Sickle cell disease subjects and mouse models have elevated nitrite and cGMP levels in blood compartments.

The hypothesis of decreased nitric oxide (NO) bioavailability in sickle cell disease (SCD) proposes that multiple factors leading to decreased NO production and increased consumption contributes to vaso-occlusion, pulmonary hypertension, and pain. The anion nitrite is central to NO physiology as it is an end product of NO metabolism and serves as a reservoir for NO formation. However, there is little data on nitrite levels in SCD patients and its relationship to pain phenotype. We measured nitrite in SCD subjects and examined its relationship to SCD pain. In SCD subjects, median whole blood, red blood cell and plasma nitrite levels were higher than in controls, and were not associated with pain burden. Similarly, Townes and BERK homozygous SCD mice had elevated blood nitrite. Additionally, in red blood cells and plasma from SCD subjects and in blood and kidney from Townes homozygous mice, levels of cyclic guanosine monophosphate (cGMP) were higher compared to controls. In vitro, hemoglobin concentration, rather than sickle hemoglobin, was responsible for nitrite metabolism rate. In vivo, inhibition of NO synthases and xanthine oxidoreductase decreased nitrite levels in homozygotes but not in control mice. Long-term nitrite treatment in SCD mice further elevated blood nitrite and cGMP, worsened anemia, decreased platelets, and did not change pain response. These data suggest that SCD in humans and animals is associated with increased nitrite/NO availability, which is unrelated to pain phenotype. These findings might explain why multiple clinical trials aimed at increasing NO availability in SCD patients failed to improve pain outcomes.

Blood collection vials and clinically used intravenous fluids contain significant amounts of nitrite.

The biology of the inorganic anion nitrite is linked to nitric oxide (NO) as nitrite can be reduced to NO and mediate its biological activities. Thus, studies of nitrite biology require sensitive and selective chemical assays. The acetic and ascorbic acids method is selective for nitrite and measures it in biological matrices. However, one of the pitfalls of nitrite measurements is its ubiquitous presence in sample collection tubes. Here, we showed high levels of nitrite in collection tubes containing EDTA, sodium citrate or sodium heparin and smaller amounts in tubes containing lithium heparin or serum clot activator. We also showed the presence of nitrite in colloid and crystalloid solutions frequently administered to patients and found variable levels of nitrite in 5% albumin, 0.9% sodium chloride, lactated ringer's, and dextrose-plus-sodium chloride solutions. These levels of nitrite varied across lots and manufacturers of the same type of fluid. Because these fluids are administered intravenously to patients (including those in shock), sometimes in large volumes (liters), it is possible that infusions of these nitrite-containing fluids may have clinical implications. A protocol for blood collection free of nitrite contamination was developed and used to examine nitrite levels in whole blood, red blood cells, plasma and urine from normal volunteers. Nitrite measurements were reproducible, had minimal variability, and did not indicate sex-differences. These findings validated a method and protocol for selective nitrite assay in biological fluids free of nitrite contamination which can be applied for study of diseases where dysfunctional NO signaling has been implicated.

High throughput in vivo functional validation of candidate congenital heart disease genes in Drosophila.

Genomic sequencing has implicated large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system is needed to validate gene associations with pathology. We developed a Drosophila-based functional system to screen candidate disease genes identified from Congenital Heart Disease (CHD) patients. 134 genes were tested in the Drosophila heart using RNAi-based gene silencing. Quantitative analyses of multiple cardiac phenotypes demonstrated essential structural, functional, and developmental roles for more than 70 genes, including a subgroup encoding histone H3K4 modifying proteins. We also demonstrated the use of Drosophila to evaluate cardiac phenotypes resulting from specific, patient-derived alleles of candidate disease genes. We describe the first high throughput in vivo validation system to screen candidate disease genes identified from patients. This approach has the potential to facilitate development of precision medicine approaches for CHD and other diseases associated with genetic factors.

Altered nocifensive behavior in animal models of autism spectrum disorder: The role of the nicotinic cholinergic system.

Caretakers and clinicians alike have long recognized that individuals with autism spectrum disorder (ASD) can have altered sensory processing, which can contribute to its core symptoms. However, the pathobiology of sensory alterations in ASD is poorly understood. Here we examined nocifensive behavior in ASD mouse models, the BTBR T+Itpr3tf/J (BTBR) and the fragile-X mental retardation-1 knockout (Fmr1-KO) mice. We also examined the effects of nicotine on nocifensive behavior given that nicotine, a nicotinic cholinergic receptor (nAChR) agonist that has antinociceptive effects, was shown to improve social deficits and decrease repetitive behaviors in BTBR mice. Compared to respective controls, both BTBR and Fmr1-KO had hyporesponsiveness to noxious thermal stimuli and electrical stimulation of C-sensory fibers, normal responsiveness to electrical stimulation of Aß- and Aδ-fiber, and hyperresponsiveness to visceral pain after acetic acid intraperitoneal injection. In BTBR, nicotine at lower doses increased, whereas at higher doses, it decreased hotplate latency compared to vehicle. In a significantly different effect pattern, in control mice, nicotine had antinociceptive effects to noxious heat only at the high dose. Interestingly, these nocifensive behavior alterations and differential responses to nicotine antinociceptive effects in BTBR mice were associated with significant downregulation of α3, α4, α5, α7, ß2, ß3, and ß4 nAChR subunits in several cerebral regions both, during embryonic development and adulthood. Taken together, these findings further implicate nAChRs in behaviors alterations in the BTBR model and lend support to the hypothesis that nAChRs may be a target for treatment of behavior deficits and sensory dysfunction in ASD.

KANK deficiency leads to podocyte dysfunction and nephrotic syndrome.

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of progressive renal function decline and affects millions of people. In a recent study, 30% of SRNS cases evaluated were the result of monogenic mutations in 1 of 27 different genes. Here, using homozygosity mapping and whole-exome sequencing, we identified recessive mutations in kidney ankyrin repeat-containing protein 1 (KANK1), KANK2, and KANK4 in individuals with nephrotic syndrome. In an independent functional genetic screen of Drosophila cardiac nephrocytes, which are equivalents of mammalian podocytes, we determined that the Drosophila KANK homolog (dKank) is essential for nephrocyte function. RNAi-mediated knockdown of dKank in nephrocytes disrupted slit diaphragm filtration structures and lacuna channel structures. In rats, KANK1, KANK2, and KANK4 all localized to podocytes in glomeruli, and KANK1 partially colocalized with synaptopodin. Knockdown of kank2 in zebrafish recapitulated a nephrotic syndrome phenotype, resulting in proteinuria and podocyte foot process effacement. In rat glomeruli and cultured human podocytes, KANK2 interacted with ARHGDIA, a known regulator of RHO GTPases in podocytes that is dysfunctional in some types of nephrotic syndrome. Knockdown of KANK2 in cultured podocytes increased active GTP-bound RHOA and decreased migration. Together, these data suggest that KANK family genes play evolutionarily conserved roles in podocyte function, likely through regulating RHO GTPase signaling.

Ras-association domain of sorting Nexin 27 is critical for regulating expression of GIRK potassium channels.

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability.