Enhanced Vaccine Effectiveness during the Delta Phase of the COVID-19 Pandemic in the Medicare Population Supports a Multilayered Prevention Approach.

Throughout the pandemic, individuals 65 years and older have contributed most COVID-19 related deaths. To best formulate effective vaccination and other prevention policies to protect older adults, large scale observational studies of these higher risk individuals are needed. We conducted a Vaccine Effectiveness (VE) study during the B.1.617.2 Delta variant phase of the pandemic in July and August 2021 in a cohort of 17 million Medicare beneficiaries of which 5.7 million were fully vaccinated. We found that individuals fully vaccinated with the Pfizer-BioNTech BNT162b2 and Moderna mRNA-1273 vaccines in January 2021 had 2.5 times higher breakthrough infections and hospitalizations than those fully vaccinated in March 2021, consistent with waning of vaccine-induced immunity. Measuring VE weekly, we found that VE against hospitalization, and even more so against infection, increased from July 2021 through August 2021, suggesting that in addition to the protective role of vaccination, increased masking or social distancing might have contributed to the unexpected increase in VE. Ongoing monitoring of Medicare beneficiaries should be a priority as new variants continue to emerge, and the VE of the new bivalent vaccines remains to be established. This could be accomplished with a large Medicare claims database and the analytics platform used for this study.

A Predictive Model for Severe COVID-19 in the Medicare Population: A Tool for Prioritizing Primary and Booster COVID-19 Vaccination.

Recommendations for prioritizing COVID-19 vaccination have focused on the elderly at higher risk for severe disease. Existing models for identifying higher-risk individuals lack the needed integration of socio-demographic and clinical risk factors. Using multivariate logistic regression and random forest modeling, we developed a predictive model of severe COVID-19 using clinical data from Medicare claims for 16 million Medicare beneficiaries and socio-economic data from the CDC Social Vulnerability Index. Predicted individual probabilities of COVID-19 hospitalization were then calculated for population risk stratification and vaccine prioritization and mapping. The leading COVID-19 hospitalization risk factors were non-white ethnicity, end-stage renal disease, advanced age, prior hospitalization, leukemia, morbid obesity, chronic kidney disease, lung cancer, chronic liver disease, pulmonary fibrosis or pulmonary hypertension, and chemotherapy. However, previously reported risk factors such as chronic obstructive pulmonary disease and diabetes conferred modest hospitalization risk. Among all social vulnerability factors, residence in a low-income zip code was the only risk factor independently predicting hospitalization. This multifactor risk model and its population risk dashboard can be used to optimize COVID-19 vaccine allocation in the higher-risk Medicare population.

Lack of association between adrenergic receptor genotypes and survival in heart failure patients treated with carvedilol or metoprolol.

OBJECTIVES: This study investigated the role of adrenergic receptor genetics on transplant-free survival in heart failure (HF). BACKGROUND: Discordant results exist for genetic associations between adrenergic receptor alleles and end points of beta-blocker response in HF patients. METHODS: We identified 637 patients enrolled in 2 U.S. cardiovascular genetic registries with HF and left ventricular systolic dysfunction who were discharged on beta-blocker, angiotensin-converting enzyme inhibitor (ACEI) or angiotensin II receptor blocker (ARB), and diuretic medications. End points were determined through the national Social Security Death Master File and transplant records. We genotyped 5 polymorphisms in 3 genes: ADRB1 (S49G, R389G), ADRB2 (G16R, Q27E), and ADRA2C (Del322-325) using 5' nuclease assays and performed a multivariable clinical-genetic analysis. RESULTS: A total of 190 events (29.8%) occurred over a median follow-up of 1,070 days. Multivariable analysis showed a significant effect of 4 clinical factors on survival: age (p = 0.006), gender (p = 0.005), ejection fraction (p = 0.0002), and hemoglobin (p = 0.00010). There was no significant effect of the polymorphisms or haplotypes analyzed on survival. CONCLUSIONS: Genotypes and haplotypes of ADRB1, ADRB2, and ADRA2C did not significantly affect survival in metoprolol-treated or carvedilol-treated HF patients in this study. These results complement the findings of 2 similarly designed previous studies, but do not replicate an association of ADRB2 haplotypes and survival. All 3 studies differ from a survival benefit reported for bucindolol-treated homozygous ADRB1 R389 individuals. This may be attributable to a drug-specific interaction between genotype and outcome with bucindolol that does not seem to occur with metoprolol or carvedilol.

