Multiple blood pressure loci with opposing blood pressure effects on rat chromosome 1 in a homologous region linked to hypertension on human chromosome 15.

Genetic dissection of blood pressure (BP) quantitative trait loci (QTLs) in rats has facilitated the fine-mapping of regions linked to the inheritance of hypertension. The goal of the current study was to further fine-map one such genomic region on rat chromosome 1 (BPQTL1b1), the homologous region of which on human chromosome 15 harbors BP QTLs, as reported by four independent studies. Of the six substrains constructed and studied, the systolic BP of two of the congenic strains were significantly lower by 36 and 27 mm Hg than that of the salt-sensitive (S) rat (P < 0.0001, P = 0.0003, respectively). The congenic segments of these two strains overlapped between 135.12 and 138.78 Mb and contained eight genes and two predicted miRNAs. None of the annotations had variants within expressed sequences. These data taken together with the previous localization resolved QTL1b1 with a 70% improvement from the original 7.39 Mb to the current 2.247 Mb interval. Furthermore, the systolic BP of one of the congenic substrains was significantly higher by 20 mm Hg (P < 0.0001) than the BP of the S rat. The limits of this newly identified QTL with a BP increasing effect (QTL1b1a) were between 134.12 and 135.76 Mb, spanning 1.64 Mb, containing two protein-coding genes, Mctp2 and Rgma, and a predicted miRNA. There were four synonymous variants within Mctp2. These data provide evidence for two independent BP QTLs with opposing BP effects within the previously identified BP QTL1b1 region. Additionally, these findings illustrate the complexity underlying the genetic mechanisms of BP regulation, wherein inherited elements beyond protein-coding sequences or known regulatory regions could be operational.

Cryptorchidism and infertility in rats with targeted disruption of the Adamts16 locus.

A Disintegrin And Metalloproteinase with ThromboSpondin motifs16 (ADAMTS-16) is a member of a family of metalloproteinases. Using a novel zinc-finger nuclease based gene-edited rat model harboring a targeted mutation of the Adamts16 locus, we previously reported this gene to be linked to blood pressure regulation. Here we document our observation with this model that Adamts16 is essential for normal development of the testis. Absence of Adamts16 in the homozygous Adamts16mutant males resulted in cryptorchidism and male sterility. Heterozygous Adamts16mutant males were normal, indicating that this is a recessive trait. Testes of homozygous Adamts16mutant males were significantly smaller with significant histological changes associated with the lack of sperm production. Temporal histological assessments of the testis demonstrated that the seminiferous tubules did not support active spermatogenesis, but progressively lost germ cells, accumulated vacuoles and did not have any sperm. These observations, taken together with our previous report of renal abnormalities observed with the same Adamts16mutant rats, suggest an important mechanistic link between Adamts16 and the functioning of the male genitourinary system.

Ciliotherapy: a novel intervention in polycystic kidney disease.

BACKGROUND: Ciliopathies are a group of diseases associated with abnormal structure or function of primary cilia. Ciliopathies include polycystic kidney disease (PKD), a pathology associated with vascular hypertension. We previously showed that cilia length regulates cilia function, and cilia function is required for nitric oxide (NO) biosynthesis in endothelial cells. Because patients with PKD show abnormal sensory cilia function, the aim of our current study was to search for a targeted therapy focused on primary cilia, which we refer to as 'ciliotherapy'. METHODS AND RESULTS: In the present studies, our in vitro analyses refined fenoldopam as an equipotent and more specific dopaminergic agonist to regulate cilia length and function. Our in vivo studies indicated that fenoldopam increased cilia length and serum NO thereby reducing blood pressure in a PKD mouse model. Our crossover, multicenter, double-blind and placebo-controlled clinical study further indicated that cilia-targeting therapy showed an overall reduction in mean arterial pressure in PKD patients. CONCLUSIONS: Overall, our studies provide the first evidence of ciliotherapy as an innovative intervention in patients with abnormal primary cilia.

Construction of two novel reciprocal conplastic rat strains and characterization of cardiac mitochondria.

