Mechanistic insight of the role of NHERF1 in cisplatin-induced acute kidney injury.

Background. Acute kidney injury (AKI) develops in 30% of patients who receive cisplatin, a widely used chemotherapeutic agent. We previously showed that NHERF1 loss resulted in increased susceptibility to cisplatin nephrotoxicity. The overarching goal of this dissertation was to elucidate mechanisms of susceptibility to cisplatin-induced AKI, specifically the effects of NHERF1 loss on tubule cell metabolism, tubule cell mitochondrial function, and alterations in oxidative state and/or renal handling of cisplatin. Methods. 2-4 month male wild type (WT) and NHERF1 knock out (KO) mice were treated with either vehicle control or cisplatin (20 mg/kg dose IP) for 4, 24, and 72 hours. Urine was collected for NGAL and kidneys were harvested for histology and the following assays: Thiobarbituric Acid Reactive Substances (TBARS) for lipid peroxidation, γ-glutamyl transferase (GGT) activity, and Western Blot for GGT and cysteine S-conjugate beta lyase (CCBL). Mitochondrial respiration was conducted via the Seahorse XF24 analyzer on non-treated isolated kidney mitochondria. LC-MS analysis was used to evaluate ATP content in non-treated kidneys. Electron microscopy was utilized to evaluate mitochondrial morphology and number in non-treated kidneys. HPLC of the reduced and oxidized forms of the small molecular weight thiols (glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys), and cystine (CySS), and cysteine-glutathione disulfide (CySSG) on plasma and kidney cortex. Statistical analysis was completed using Student’s t-test for LC-MS, mitochondrial number, and mitochondrial respiration and Two-way ANOVA was used for all other analysis. P-values of Results. Chapter III demonstrates that WT and NHERF1 KO mice do not exhibit metabolic changes or changes in ATP content that would definitively sensitize the KO mice to cisplatin injury. Chapter IV shows that NHERF1 loss does not affect mitochondrial morphology or mitochondrial number, or oxidative phosphorylation capacity via Seahorse XF24 analysis. Thus, mitochondrial dysfunction does not appear to sensitize the KO mice to cisplatin injury. Lastly, Chapter V reveals that NHERF1 KO mouse kidneys do not exhibit changes in lipid peroxidation, oxidative stress, GGT or CCBL protein levels that would sensitize these animals to cisplatin. However, NHERF1 KO kidneys appear to respond differently to the cisplatin insult itself, characterized by differences in GGT activity in response to cisplatin. Conclusions. In conclusion, the work presented in this dissertation reveals that metabolic stress and mitochondrial dysfunction are not the mechanisms of susceptibility to cisplatin in NHERF1 KO mice. Furthermore, NHERF1 loss does not lead to changes in kidney GSH metabolism. In conclusion, these data do not support NHERF1 loss resulting in a fundamental metabolic defect that increases susceptibility to cisplatin injury. Instead NHERF1 loss appears to influence either the handling, the initial insult, or the response to injury resulting in exacerbated injury

Breadth of Vaccinated Cancer Patient Humoral Response to SARS-CoV-2 Spike Protein and RBD Variants

SARS-CoV-2, the virus responsible for the COVID-19 of which several variants have emerged, such as the B.1.351 SARS-CoV-2 variant. The Receptor Binding Domain (RBD), located within the Spike protein is an immunogenic epitope for potent neutralizing antibodies. Current mRNA vaccines encode for the Spike protein, allowing the body to build antigen-specific antibodies. Assays measuring protective antibodies are essential to manage the COVID-19 pandemic and can be used as a platform for variant screening. RBD-foldon 2.2 is a novel antigen produced by fusing RBD with the trimerization domain Fibritin from Bacteriophage T4. Its amino acid sequence is based on the original Wuhan strain. (Breckenridge, 2021). B.1.351 RBD-foldon 2.2 antigen is identical to RBD-foldon 2.2, except it uses the B.1.351 variant RBD sequence. Using cancer patient sera samples, the breadth and robustness of response was examined in comparison to patients that indicated “no chronic conditions”. We hypothesized there would be a difference in humoral response to RBD-variant antigens in COVID-19 vaccinated cancer patients undergoing treatment vs patients with no chronic conditions. For sample selection, cancer patients were age/sex matched to individuals with no underlying health conditions, that received the same mRNA vaccine within 2 weeks of each other. To quantify antibody levels, ELISA end-point titers were performed. ELISAs detected levels of IgG and IgA antibodies against Spike, RBD-foldon, RBD-foldon 2.2, and RBD-foldon B.1.351. (Bushau, 2021). The statistical analysis used was a two-tailed student’s t-test to compare mean value of end-point titers between experimental and control groups. No significant difference between experimental and control groups for any antibody-antigen combination. B.1.351 RBD-foldon appears to elicit a lower response than RBD-foldon 2.2. Lower response may be explained by the mRNA sequence used in current vaccines encodes for original Wuhan SARS-CoV-2 spike protein. The platform is predictive of the level of antibody protection for variant screening

Public awareness and support for use of wastewater for SARS-CoV-2 monitoring: A community survey in Louisville, Kentucky