Protein kinase X (PRKX) can rescue the effects of polycystic kidney disease-1 gene (PKD1) deficiency.

Autosomal dominant polycystic kidney disease (ADPKD) is a common, genetically determined developmental disorder of the kidney that is characterized by cystic expansion of renal tubules and is caused by truncating mutations and haplo-insufficiency of the PKD1 gene. Several defects in cAMP-mediated proliferation and ion secretion have been detected in ADPKD cyst-lining epithelia. Unlike the ubiquitous PKA, the cAMP-dependent CREB-kinase, Protein Kinase X (PRKX) is developmentally regulated, tissue restricted and induces renal epithelial cell migration, and tubulogenesis in vitro as well as branching morphogenesis of ureteric bud in developing kidneys. The possibility of functional interactions between PKD1-encoded polycystin-1 and PRKX was suggested by the renal co-distribution of PRKX and polycystin-1 and the binding and phosphorylation of the C-terminal of polycystin-1 by PRKX at S4166 in vitro. Early consequences of PKD1 mutation include increased tubule epithelial cell-matrix adhesion, decreased migration, reduced ureteric bud branching and aberrant renal tubule dilation. To determine whether PRKX might counteract the adverse effects of PKD1 mutation, human ADPKD epithelial cell lines were transfected with constitutively active PRKX and shown to rescue characteristic adhesion and migration defects. In addition, the co-injection of constitutively active PRKX with inhibitory pMyr-EGFP-PKD1 into the ureteric buds of mouse embryonic kidneys in organ culture resulted in restoration of normal branching morphogenesis without cystic tubular dilations. These results suggest that PRKX can restore normal function to PKD1-deficient kidneys and have implications for the development of preventative therapy for ADPKD.

Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo.

The metanephric kidney is a mesodermal organ that develops as a result of reciprocal interactions between the ureteric bud and the blastema. The generation of embryonic stem (ES) cell-derived progenitors offers potential for regenerative therapies but is often limited by development of tumor formation. Because brachyury (T) denotes mesoderm specification, a mouse ES cell line with green fluorescence protein (GFP) knocked into the functional T locus as well as lacZ in the ROSA26 locus (LacZ/T/GFP) was used in cell selection and lineage tracing. In the absence of leukemia inhibitory factor, mouse ES cells give rise to embryoid bodies that can differentiate into mesoderm. Culture conditions were optimized (4 d, 10 ng/ml Activin-A) to generate maximal numbers of renal progenitor populations identified by expression of the specific combination of renal markers cadherin-11, WT-1, Pax-2, and Wnt-4. LacZ/T/GFP+ cells were further enriched by FACS selection. Five days after injection of LacZ/T/GFP+ cells into embryonic kidney explants in organ culture, beta-galactosidase immunohistochemistry showed incorporation into blastemal cells of the nephrogenic zone. After a single injection into developing live newborn mouse kidneys, co-localization studies showed that the LacZ/T/GFP+ cells were stably integrated into proximal tubules with normal morphology and normal polarization of alkaline phosphatase and aquaporin-1 for 7 mo, without teratoma formation. It is concluded that defined differentiation of ES cells into embryoid bodies with Activin-A and selection for T expression provides a means to isolate and purify renal proximal tubular progenitor cells with the potential for safe use in regenerative therapies.