Because of the lack of appropriate animal models, the potentially causal contributions of inherited mitochondrial genomic factors to complex traits are less well studied compared with inherited nuclear genomic factors. We previously detected variations between the mitochondrial DNA (mtDNA) of the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR). Specifically, multiple variations were detected in mitochondrial genes coding for subunits of proteins essential for electron transport, in mitochondrial reactive oxygen species production, and within the D-loop region. To evaluate the effects of these mtDNA variations in the absence of the corresponding nuclear genomic factors as confounding variables, novel reciprocal strains of S and SHR were constructed and characterized. When compared with that of the S rat, the heart tissue from the S.SHR(mt) conplastic strain wherein the mtDNA of the S rat was substituted with that of the SHR had a significant increase in mtDNA copy number and decrease in mitochondrial reactive oxygen species production. A corresponding increase in aerobic treadmill running capacity and a significant increase in survival that was not related to changes in blood pressure were observed in the S.SHR(mt) rats compared with the S rat. The reciprocal SHR.S(mt) rats did not differ from the SHR in any phenotype tested, suggesting lower penetrance of the S mtDNA on the nuclear genomic background of the SHR. These novel conplastic strains serve as invaluable tools to further dissect the relationship between heart function, aerobic fitness, cardiovascular disease progression, and mortality.

Targeted disruption of Adamts16 gene in a rat genetic model of hypertension.

A disintegrin-like metalloproteinase with thrombospondin motifs-16 (Adamts16) is an important candidate gene for hypertension. The goal of the present study was to further assess the candidacy of Adamts16 by targeted disruption of this gene in a rat genetic model of hypertension. A rat model was generated by manipulating the genome of the Dahl Salt-sensitive (S) rat using zinc-finger nucleases, wherein the mutant rat had a 17 bp deletion in the first exon of Adamts16, introducing a stop codon in the transcript. Systolic blood pressure (BP) of the homozygous Adamts16(mutant) rats was lower by 36 mmHg compared with the BP of the S rats. The Adamts16(mutant) rats exhibited significantly lower aortic pulse wave velocity and vascular media thickness compared with S rats. Scanning electron and fluorescence microscopic studies indicated that the mechanosensory cilia of vascular endothelial cells from the Adamts16(mutant) rats were longer than that of the S rats. Furthermore, Adamts16(mutant) rats showed splitting and thickening of glomerular capillaries and had a longer survival rate, compared with the S rats. Taken together, these physiological observations functionally link Adamts16 to BP regulation and suggest the vasculature as the potential site of action of Adamts16 to lower BP.

Cilostazol prevents endothelin-induced smooth muscle constriction and proliferation.

Cilostazol is a phosphodiesterase inhibitor that has been shown to inhibit platelet activation. Endothelin is known to be the most potent endogenous growth promoting and vasoactive peptide. In patients and animal models with stroke, the level of circulating endothelin increases and complicates the recovery progress contributed by vascular constriction (an immediate pathology) and vascular proliferation (a long-term pathology). However, the effects of cilostazol on endothelin have not been explored. To demonstrate the dual-antagonizing effects of cilostazol on vasoconstriction and cell proliferation induced by endothelin, we used primary culture of mouse vascular smooth muscle cells in vitro, mouse femoral artery ex vivo, and intracranial basilar artery ex vivo. We show that the dual-inhibition effects of cilostazol are mediated by blocking endothelin-induced extracellular calcium influx. Although cilostazol does not inhibit endothelin-induced intraorganellar calcium release, blockade of extracellular calcium influx is sufficient to blunt endothelin-induced vasoconstriction. We also show that cilostazol inhibits endothelin-induced cellular proliferation by blocking extracellular calcium influx. Inhibition of cAMP-dependent protein kinase (PKA) can block anti-proliferation activity of cilostazol, confirming the downstream role of PKA in cellular proliferation. To further demonstrate the selectivity of the dual-antagonizing effects of cilostazol, we used a different phosphodiesterase inhibitor. Interestingly, sildenafil inhibits endothelin-induced vasoconstriction but not cellular proliferation in smooth muscle cells. For the first time, we show selective dual-antagonizing effects of cilostazol on endothelin. We propose that cilostazol is an excellent candidate to treat endothelin-associated diseases, such as stroke.

