Intraperitoneal pyrophosphate treatment reduces renal calcifications in Npt2a null mice

Mutations in the proximal tubular sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis, however the relative contribution of genotype, dietary calcium and phosphate, and modifiers of mineralization such as pyrophosphate (PPi) to the formation of renal mineral deposits is unclear. In the present study, we used Npt2a-/- mice to model the renal calcifications observed in these disorders. We observed elevated urinary excretion of PPi in Npt2a-/- mice when compared to WT mice. Presence of two hypomorphic Extracellular nucleotide pyrophosphatase phosphodiesterase 1 (Enpp1asj/asj) alleles decreased urine PPi and worsened renal calcifications in Npt2a-/- mice. These studies suggest that PPi is a thus far unrecognized factor protecting Npt2a-/- mice from the development of renal mineral deposits. Consistent with this conclusion, we next showed that renal calcifications in these mice can be reduced by intraperitoneal administration of sodium pyrophosphate. If confirmed in humans, urine PPi could therefore be of interest for developing new strategies to prevent the nephrocalcinosis and nephrolithiasis seen in phosphaturic disorders

The cardiomyocyte disrupts pyrimidine biosynthesis in non-myocytes to regulate heart repair

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair

Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites

Improving the Pharmacodynamics and In Vivo Activity of ENPP1‐Fc Through Protein and Glycosylation Engineering

Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase‐1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three‐prong strategy. First, we added new N‐glycans to ENPP1; second, we optimized pH‐dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two‐step process to improve sialylation by first producing ENPP1‐Fc in cells stably transfected with human α‐2,6‐sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4‐ O ‐Bu 3 ManNAc. These steps sequentially increased the half‐life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N‐glycan, to ~ 96 hours with optimized pH‐dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6‐overexpressing cells with 1,3,4‐ O ‐Bu 3 ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1‐deficient mice when the optimized biologic was administered at a 10‐fold lower mass dose less frequently than the parent compound—once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics

Urine PPi concentration and renal gene expression in <i>Npt2a</i>^<i>-/-</i> mice.

<p>Urine pyrophosphate concentration (U-PPi, <b>A</b>) following an overnight fast and renal gene expression as indicated on the y-axis for <i>ectonucleotide pyrophosphatase/phosphodiesterase 1</i> (<i>Enpp1</i>, <b>B</b>), <i>progressive ankylosis</i> (<i>Ank</i>, <b>C</b>), <i>ectonucleoside triphosphate diphosphohydrolase 5</i> (<i>Entpd5</i>, <b>D</b>), <i>tissue nonspecific alkaline phosphatase</i> (<i>Tnsalp</i>, <b>E</b><i>)</i> in mice fed regular chow for 10 weeks. The data represent mean±SEM of 4–19 mice, p-values shown above the lines of comparisons were calculated by one-way ANOVA using Tukey’s adjustment for multiple comparisons (<b>A</b>) and Student’s t-test (<b>B-E</b>).</p

Intraperitoneal injection of Na-pyrophosphate reduces cortical and medulary renal mineralization in <i>Npt2a</i>^<i>-/-</i> mice.

<p>Light micrographs of 10 um renal sections prepared from paraffin-embedded kidneys, obtained from mice with various genotypes fed regular chow for 10 weeks (<b>A, upper panels:</b> von Kossa, methylene green staining, 4X, and <b>A, lower panels:</b> von Kossa, hematoxylin and eosin staining, 40X); Transmission electron micrographs showing microspheres in double mutant mice on regular chow, inset with larger magnification shown to the right (<b>B</b>); Two weeks old <i>Npt2a</i>^<i>-/-</i> pups treated with i.p. injections of vehicle or sodium pyrophosphate (160 micromole/Kg/day) for two weeks (<b>C</b>); Histomorphometric analysis of renal mineralization (Êlcified area = 100*mineralization area/tissue area, (<b>D)</b>; calcification size = mineralization area/number of calcifications, um², (<b>E</b>), and plasma pyrophosphate levels (<b>F</b>) and urine pyrophosphate (U-PPi) (<b>G</b>) of two weeks old <i>Npt2a</i>^<i>-/-</i> pups treated with i.p. injections of vehicle or sodium pyrophosphate (160 micromole/Kg/day) for two weeks, measured after overnight fast and 18–24 hrs. following the last treatment. The data represent individual animals (closed circles) with the means±SEM, p-values shown above the lines of comparisons were calculated by Student’s t-test.</p

Lonafarnib improves cardiovascular function and survival in a mouse model of Hutchinson-Gilford progeria syndrome

Clinical trials have demonstrated that lonafarnib, a farnesyltransferase inhibitor, extends the lifespan in patients afflicted by Hutchinson-Gilford progeria syndrome, a devastating condition that accelerates many characteristics of aging and results in premature death due to cardiovascular sequelae. The US Food and Drug Administration approved Zokinvy (lonafarnib) in November 2020 for treating these patients, yet a detailed examination of drug-associated effects on cardiovascular structure, properties, and function has remained wanting. In this paper, we report encouraging outcomes of daily post-weaning treatment with lonafarnib on the composition and biomechanical phenotype of elastic and muscular arteries as well as associated cardiac function in a well-accepted mouse model of progeria that exhibits severe perimorbid cardiovascular disease. Lonafarnib resulted in 100% survival of the treated progeria mice to the study end-point (time of 50% survival of untreated mice), with associated improvements in arterial structure and function working together to significantly reduce pulse wave velocity and improve left ventricular diastolic function. By contrast, neither treatment with the mTOR inhibitor rapamycin alone nor dual treatment with lonafarnib plus rapamycin improved outcomes over that achieved with lonafarnib monotherapy

Urinary pyrophosphate concentration is inversely associated with renal mineralization size in a combined bivariate linear regression analysis of all mice.

<p>All experimental WT and mutant mice from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180098#pone.0180098.g002" target="_blank">Fig 2</a> (n = 28) for which urine was available were evaluated using linear regression analysis to determine the association of renal mineralization with the urine pyrophosphate concentration (U-PPi) (% calcified area = 100*calcified area/total area <b>A</b> and calcification size = calcified area/number of mineralization <b>B</b>). Data points represent values of individual animals. Results of the linear regression analysis are shown as solid line with 95% confidence interval (stippled lines), R² and p-values.</p