Inhibition of HER-2(neu/ErbB2) restores normal function and structure to polycystic kidney disease (PKD) epithelia.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a very common lethal monogenetic disease with significant morbidities and a high likelihood of progression to renal failure for which there is no proven disease-specific therapy currently available for clinical use. Human ADPKD cystic epithelia have proliferative abnormalities mediated by EGFR over-expression and mispolarization leading autocrine response to EGF family ligands. We now show that apical localization of EGFR complexes in normal fetal and ADPKD epithelia is associated with heterodimerization of EGFR(HER-1) with HER-2(neu/ErbB2), while basal membrane localization in normal adult renal epithelia is associated with EGFR(HER-1) homodimers. Since ADPKD epithelial cells have reduced migratory function, this was used as a bioassay to evaluate the ability of compounds to rescue the aberrant human ADPKD phenotype. General tyrosine kinase inhibition by herbimycin and specific inhibition of HER-2(neu/ErbB2) by AG825 or pretreatment with ErbB2 siRNA reversed the migration defect of ADPKD epithelia. Selective inhibition of EGFR(HER-1) showed partial rescue. Increased ADPKD cell migration after inhibition of p38MAP kinase but not of PI3-kinase implicated p38MAPK downstream of HER-2(neu/ErbB2) stimulation. Daily administration of AG825 to PKD1 null heterozygous mice significantly inhibited the development of renal cysts. These studies implicate HER2(neu/ErbB2) as an effector of apical EGFR complex mispolarization and that its inhibition should be considered a candidate for clinical therapy of ADPKD.

Protein kinase X activates ureteric bud branching morphogenesis in developing mouse metanephric kidney.

The human protein kinase X (PRKX) gene was identified previously as a cAMP-dependent serine/threonine kinase that is aberrantly expressed in autosomal dominant polycystic disease kidneys and normally expressed in fetal kidneys. The PRKX kinase belongs to a serine/threonine kinase family that is phylogenetically and functionally distinct from classical protein kinase A kinases. Expression of PRKX activates cAMP-dependent renal epithelial cell migration and tubular morphogenesis in cell culture, suggesting that it might regulate branching growth of the collecting duct system in the fetal kidney. With the use of a mouse embryonic kidney organ culture system that recapitulates early kidney development in vitro, it is demonstrated that lentiviral vector-driven expression of a constitutively active, cAMP-independent PRKX in the ureteric bud epithelium stimulates branching morphogenesis and results in a 2.5-fold increase in glomerular number. These results suggest that PRKX stimulates epithelial branching morphogenesis by activating cell migration and support a role for this kinase in the regulation of nephrogenesis and of collecting system development in the fetal kidney.

Disruption of polycystin-1 function interferes with branching morphogenesis of the ureteric bud in developing mouse kidneys.

The polycystic kidney disease (PKD1) gene-encoded protein, polycystin-1, is developmentally regulated, with highest expression levels seen in normal developing kidneys, where it is distributed in a punctate pattern at the basal surface of ureteric bud epithelia. Overexpression in ureteric epithelial cell membranes of an inhibitory pMyr-GFP-PKD1 fusion protein via a retroviral (VVC) delivery system and microinjection into the ureteric bud lumen of embryonic day 11 mouse metanephric kidneys resulted in disrupted branching morphogenesis. Using confocal quantitative analysis, significant reductions were measured in the numbers of ureteric bud branch points and tips, as well as in the total ureteric bud length, volume and area, while significant increases were seen as dilations of the terminal branches, where significant increases in outer diameter and volumes were measured. Microinjection of an activating 5TM-GFP-PKD1 fusion protein had an opposite effect and showed significant increases in ureteric bud length and area. These are the first studies to experimentally manipulate polycystin-1 expression by transduction in the embryonic mouse kidney and suggest that polycystin-1 plays a critical role in the regulation of epithelial morphogenesis during renal development.

The exocyst localizes to the primary cilium in MDCK cells.

Primary cilia play a role in the maintenance of tubular epithelial differentiation and ciliary dysfunction can result in abnormal cyst formation, such as occurs in autosomal dominant polycystic kidney disease (ADPKD). We previously showed that the exocyst, an eight-protein complex involved in the biogenesis of polarity from yeast to mammals, is centrally involved in cyst formation [Mol. Biol. Cell. 11 (2000) 4259]. Here we show that the exocyst complex localizes to the primary cilium in Madin-Darby canine kidney (MDCK) tubular epithelial cells. We further show that the exocyst is overexpressed in both cell lines and primary cell cultures of ADPKD origin, suggesting that the exocyst may be involved in the pathogenesis of ADPKD.

