Protocol for assessing myogenic tone and perfusion pressure in isolated mouse kidneys.

The isolated perfused kidney is a classic ex vivo preparation for studying renal physiology in general and vascular function. Here, we present a protocol for assessing myogenic tone in isolated mouse kidneys as well as vasodilatory and vasoconstrictive responses, expressed as perfusion pressure. We describe steps for pre-operative preparation, kidney and renal artery isolation, and connection of renal artery with glass cannula. We then detail how to measure pressure changes in perfused kidneys and the myogenic tone. For complete details on the use and execution of this protocol, please refer to Cui et al.1.

Myogenic Vasoconstriction Requires Canonical Gq/11 Signaling of the Angiotensin II Type 1 Receptor.

Background Blood pressure and tissue perfusion are controlled in part by the level of intrinsic (myogenic) arterial tone. However, many of the molecular determinants of this response are unknown. We previously found that mice with targeted disruption of the gene encoding the angiotensin II type 1a receptor (AT1AR) (Agtr1a), the major murine angiotensin II type 1 receptor (AT1R) isoform, showed reduced myogenic tone; however, uncontrolled genetic events (in this case, gene ablation) can lead to phenotypes that are difficult or impossible to interpret. Methods and Results We tested the mechanosensitive function of AT1R using tamoxifen-inducible smooth muscle-specific AT1aR knockout (smooth muscle-Agtr1a-/-) mice and studied downstream signaling cascades mediated by Gq/11 and/or ß-arrestins. FR900359, Sar1Ile4Ile8-angiotensin II (SII), TRV120027 and TRV120055 were used as selective Gq/11 inhibitor and biased agonists to activate noncanonical ß-arrestin and canonical Gq/11 signaling of the AT1R, respectively. Myogenic and Ang II-induced constrictions were diminished in the perfused renal vasculature, mesenteric and cerebral arteries of smooth muscle-Agtr1a-/- mice. Similar effects were observed in arteries of global mutant Agtr1a-/- but not Agtr1b-/- mice. FR900359 decreased myogenic tone and angiotensin II-induced constrictions whereas selective biased targeting of AT1R-ß-arrestin signaling pathways had no effects. Conclusions This study demonstrates that myogenic arterial constriction requires Gq/11-dependent signaling pathways of mechanoactivated AT1R but not G protein-independent, noncanonical pathways in smooth muscle cells.

Phosphodiesterase 3A and Arterial Hypertension.

BACKGROUND: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A (PDE3A); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking. METHODS: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways. RESULTS: We describe a novel mutation within a 15 base pair (bp) region of the PDE3A gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3θ adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function. CONCLUSIONS: The mutated PDE3A gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.

Role of Ryanodine Type 2 Receptors in Elementary Ca2+ Signaling in Arteries and Vascular Adaptive Responses.

Background Hypertension is the major risk factor for cardiovascular disease, the most common cause of death worldwide. Resistance arteries are capable of adapting their diameter independently in response to pressure and flow-associated shear stress. Ryanodine receptors (RyRs) are major Ca2+-release channels in the sarcoplasmic reticulum membrane of myocytes that contribute to the regulation of contractility. Vascular smooth muscle cells exhibit 3 different RyR isoforms (RyR1, RyR2, and RyR3), but the impact of individual RyR isoforms on adaptive vascular responses is largely unknown. Herein, we generated tamoxifen-inducible smooth muscle cell-specific RyR2-deficient mice and tested the hypothesis that vascular smooth muscle cell RyR2s play a specific role in elementary Ca2+ signaling and adaptive vascular responses to vascular pressure and/or flow. Methods and Results Targeted deletion of the Ryr2 gene resulted in a complete loss of sarcoplasmic reticulum-mediated Ca2+-release events and associated Ca2+-activated, large-conductance K+ channel currents in peripheral arteries, leading to increased myogenic tone and systemic blood pressure. In the absence of RyR2, the pulmonary artery pressure response to sustained hypoxia was enhanced, but flow-dependent effects, including blood flow recovery in ischemic hind limbs, were unaffected. Conclusions Our results establish that RyR2-mediated Ca2+-release events in VSCM s specifically regulate myogenic tone (systemic circulation) and arterial adaptation in response to changes in pressure (hypoxic lung model), but not flow. They further suggest that vascular smooth muscle cell-expressed RyR2 deserves scrutiny as a therapeutic target for the treatment of vascular responses in hypertension and chronic vascular diseases.

Possible Digenic Disease in a Caucasian Family with COL4A3 and COL4A5 Mutations.

Microscopic hematuria is a common feature of patients with Alport syndrome, a familial nephropathy due to mutations in COL4A3, COL4A4 or COL4A5. These genes encode for α3, α4, and α5 type IV collagen polypeptide chains (collagen IV α345), crucial for the structural component of the glomerular basement membrane. Even patients with mild phenotype, namely isolated microhematuria (X-linked females with thin basement membrane on electron microscopy or heterozygous carriers of COL4A3 or COL4A4 mutations), can potentially progress to proteinuria and to end-stage renal disease. Recent pedigree analyses provided evidence for digenic inheritance of Alport syndrome by concomitant mutations in COL4A3/COL4A4 or COL4A4/COL4A5. We describe a Caucasian family with concomitant COL4A3 and COL4A5 mutations, consisting of a novel c.4484A>G COL4A3 (p.Gln1495Arg) mutation and a previously reported c.1871G>A COL4A5 (p.Gly624Asp) mutation. Our segregation analysis raises the possibility that Alport syndrome resembles also digenic inheritance by COL4A3/COL4A5.

