Bias in IGF-1 concentrations and interpretation across three different clinical laboratory assays.

In this study, we compared the DiaSorin LiaisonXL IGF-1 immunoassay to both the Roche Elecsys IGF-1 immunoassay and to the liquid chromatography-high resolution mass spectrometry (LC-MS) IGF-1 assay. Our study shows a constant positive bias in DiaSorin compared to the Roche immunoassay (mean 42 µg/L, 24%), and a proportional positive bias in DiaSorin compared to the LC-MS method (mean 49 µg/L, 29%). Further, we demonstrate the potential clinical impact of this bias by evaluating 43 adult samples, collected over a 2-month period, which were shown to be discrepant based on a chart review. Despite the positive analytical bias in the Diasorin assay compared to the LC-MS assay, the Diasorin assay upper reference limits were lower than those of the LC-MS assay. This effect caused nine out of forty-three samples to show falsely elevated results when they were clinically diagnosed as negative for acromegaly. Discussed in the context of previous literature, our findings emphasize the importance of adjusting reference intervals for IGF-1 assays based on the clinical needs of a patient population.

Assessment of SARS-CoV-2 Seropositivity During the First and Second Viral Waves in 2020 and 2021 Among Canadian Adults.

Importance: The incidence of infection during SARS-CoV-2 viral waves, the factors associated with infection, and the durability of antibody responses to infection among Canadian adults remain undocumented. Objective: To assess the cumulative incidence of SARS-CoV-2 infection during the first 2 viral waves in Canada by measuring seropositivity among adults. Design, Setting, and Participants: The Action to Beat Coronavirus study conducted 2 rounds of an online survey about COVID-19 experience and analyzed immunoglobulin G levels based on participant-collected dried blood spots (DBS) to assess the cumulative incidence of SARS-CoV-2 infection during the first and second viral waves in Canada. A sample of 19 994 Canadian adults (aged ≥18 years) was recruited from established members of the Angus Reid Forum, a public polling organization. The study comprised 2 phases (phase 1 from May 1 to September 30, 2020, and phase 2 from December 1, 2020, to March 31, 2021) that generally corresponded to the first (April 1 to July 31, 2020) and second (October 1, 2020, to March 1, 2021) viral waves. Main Outcomes and Measures: SARS-CoV-2 immunoglobulin G seropositivity (using a chemiluminescence assay) by major geographic and demographic variables and correlation with COVID-19 symptom reporting. Results: Among 19 994 adults who completed the online questionnaire in phase 1, the mean (SD) age was 50.9 (15.4) years, and 10 522 participants (51.9%) were female; 2948 participants (14.5%) had self-identified racial and ethnic minority group status, and 1578 participants (8.2%) were self-identified Indigenous Canadians. Among participants in phase 1, 8967 had DBS testing. In phase 2, 14 621 adults completed online questionnaires, and 7102 of those had DBS testing. Of 19 994 adults who completed the online survey in phase 1, fewer had an educational level of some college or less (4747 individuals [33.1%]) compared with the general population in Canada (45.0%). Survey respondents were otherwise representative of the general population, including in prevalence of known risk factors associated with SARS-CoV-2 infection. The cumulative incidence of SARS-CoV-2 infection among unvaccinated adults increased from 1.9% in phase 1 to 6.5% in phase 2. The seropositivity pattern was demographically and geographically heterogeneous during phase 1 but more homogeneous by phase 2 (with a cumulative incidence ranging from 6.4% to 7.0% in most regions). The exception was the Atlantic region, in which cumulative incidence reached only 3.3% (odds ratio [OR] vs Ontario, 0.46; 95% CI, 0.21-1.02). A total of 47 of 188 adults (25.3%) reporting COVID-19 symptoms during phase 2 were seropositive, and the OR of seropositivity for COVID-19 symptoms was 6.15 (95% CI, 2.02-18.69). In phase 2, 94 of 444 seropositive adults (22.2%) reported having no symptoms. Of 134 seropositive adults in phase 1 who were retested in phase 2, 111 individuals (81.8%) remained seropositive. Participants who had a history of diabetes (OR, 0.58; 95% CI, 0.38-0.90) had lower odds of having detectable antibodies in phase 2. Conclusions and Relevance: The Action to Beat Coronavirus study found that the incidence of SARS-CoV-2 infection in Canada was modest until March 2021, and this incidence was lower than the levels of population immunity required to substantially reduce transmission of the virus. Ongoing vaccination efforts remain central to reducing viral transmission and mortality. Assessment of future infection-induced and vaccine-induced immunity is practicable through the use of serial online surveys and participant-collected DBS.