Midkine promotes selective expansion of the nephrogenic mesenchyme during kidney organogenesis.

During kidney development, the growth and development of the stromal and nephrogenic mesenchyme cell populations and the ureteric bud epithelium is tightly coupled through intricate reciprocal signaling mechanisms between these three tissue compartments. Midkine, a target gene activated by retinoid signaling in the metanephros, encodes a secreted polypeptide with mitogenic and anti-apoptotic activities in a wide variety of cell types. Using immmunohistochemical methods we demonstrated that Midkine is found in the uninduced mesenchyme at the earliest stages of metanephric kidney development and only subsequently concentrated in the ureteric bud epithelium and basement membrane. The biological effects of purified recombinant Midkine were analyzed in metanephric organ culture experiments carried out in serum-free defined media. These studies revealed that Midkine selectively promoted the overgrowth of the Pax-2 and N-CAM positive nephrogenic mesenchymal cells, failed to stimulate expansion of the stromal compartment and suppressed branching morphogenesis of the ureteric bud. Midkine suppressed apoptosis and stimulated cellular proliferation of the nephrogenic mesenchymal cells, and was capable of maintaining the viability of isolated mesenchymes cultured in the absence of the ureteric bud. These results suggest that Midkine may regulate the balance of epithelial and stromal progenitor cell populations of the metanephric mesenchyme during renal organogenesis.

Spectrum and prevalence of cardiac sodium channel variants among black, white, Asian, and Hispanic individuals: implications for arrhythmogenic susceptibility and Brugada/long QT syndrome genetic testing.

OBJECTIVES: The purpose of this study was to determine the prevalence and spectrum of nonsynonymous polymorphisms (amino acid variants) in the cardiac sodium channel among healthy subjects. BACKGROUND: Pathogenic mutations in the cardiac sodium channel gene, SCN5A, cause approximately 15 to 20% of Brugada syndrome (BrS1), 5 to 10% of long QT syndrome (LQT3), and 2 to 5% of sudden infant death syndrome. METHODS: Using single-stranded conformation polymorphism, denaturing high-performance liquid chromatography, and/or direct DNA sequencing, mutational analysis of the protein-encoding exons of SCN5A was performed on 829 unrelated, anonymous healthy subjects: 319 black, 295 white, 112 Asian, and 103 Hispanic. RESULTS: In addition to the four known common polymorphisms (R34C, H558R, S1103Y, and R1193Q), four relatively ethnic-specific polymorphisms were identified: R481W, S524Y, P1090L, and V1951L. Overall, 39 distinct missense variants (28 novel) were elucidated. Nineteen variants (49%) were found only in the black cohort. Only seven variants (18%) localized to transmembrane-spanning domains. Four variants (F1293S, R1512W, and V1951L cited previously as BrS1-causing mutations and S1787N previously published as a possible LQT3-causing mutation) were identified in this healthy cohort. CONCLUSIONS: This study provides the first comprehensive determination of the prevalence and spectrum of cardiac sodium channel variants in healthy subjects from four distinct ethnic groups. This compendium of SCN5A variants is critical for proper interpretation of SCN5A genetic testing and provides an essential hit list of targets for future functional studies to determine whether or not any of these variants mediate genetic susceptibility for arrhythmias in the setting of either drugs or disease.

Ethnic differences in cardiac potassium channel variants: implications for genetic susceptibility to sudden cardiac death and genetic testing for congenital long QT syndrome.

