Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe

Immunoprecipitation performance and affinity of the PARP1 nanobody.

<p>(A) Immunoprecipitation of endogenous hPARP1 from whole-cell lysates of HEK293T cells with PARP1 nanotrap. Input (I), flow-through (FT) and bound (B) fractions were analyzed by SDS-PAGE followed by Coomassie Blue staining (left) and western blotting with anti-PARP1 antibody (right). (B) Affinity measurement of the PARP1 nanobody with Biacore SPR. The sensorgrams for the nanobody at different concentrations of hPARP1 are indicated.</p

Recruitment of endogenous PARP1 to the DNA damage sites as visualized by the PARP1 chromobody in live human cells.

<p>(A) Live-cell imaging of laser-microirradiated (405 nm laser, 100% power, 1 s) human HeLa, human PC3 cells and hamster BHK cells transiently expressing PARP1 chromobody. Time-lapse imaging was carried out at 1 frame per second with the spinning disc microscope acquiring 9 pre-irradiation and 100 post-irradiation frames. Selected time-frames are shown, yellow circles depict the regions of microirradiation (Ø 1 μm), yellow arrow-heads mark the sites before and after irradiation. Scale bar, 5 μm. (B) Live-cell imaging of carbon ion-irradiated HeLa cells (300 ions per point) transiently expressing PARP1 chromobody. The cells were irradiated with accelerated 55 MeV (total energy) carbon ions (LET in water: 310 KeV/μm). At 0 s yellow dots in a cross-shape mark the prospective sites of irradiation. After irradiation images were acquired every ~4 s. Selected time points are shown. Scale bar, 10 μm.</p

Determination of the PARP family selectivity and species reactivity of the PARP1 nanobody.

<p>(A) Immunoprecipitation of GFP-tagged hPARP1 (141 kDa), hPARP2 (93 kDa), hPARP3 (87 kDa), hPARP9 (123 kDa) and GFP (27 kDa, negative control) with the PARP1 nanotrap from transiently transfected HEK293T cells. RFP-Trap was used as control. Input (I), flow-through (FT) and bound (B) fractions were separated by SDS-PAGE followed by immunoblotting with anti-GFP antibody. (B) Immunoprecipitation of endogenous PARP1 from mouse (MEF) and hamster (BHK) cells with the PARP1 nanotrap. The fractions were analyzed by SDS-PAGE and immunoblotting with anti-PARP1 antibody.</p

Intracellular F2H analysis of the PARP1 chromobody.

<p>BHK-F2H cells were pairwise co-transfected with the PARP1 chromobody fused to TagRFP and one of the GFP-tagged bait constructs: GFP alone, GFP-hPARP1, GFP-hPARP2, GFP-hPARP3, GFP-hPARP9, wild-type ZnF2-GFP and ZnF2mut-GFP. The cells were fixed, stained with DAPI and subjected to fluorescence microscopy. Upper row, green channel: GFP-fusion proteins are enriched at the “spot” in the nuclei of transfected BHK-F2H cells (arrows). Middle row, red channel: binding of the PARP1 chromobody to the full-length GFP-hPARP1 and to the wild-type ZnF2-GFP is visible as local enrichments of the red fluorescent signals (arrows). Neither interaction of the PARP1 chromobody with hPARP2, 3, or 9, nor interaction with the mutant ZnF2mut-GFP construct (G161T, A188S and T189A) can be observed. Co-transfection with GFP (first column) served as negative control to exclude non-specific binding of the PARP1 chromobody to GFP. Scale bar, 5 μm.</p

On-bead pADPr chain synthesis with the endogenous hPARP1 immunoprecipitated with the PARP1 nanotrap.

<p>Gel electrophoresis and silver staining of pADPr fractions from <i>in vitro</i> synthesis. Lanes 1–7: commercially available pADPr chains (lane 1, control); reaction with the purified recombinant hPARP1 with NAD+ (lane 2) or without NAD+ (lane 3); on-bead reaction with PARP1 nanotrap-precipitated endogenous hPARP1 with NAD+ (lane 4) or without NAD+ (lane 5); on-bead reaction with GFP-Trap with NAD+ (lane 6) or without NAD+ (lane 7).</p

PARP1 chromobody enables visualization of hPARP1 in human HeLa cells.

<p>(A) PARP1 chromobody fused to TagRFP (red) co-localizes with GFP-PARP1 (green) in nucleoli and nucleoplasm. (B-C) PARP1 chromobody fused to TagGFP (B, green) or fused to TagRFP (C, red) visualizes endogenous hPARP1 in nucleoli and nucleoplasm. Cells were fixed, stained with DAPI (blue) and subjected to epifluorescence imaging. Scale bar, 10 μm. (D-F) Live-cell imaging with the PARP1 chromobody upon compound treatment. HeLa cells transiently expressing either PARP1 chromobody, TagRFP-hPARP1 or TagRFP alone were treated for 2 h with 10 μM camptothecin (D), 0.01 μM actinomycin D (E), or 0.01 μM 4-NQO (F). After subjecting cells to incubation with the compounds, cells were washed and allowed to recover for another 2 h. Time-lapse epifluorescence imaging was carried out in an automated fashion every 15 min during treatments and during recovery. The panels show selected frames of the cells before treatments, after 2 h of treatment and after 2 h of recovery. Scale bar, 5 μm.</p

Endogenous hPARP1 co-precipitates together with the intracellularly expressed PARP1 chromobody.

<p>PARP1 chromobody fused to TagRFP was precipitated using the RFP-affinity resin (RFP-Trap) from a whole-cell lysate of HeLa cells stably expressing the chromobody. TagRFP-transfected HeLa cells served as negative control for non-specific binding. Input (I), flow-through (FT) and bound (B) fractions were subjected to SDS-PAGE and immunoblotting with anti-PARP1 antibody, followed by anti-TagRFP antibody and anti-GAPDH antibody as loading control.</p