TDAG51 induces renal interstitial fibrosis through modulation of TGF-ß receptor 1 in chronic kidney disease.

Chronic kidney disease (CKD) is characterized by the gradual loss of renal function and is a major public health concern. Risk factors for CKD include hypertension and proteinuria, both of which are associated with endoplasmic reticulum (ER) stress. ER stress-induced TDAG51 protein expression is increased at an early time point in mice with CKD. Based on these findings, wild-type and TDAG51 knock-out (TDKO) mice were used in an angiotensin II/deoxycorticosterone acetate/salt model of CKD. Both wild-type and TDKO mice developed hypertension, increased proteinuria and albuminuria, glomerular injury, and tubular damage. However, TDKO mice were protected from apoptosis and renal interstitial fibrosis. Human proximal tubular cells were used to demonstrate that TDAG51 expression induces apoptosis through a CHOP-dependent mechanism. Further, a mouse model of intrinsic acute kidney injury demonstrated that CHOP is required for ER stress-mediated apoptosis. Renal fibroblasts were used to demonstrate that TGF-ß induces collagen production through an IRE1-dependent mechanism; cells treated with a TGF-ß receptor 1 inhibitor prevented XBP1 splicing, a downstream consequence of IRE1 activation. Interestingly, TDKO mice express significantly less TGF-ß receptor 1, thus, preventing TGF-ß-mediated XBP1 splicing. In conclusion, TDAG51 induces apoptosis in the kidney through a CHOP-dependent mechanism, while contributing to renal interstitial fibrosis through a TGF-ß-IRE1-XBP1 pathway.

Association between the renin-angiotensin system and chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) is the major cause of death after lung transplantation. Angiotensin II (AngII), the main effector of the renin-angiotensin system, elicits fibrosis in both kidney and lung. We identified six AngII-regulated proteins (Ras homolog family member B (RHOB), bone marrow stromal cell antigen 1 (BST1), lysophospholipase 1 (LYPA1), glutamine synthetase (GLNA), thrombospondin 1 (TSP1) and laminin subunit ß2 (LAMB2)) that were increased in urine of patients with kidney allograft fibrosis. We hypothesised that the renin-angiotensin system is active in CLAD and that AngII-regulated proteins are increased in bronchoalveolar lavage fluid (BAL) of CLAD patients.We performed immunostaining of AngII receptors (AGTR1 and AGTR2), TSP1 and GLNA in 10 CLAD lungs and five controls. Using mass spectrometry, we quantified peptides corresponding to AngII-regulated proteins in BAL of 40 lung transplant recipients (stable, acute lung allograft dysfunction (ALAD) and CLAD). Machine learning algorithms were developed to predict CLAD based on BAL peptide concentrations.Immunostaining demonstrated significantly more AGTR1+ cells in CLAD versus control lungs (p=0.02). TSP1 and GLNA immunostaining positively correlated with the degree of lung fibrosis (R2=0.42 and 0.57, respectively). In BAL, we noted a trend towards higher concentrations of AngII-regulated peptides in patients with CLAD at the time of bronchoscopy, and significantly higher concentrations of BST1, GLNA and RHOB peptides in patients that developed CLAD at follow-up (p<0.05). The support vector machine classifier discriminated CLAD from stable and ALAD patients at the time of bronchoscopy (area under the curve (AUC) 0.86) and accurately predicted subsequent CLAD development (AUC 0.97).Proteins involved in the renin-angiotensin system are increased in CLAD lungs and BAL. AngII-regulated peptides measured in BAL may accurately identify patients with CLAD and predict subsequent CLAD development.