The Case: Chronic Kidney Disease Unmasked by Single-Subject Research.

We present a 42-year-old man with a BMI of 32, who was referred because of proteinuria and decreased renal function. We were impressed by his markedly muscular physique. A renal biopsy was performed, which showed focal segmental glomerular sclerosis (FSGS). Is this patient merely an obese person with FSGS or is something else going on here? We performed extensive clinical and laboratory examinations, genetic testing, and anthropometric data monitoring over time. We transferred our methodology for routine FSGS mutation screening (Sanger sequencing) to the Ion Torrent PGM platform with a new custom-targeted NGS gene panel (Ion Ampliseq FSGS panel) and tested the performance of the system in two cohorts of patients with FSGS. We discuss FSGS in bodybuilders, including possible mechanisms, and review the literature.

Late-onset Bartter syndrome type II.

Mutations in the ROMK1 potassium channel gene (KCNJ1) cause antenatal/neonatal Bartter syndrome type II (aBS II), a renal disorder that begins in utero, accounting for the polyhydramnios and premature delivery that is typical in affected infants, who develop massive renal salt wasting, hypokalaemic metabolic alkalosis, secondary hyperreninaemic hyperaldosteronism, hypercalciuria and nephrocalcinosis. This BS type is believed to represent a disorder of the infancy, but not in adulthood. We herein describe a female patient with a remarkably late-onset and mild clinical manifestation of BS II with compound heterozygous KCNJ1 missense mutations, consisting of a novel c.197T > A (p.I66N) and a previously reported c.875G > A (p.R292Q) KCNJ1 mutation. We implemented and evaluated the performance of two different bioinformatics-based approaches of targeted massively parallel sequencing [next generation sequencing (NGS)] in defining the molecular diagnosis. Our results demonstrate that aBS II may be suspected in patients with a late-onset phenotype. Our experimental approach of NGS-based mutation screening combined with Sanger sequencing proved to be a reliable molecular approach for defining the clinical diagnosis in our patient, and results in important differential diagnostic and therapeutic implications for patients with BS. Our results could have a significant impact on the diagnosis and methodological approaches of genetic testing in other patients with clinical unclassified phenotypes of nephrocalcinosis and congenital renal electrolyte abnormalities.

A CD2AP Mutation Associated with Focal Segmental Glomerulosclerosis in Young Adulthood.

Mutations in CD2-associated protein (CD2AP) have been identified in patients with focal segmental glomerulosclerosis (FSGS); however, reports of CD2AP mutations remain scarce. We performed Sanger sequencing in a patient with steroid-resistant FSGS and identified a heterozygous CD2AP mutation (p.T374A, c.1120 A > G). Our patient displayed mild cognitive decline, a phenotypic characteristic not previously associated with CD2AP-associated FSGS. His proteinuria was remarkably reduced by treatment with cyclosporine A. Our findings expand the genetic spectrum of CD2AP-associated disorders and broaden the associated phenotype with the co-occurrence of cognitive decline. Our case shows that cyclosporin A is a treatment option for CD2AP-associated nephropathy.

Stretch-activation of angiotensin II type 1a receptors contributes to the myogenic response of mouse mesenteric and renal arteries.

RATIONALE: Vascular wall stretch is the major stimulus for the myogenic response of small arteries to pressure. The molecular mechanisms are elusive, but recent findings suggest that G protein-coupled receptors can elicit a stretch response. OBJECTIVE: To determine whether angiotensin II type 1 receptors (AT1R) in vascular smooth muscle cells exert mechanosensitivity and identify the downstream ion channel mediators of myogenic vasoconstriction. METHODS AND RESULTS: We used mice deficient in AT1R signaling molecules and putative ion channel targets, namely AT1R, angiotensinogen, transient receptor potential channel 6 (TRPC6) channels, or several subtypes of the voltage-gated K+ (Kv7) gene family (KCNQ3, 4, or 5). We identified a mechanosensing mechanism in isolated mesenteric arteries and in the renal circulation that relies on coupling of the AT1R subtype a to a Gq/11 protein as a critical event to accomplish the myogenic response. Arterial mechanoactivation occurs after pharmacological block of AT1R and in the absence of angiotensinogen or TRPC6 channels. Activation of AT1R subtype a by osmotically induced membrane stretch suppresses an XE991-sensitive Kv channel current in patch-clamped vascular smooth muscle cells, and similar concentrations of XE991 enhance mesenteric and renal myogenic tone. Although XE991-sensitive KCNQ3, 4, and 5 channels are expressed in vascular smooth muscle cells, XE991-sensitive K+ current and myogenic contractions persist in arteries deficient in these channels. CONCLUSIONS: Our results provide definitive evidence that myogenic responses of mouse mesenteric and renal arteries rely on ligand-independent, mechanoactivation of AT1R subtype a. The AT1R subtype a signal relies on an ion channel distinct from TRPC6 or KCNQ3, 4, or 5 to enact vascular smooth muscle cell activation and elevated vascular resistance.