Biomolecular Markers for Safe Minimization of Immunosuppression

We seek additional funds for research responsive to the SARS-CoV-2/COVID-19 outbreak that is in scope of our ongoing grant R37AI051652 “Biomolecular Markers for Safe Minimization of Immuno-suppression.” From March 13, 2020 to April 20, 2020, we hospitalized 39 kidney allograft recipients positive for SARS-CoV-2 and with Covid-19 symptoms. Among these patients, 20 (51%) developed acute kidney injury (AKI). Importantly, graft dysfunction due to AKI recovered in 9 patients only and did not recover in 11 patients as of May 15, 2020. None underwent a diagnostic allograft biopsy because of biopsy-associated complications such as bleeding are potentially much more serious in this cohort and also to limit potential exposure of healthcare workers to SARS- CoV-2 during the invasive biopsy procedure. In the absence of a diagnostic biopsy, none received anti-rejection therapy. Whether the graft dysfunction was reversible or nonreversible could not be predicted at the time of graft dysfunction diagnosis. The dynamics of anti-allograft response from the reductions in their immunosuppressive therapy could not be captured with the available clinical analytes. To address these existing challenges, we propose the following: Specific Aim 1. To perform RNA sequencing of urinary cells and investigate whether the urinary cell transcriptome, ascertained at the time of graft dysfunction, is prognostic of allograft dysfunction. Urine will be collected at the time of graft dysfunction diagnosis and RNA isolated from urinary cells. RNA from 30 patients with reversible graft dysfunction; RNA from 30 patients with nonreversible graft dysfunction; and RNA from 30 patients with no graft dysfunction during the 3 months since Covid-19 diagnosis will be RNA sequenced and bioinformatics performed. The goal is to determine whether the urinary cell transcriptome profile, ascertained at the time of graft dysfunction, is prognostic of graft dysfunction and distinguishes those with reversible graft dysfunction from those with nonreversible graft dysfunction. Specific Aim 2. To measure urinary cell 3-gene signature score in sequential urine samples from Covid-19 kidney allograft recipients. Urine will be collected at baseline and sequentially every two weeks for 3 months since Covid-19 diagnosis. We will retrieve 210 sequential samples from 30 Covid-19 kidney allograft recipients (Baseline and 6 sequential samples from each patient) who developed reversible graft dysfunction; 210 sequential samples from 30 Covid-19 kidney allograft recipients who developed nonreversible graft dysfunction; and 30 Covid-19 kidney allograft recipients who did not develop graft dysfunction during the 3-months since Covid-19 diagnosis. RNA isolated from urinary cells will be reverse transcribed to cDNA and absolute copy numbers of CD3E mRNA, CXCL10 mRNA and 18S rRNA will be measured using customized PCR assays and urinary cell 3-gene signature score will be computed using a validated regression equation. The objective is to determine whether the urinary cell 3-gene scores in sequential samples anticipate those who develop nonreversible graft dysfunction rom those who develop reversible graft dysfunction.

We seek additional funds for research responsive to the SARS-CoV-2/COVID-19 outbreak that is in scope of our ongoing grant R37AI051652 “Biomolecular Markers for Safe Minimization of Immuno-suppression.” From March 13, 2020 to April 20, 2020, we hospitalized 39 kidney allograft recipients positive for SARS-CoV-2 and with Covid-19 symptoms. Among these patients, 20 (51%) developed acute kidney injury (AKI). Importantly, graft dysfunction due to AKI recovered in 9 patients only and did not recover in 11 patients as of May 15, 2020. None underwent a diagnostic allograft biopsy because of biopsy-associated complications such as bleeding are potentially much more serious in this cohort and also to limit potential exposure of healthcare workers to SARS- CoV-2 during the invasive biopsy procedure. In the absence of a diagnostic biopsy, none received anti-rejection therapy. Whether the graft dysfunction was reversible or nonreversible could not be predicted at the time of graft dysfunction diagnosis. The dynamics of anti-allograft response from the reductions in their immunosuppressive therapy could not be captured with the available clinical analytes. To address these existing challenges, we propose the following: Specific Aim 1. To perform RNA sequencing of urinary cells and investigate whether the urinary cell transcriptome, ascertained at the time of graft dysfunction, is prognostic of allograft dysfunction. Urine will be collected at the time of graft dysfunction diagnosis and RNA isolated from urinary cells. RNA from 30 patients with reversible graft dysfunction; RNA from 30 patients with nonreversible graft dysfunction; and RNA from 30 patients with no graft dysfunction during the 3 months since Covid-19 diagnosis will be RNA sequenced and bioinformatics performed. The goal is to determine whether the urinary cell transcriptome profile, ascertained at the time of graft dysfunction, is prognostic of graft dysfunction and distinguishes those with reversible graft dysfunction from those with nonreversible graft dysfunction. Specific Aim 2. To measure urinary cell 3-gene signature score in sequential urine samples from Covid-19 kidney allograft recipients. Urine will be collected at baseline and sequentially every two weeks for 3 months since Covid-19 diagnosis. We will retrieve 210 sequential samples from 30 Covid-19 kidney allograft recipients (Baseline and 6 sequential samples from each patient) who developed reversible graft dysfunction; 210 sequential samples from 30 Covid-19 kidney allograft recipients who developed nonreversible graft dysfunction; and 30 Covid-19 kidney allograft recipients who did not develop graft dysfunction during the 3-months since Covid-19 diagnosis. RNA isolated from urinary cells will be reverse transcribed to cDNA and absolute copy numbers of CD3E mRNA, CXCL10 mRNA and 18S rRNA will be measured using customized PCR assays and urinary cell 3-gene signature score will be computed using a validated regression equation. The objective is to determine whether the urinary cell 3-gene scores in sequential samples anticipate those who develop nonreversible graft dysfunction rom those who develop reversible graft dysfunction.