) 3 is required for T cell development. T cell development in CD2-icre HDAC3 conditional knockout (cKO) mice (HDAC3-cKO) was blocked at positive selection, resulting in few CD4 and CD8 T cells, and it could not be rescued by a TCR transgene. These single-positive thymocytes failed to upregulate Bcl-2, leading to increased apoptosis. HDAC3-cKO mice failed to downregulate retinoic acid–related orphan receptor (ROR) γt during positive selection, similar to the block in positive selection in RORγt transgenic mice. In the absence of HDAC3, the RORC promoter was hyperacetylated. In the periphery, the few CD4 T cells present were skewed toward RORγt+ IL-17–producing Th17 cells, leading to inflammatory bowel disease. Positive selection of CD8 single-positive thymocytes was restored in RORγt-KO Bcl-xL transgenic HDAC3-cKO mice, demonstrating that HDAC3 is required at positive selection to downregulate RORγt.

Abstract Recent thymic emigrants are newly generated T cells that need to undergo postthymic maturation to gain functional competency and enter the long-lived naive T cell pool. The mechanism of T cell maturation remains incompletely understood. Previously, we demonstrated that the transcriptional repressor NKAP is required for T cell maturation. Because NKAP associates with histone deacetylase 3 (HDAC3), we examined whether HDAC3 is also required for T cell maturation. Although thymic populations are similar in CD4-cre HDAC3 conditional knockout mice compared with wild-type mice, the peripheral numbers of CD4+ and CD8+ T cells are dramatically decreased. In the periphery, the majority of HDAC3-deficient naive T cells are recent thymic emigrants, indicating a block in T cell maturation. CD55 upregulation during T cell maturation is substantially decreased in HDAC3-deficient T cells. Consistent with a block in functional maturation, HDAC3-deficient peripheral T cells have a defect in TNF licensing after TCR/CD28 stimulation. CD4-cre HDAC3 conditional knockout mice do not have a defect in intrathymic migration, thymic egress, T cell survival, or homeostasis. In the periphery, similar to immature NKAP-deficient peripheral T cells, HDAC3-deficient peripheral T cells were bound by IgM and complement proteins, leading to the elimination of these cells. In addition, HDAC3-deficient T cells display decreases in the sialic acid modifications on the cell surface that recruit natural IgM to initiate the classical complement pathway. Therefore, HDAC3 is required for T cell maturation.