OBJECTIVE: To determine the spectrum, frequency, and ethnic-specificity of channel variants in the potassium channel genes implicated in congenital long QT syndrome (LQTS) among healthy subjects. SUBJECTS AND METHODS: Genomic DNA from 744 apparently healthy individuals-305 black, 187 white, 134 Asian, and 118 Hispanic--was subject to a comprehensive mutational analysis of the 4 LQTS-causing potassium channel genes: KCNQ1 (LQT1), KCNH2 (LQT2), KCNE1 (LQT5), and KCNE2 (LQT6). RESULTS: Overall, 49 distinct amino acid-altering variants (36 novel) were identified: KCNQ1 (n = 16), KCNH2 (n = 25),KCNE1 (n = 5), and KCNE2 (n = 3). More than half of these variants (26/49) were found exclusively in black subjects. The known K897T-HERG and the G38S-min K common polymorphisms were identified in all 4 ethnic groups. Excluding these 2 common polymorphisms, 25% of black subjects had at least 1 nonsynonymous potassium channel variant compared with 14% of white subjects (P < .01). CONCLUSIONS: To our knowledge, this study represents the first comprehensive determination of the frequency and spectrum of cardiac channel variants found among healthy subjects from 4 major ethnic groups. Defining the population burden of genetic variants in these critical cardiac ion channels is crucial for proper interpretation of genetic test results of individuals at risk for LQTS. This compendium provides a resource for epidemiological and functional investigation of variant effects on the repolarization properties of cardiac tissues, including susceptibility to lethal cardiac arrhythmias.

Na transport in autosomal recessive polycystic kidney disease (ARPKD) cyst lining epithelial cells.

Autosomal dominant (ADPKD) and recessive (ARPKD) polycystic kidney disease are characterized by the progressive growth and expansion of cysts or ectatic collecting ducts, respectively, that ultimately destroy the normal renal parenchyma. Evidence from experimental models of ADPKD suggests that transepithelial Na and fluid secretion contribute to cyst growth, yet little is known about solute transport in ARPKD. This purpose of this study was to begin to characterize the expression and polarity of transport proteins involved in vectorial Na movement in ARPKD epithelium. Immunodetectable alpha1 and beta2 subunits of the Na/K-ATPase localized to the apical membrane of collecting duct cysts in tissue sections of human fetal ARPKD nephrectomy specimens and conditionally immortalized cells derived from these cysts. Measurements of transepithelial (22)Na transport performed on monolayers of ARPKD and age-matched collecting tubule (HFCT) cells grown on permeable supports revealed net Na absorption in both models. However, ARPKD cells absorbed Na at a rate approximately 50% greater than that of HFCT. Furthermore, Na absorption in ARPKD cells was partially inhibited by 100 micro M apical amiloride or 1 mM basolateral but not apical ouabain. Northern blot analyses of ARPKD whole kidney and Western immunoblot of ARPKD cells showed approximately twofold greater expression of the alpha-subunit of the epithelial Na channel (ENaC) compared with age-matched controls. These results suggest that, despite the presence of apical Na/K-ATPase, ARPKD cyst-lining cells absorb Na by a pathway that is modestly amiloride-sensitive. Whether Na absorption is mediated by ENaC, perhaps of nonclassical subunit composition, or another amiloride-sensitive transporter remains to be determined.

Towards understanding the polycystins.

Autosomal dominant polycystic kidney disease (ADPKD) is a very common inherited disease caused by mutations in PKD1 or PKD2 genes characterized by progressive enlargement of fluid-filled cysts and loss of renal function [1]. Previous studies proposed a role for human polycystin-1 in renal morphogenesis acting as a matrix receptor in focal adhesions and for polycystin-2 as a putative calcium channel [2, 3]. The genome of Caenorhabditis elegans contains 2 new members of the polycystin family: lov-1, the homolog for PKD1; and pkd-2, the homolog for PKD2 [4; this paper]. Mutation analysis in C. elegans showed similarly compromised male mating behaviors in all single and double lov-1 and pkd-2 mutants, indicating their participation in a single genetic pathway. Expression analysis localized LOV-1 and PKD-2 to the ends of sensory neurons in male tails and to the tips of CEM neurons in the head, consistent with functions as chemo- or mechanosensors. Human and C. elegans PKD1 and PKD2 homologs, transfected into mammalian renal epithelial cells, co-localized with paxillin in focal adhesions suggesting function in a single biological pathway. Based on the role of polycystins in C. elegans sensory neuron function and the conservation of PKD pathways we suggest that polycystins act as sensors of the extracellular environment, initiating, via focal adhesion assembly, intracellular transduction events in neuronal or morphogenetic processes.