Mechanisms regulating cilia growth and cilia function in endothelial cells.

The primary cilium is an important sensory organelle present in most mammalian cells. Our current studies aim at examining intracellular molecules that regulate cilia length and/or cilia function in vitro and ex vivo. For the first time, we show that intracellular cAMP and cAMP-dependent protein kinase (PKA) regulate both cilia length and function in vascular endothelial cells. Although calcium-dependent protein kinase modulates cilia length, it does not play a significant role in cilia function. Cilia length regulation also involves mitogen-activated protein kinase (MAPK), protein phosphatase-1 (PP-1), and cofilin. Furthermore, cofilin regulates cilia length through actin rearrangement. Overall, our study suggests that the molecular interactions between cilia function and length can be independent of one another. Although PKA regulates both cilia length and function, changes in cilia length by MAPK, PP-1, or cofilin do not have a direct correlation to changes in cilia function. We propose that cilia length and function are regulated by distinct, yet complex intertwined signaling pathways.

Dopamine receptor type 5 in the primary cilia has dual chemo- and mechano-sensory roles.

Polycystic kidney disease is characterized by cardiovascular irregularities, including hypertension. Dopamine, a circulating hormone, is implicated in essential hypertension in humans and animal models. Vascular endothelial primary cilia are known to function as mechano-sensory organelles. Although both primary cilia and dopamine receptors play important roles in vascular hypertension, their relationship has never been explored. To determine the roles of the dopaminergic system and mechano-sensory cilia, we studied the effects of dopamine on ciliary length and function in wild-type and mechano-insensitive polycystic mutant cells (Pkd1(-/)(-) and Tg737(orpk/orpk)). We show for the first time that mouse vascular endothelia exhibit dopamine receptor-type 5 (DR5), which colocalizes to primary cilia in cultured cells and mouse arteries in vivo. DR5 activation increases cilia length in arteries and endothelial cells through cofilin and actin polymerization. DR5 activation also restores cilia function in the mutant cells. In addition, silencing DR5 completely abolishes mechano-ciliary function in WT cells. We found that DR5 plays very important roles in ciliary length and function. Furthermore, the chemo-sensory function of cilia can alter the mechano-sensory function through changes in sensitivity to fluid-shear stress. We propose that ciliary DR5 has functional chemo- and mechano-sensory roles in endothelial cells.

Calcium-mediated mechanisms of cystic expansion.

In this review, we will discuss several well-accepted signaling pathways toward calcium-mediated mechanisms of cystic expansion. The second messenger calcium ion has contributed to a vast diversity of signal transduction pathways. We will dissect calcium signaling as a possible mechanism that contributes to renal cyst formation. Because cytosolic calcium also regulates an array of signaling pathways, we will first discuss cilia-induced calcium fluxes, followed by Wnt signaling that has attributed to much-discussed planar cell polarity. We will then look at the relationship between cytosolic calcium and cAMP as one of the most important aspects of cyst progression. The signaling of cAMP on MAPK and mTOR will also be discussed. We infer that while cilia-induced calcium fluxes may be the initial signaling messenger for various cellular pathways, no single signaling mediator or pathway is implicated exclusively in the progression of the cystic expansion. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

Polycystic diseases in visceral organs.

Primary cilia are nonmotile, microtubule-based, antenna-like organelles projecting from the apical surface of most mammalian cells. Elegant studies have established the importance of ciliary structure and function in signal transduction and the sensory roles of cilia in maintaining healthy cellular state. In particular, dysfunctional cilia have been implicated in a large number of diseases mainly characterized by the presence of fluid-filled cysts in various organs. Aside from polycystic kidney disease (PKD), however, the roles of cilia in polycystic liver disease (PLD), polycystic pancreas disease (PPD), and polycystic ovarian syndrome (PCOS) are still very vague. In addition, although gender and sex hormones are known to regulate cyst formation, their roles in regulating physiological functions of cilia need to be further explored.