The majority of sewer systems in the United States and other countries, are operated by public utilities. In the absence of any regulation, public perception of monitoring wastewater for population health biomarkers is an important consideration for a public utility commission when allocating resources for this purpose. In August 2021, we conducted a survey as part of an ongoing COVID-19 community prevalence study in Louisville/Jefferson County, KY. The survey comprised of seven questions about awareness of and privacy concerns and was sent to 32,000 households randomly distributed within the county. A total of 1,220 sampled adults participated in the probability sample, and 981 were used in analysis. A total of 2,444 adults additionally responded in the convenience sample, and 1,751 were used in analysis. The samples were weighted to produce estimates representative of all adults in the county. Public awareness of tracking COVID-19 virus in the sewers was low. Opinions about how data from this activity are shared strongly supported public disclosure of monitoring results. Responses showed more support for measuring the largest areas (\u3e30,000 to 50,000 households) typically representing population levels found in a community or regional wastewater treatment plant. Those who had a history of COVID-19 infection were more likely to support highly localized monitoring. Understanding wastewater surveillance strategies and thresholds of privacy concerns requires in-depth, comprehensive analysis of public opinion for continued success and efficacy of public health monitoring

Pre-Existing Comorbidities Diminish the Likelihood of Seropositivity after SARS-CoV-2 Vaccination

Background: The impact of chronic health conditions (CHCs) on serostatus post-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is unknown. Methods: We assessed serostatus post-SARS-CoV-2 vaccination among fully vaccinated adult residents of Jefferson County, Kentucky, USA, from April 2021 to August 2021. Serostatus was determined by qualitative analysis of SARS-CoV-2-specific Spike IgG antibodies via enzyme-linked immunoassay (ELISA) in peripheral blood samples. Results: Of the 5178 fully vaccinated participants, 51 were seronegative and 5127 were seropositive. Chronic kidney disease (CKD) and autoimmune disease showed the highest association with negative serostatus in fully vaccinated individuals. The absence of any CHC was strongly associated with positive serostatus. The risk of negative serostatus increased as the total number of pre-existing CHCs increased. Similarly, the use of two or more CHC-related medications was associated with seronegative status. Conclusions: The presence of any CHC, especially CKD or autoimmune disease, increased the likelihood of seronegative status among individuals who were fully vaccinated to SAR-CoV-2. This risk increased with a concurrent increase in number of comorbidities, especially with multiple medications. The absence of any CHC was protective and increased the likelihood of a positive serological response. These results will help develop appropriate guidelines for booster doses and targeted vaccination programs

Na/h exchange regulatory factor 1 deficient mice show evidence of oxidative stress and altered cisplatin pharmacokinetics

(1) Background: One third of patients who receive cisplatin develop an acute kidney injury. We previously demonstrated the Na/H Exchange Regulatory Factor 1 (NHERF1) loss resulted in increased kidney enzyme activity of the pentose phosphate pathway and was associated with more severe cisplatin nephrotoxicity. We hypothesized that changes in proximal tubule biochemical pathways associated with NHERF1 loss alters renal metabolism of cisplatin or response to cisplatin, resulting in exacerbated nephrotoxicity. (2) Methods: 2–4 month-old male wild-type and NHERF1 knock out littermate mice were treated with either vehicle or cisplatin (20 mg/kg dose IP), with samples taken at either 4, 24, or 72 h. Kidney injury was determined by urinary neutrophil gelatinase-associated lipocalin and histology. Glutathione metabolites were measured by HPLC and genes involved in glutathione synthesis were measured by qPCR. Kidney handling of cisplatin was assessed by a kidney cortex measurement of γ-glutamyl transferase activity, Western blot for γ-glutamyl transferase and cysteine S-conjugate beta lyase, and ICP-MS for platinum content. (3) Re-sults: At 24 h knock out kidneys show evidence of greater tubular injury after cisplatin and exhibit a decreased reduced/oxidized glutathione ratio under baseline conditions in comparison to wild-type. KO kidneys fail to show an increase in γ-glutamyl transferase activity and experience a more rapid decline in tissue platinum when compared to wild-type. (4) Conclusions: Knock out kidneys show evidence of greater oxidative stress than wild-type accompanied by a greater degree of early injury in response to cisplatin. NHERF1 loss has no effect on the initial accumulation of cisplatin in the kidney cortex but is associated with an altered redox status which may alter the activity of enzymes involved in cisplatin metabolism

Quantifying the relationship between sub-population wastewater samples and community-wide SARS-CoV-2 seroprevalence

Robust epidemiological models relating wastewater to community disease prevalence are lacking. Assessments of SARS-CoV-2 infection rates have relied primarily on convenience sampling, which does not provide reliable estimates of community disease prevalence due to inherent biases. This study conducted serial stratified randomized samplings to estimate the prevalence of SARS-CoV-2 antibodies in 3717 participants, and obtained weekly samples of community wastewater for SARS-CoV-2 concentrations in Jefferson County, KY (USA) from August 2020 to February 2021. Using an expanded Susceptible-Infected-Recovered model, the longitudinal estimates of the disease prevalence were ob-tained and compared with the wastewater concentrations using regression analysis. The model analysis revealed sig-nificant temporal differences in epidemic peaks. The results showed that in some areas, the average incidence rate, based on serological sampling, was 50 % higher than the health department rate, which was based on convenience sampling. The model-estimated average prevalence rates correlated well with the wastewater (correlation = 0.63, CI (0.31,0.83)). In the regression analysis, a one copy per ml-unit increase in weekly average wastewater concentration of SARS-CoV-2 corresponded to an average increase of 1-1.3 cases of SARS-CoV-2 infection per 100,000 residents. The analysis indicates that wastewater may provide robust estimates of community spread of infection, in line with the modeled prevalence estimates obtained from stratified randomized sampling, and is therefore superior to publicly available health data.11Nsciescopu