PRKX, a phylogenetically and functionally distinct cAMP-dependent protein kinase, activates renal epithelial cell migration and morphogenesis.

The human protein kinase X gene (PRKX) is a member of an ancient family of cAMP-dependent serine/threonine kinases here shown to be phylogenetically distinct from the classical PKA, PKB/Akt, PKC, SGK, and PKG gene families. Renal expression of the PRKX gene is developmentally regulated and restricted to the ureteric bud epithelium of the fetal metanephric kidney. Aberrant adult kidney expression of PRKX was found in autosomal dominant polycystic kidney disease. PRKX kinase expression markedly activated migration of cultured renal epithelial cells in the presence of cAMP; this effect was blocked by cell treatment with the PKA inhibitor H89 and was not observed in PKA-transfected cells. In addition, expression of PRKX kinase activated branching morphogenesis of Madin-Darby canine kidney cells in collagen gels even in the absence of cAMP and/or hepatocyte growth factor, an effect not seen with either PKA expression or expression of a mutant, kinase-inactivated PRKX. These results suggest that the PRKX kinase may regulate epithelial morphogenesis during mammalian kidney development. Because another member of the PRKX gene family (the Dictyostelium discoideum gene KAPC-DICDI) also plays a role in cellular migration, these studies suggest that regulation of morphogenesis may be a distinctive property of these genes that has been conserved in evolution that is not shared with PKA family genes.

Herpes simplex virus as a model vector system for gene therapy in renal disease.

The past decade has been marked by significant advances in the application of gene transfer into living cells of animals and humans. These approaches have been tested in a few animal models of inherited and acquired renal diseases, including carbonic anhydrase II deficiency [1] and experimental glomerulonephritis [2, 3]. Gene transfer into proximal tubular cells has been successfully accomplished by intrarenal arterial infusion of a liposomal complex [4] or an adenoviral vector [5]. Tubular cells from the papilla and medulla have been selectively transduced by retrograde infusion into the pelvi-calyceal system of an adenoviral vector containing a reporter for beta-galactosidase [5]. Although the results of these initial studies are promising, further studies to optimize viral vectors, maximize gene delivery, minimize side-effects, and develop cell-specific and long-term regulated gene expression are critical to the success of gene therapy targeted to specific compartments of the kidney. Our recent efforts have focused on defining the cellular pathways responsible for viral entry and infection into renal epithelial cells using herpes simplex virus (HSV) as a model vector. We anticipate that a solid understanding of the basic scientific principles underlying viral entry and gene expression into specific populations of renal cells will facilitate the design of successful therapeutic viral-based gene transfer strategies.

Polarity of alpha-galactosidase A uptake by renal tubule cells.

BACKGROUND: Congenital absence of alpha-galactosidase in Fabry disease leads eventually to renal failure. Fabry disease is an attractive candidate for gene therapy, but uptake mechanisms of the enzyme must be understood for it to be used in treating patients with Fabry disease. METHODS: Immortalized human renal epithelial cells from three regions of the tubule were grown in culture on collagen-coated Transwell filters and were incubated with recombinant alpha-galactosidase protein placed at either the luminal or basolateral side of the cells. Uptake into cells was measured, and kinetic studies were performed. Blocking experiments were done with mannose 6-phosphate. RESULTS: Uptake from the basolateral side of the filters predominated in all three cell types. Only in distal tubule cells was mannose 6-phosphate able to block uptake to any degree. The kinetic data reveal a high Km for both luminal and basolateral cell surfaces. CONCLUSIONS: These data suggest that to correct the renal phenotype in Fabry disease, high levels of the enzyme will be need to be delivered to kidney cells. This will likely best be achieved with local administration of a vector containing the transgene directly to the kidney.