Performance evaluation of all analytes on the epoc® Blood Analysis System for use in hospital surgical and intensive care units.

OBJECTIVE: To evaluate the performance of the epoc hand-held analyzer against the RAPIDPoint 500 blood gas analyzer and laboratory analyzers where applicable. METHODS: Venous or arterial whole blood samples collected in balanced heparinized syringes were obtained from 69 patients (35 females, 34 males) predominantly (77%) from the surgical unit and intensive care unit (ICU). Method comparison was performed for all analytes on the epoc System against the RAPIDPoint 500 Blood gas analyzer or laboratory analyzers where applicable. Results: Mean bias was <5% for blood gases, electrolytes, lactate and glucose. Hematocrit showed a bias of -6.76% (95% CI â€‹= â€‹-8.91, - 4.61) compared to the HemataSTAT-II method, whereas calculated total hemoglobin showed a bias of 1.51% (95% CI â€‹= â€‹-1.04, 4.06) against the Sysmex XN-10 hematology analyzer. Creatinine showed the largest bias relative to laboratory analyzers, Abbott Architect c8000 Jaffe method (13.54%, 95% CI â€‹= â€‹5.43, 21.65) and Roche Cobas c702 enzymatic method (30.01%, 95% CI â€‹= â€‹12.64, 47.38). Conclusions: The epoc system is fit for use in the surgical and ICU setting for the measurement of all analytes except for creatinine.

Urine Angiotensin II Signature Proteins as Markers of Fibrosis in Kidney Transplant Recipients.

BACKGROUND: Interstitial fibrosis/tubular atrophy (IFTA) is an important cause of kidney allograft loss; however, noninvasive markers to identify IFTA or guide antifibrotic therapy are lacking. Using angiotensin II (AngII) as the prototypical inducer of IFTA, we previously identified 83 AngII-regulated proteins in vitro. We developed mass spectrometry-based assays for quantification of 6 AngII signature proteins (bone marrow stromal cell antigen 1, glutamine synthetase [GLNA], laminin subunit beta-2, lysophospholipase I, ras homolog family member B, and thrombospondin-I [TSP1]) and hypothesized that their urine excretion will correlate with IFTA in kidney transplant patients. METHODS: Urine excretion of 6 AngII-regulated proteins was quantified using selected reaction monitoring and normalized by urine creatinine. Immunohistochemistry was used to assess protein expression of TSP1 and GLNA in kidney biopsies. RESULTS: The urine excretion rates of AngII-regulated proteins were found to be increased in 15 kidney transplant recipients with IFTA compared with 20 matched controls with no IFTA (mean log2[fmol/µmol of creatinine], bone marrow stromal cell antigen 1: 3.8 versus 3.0, P = 0.03; GLNA: 1.2 versus -0.4, P = 0.03; laminin subunit beta-2: 6.1 versus 5.4, P = 0.06; lysophospholipase I: 2.1 versus 0.6, P = 0.002; ras homolog family member B: 1.2 versus -0.1, P = 0.006; TSP1_GGV: 2.5 versus 1.9; P = 0.15; and TSP1_TIV: 2.0 versus 0.6, P = 0.0006). Receiver operating characteristic curve analysis demonstrated an area under the curve = 0.86 for the ability of urine AngII signature proteins to discriminate IFTA from controls. Urine excretion of AngII signature proteins correlated strongly with chronic IFTA and total inflammation. In a separate cohort of 19 kidney transplant recipients, the urine excretion of these 6 proteins was significantly lower following therapy with AngII inhibitors (P < 0.05). CONCLUSIONS: AngII-regulated proteins may represent markers of IFTA and guide antifibrotic therapies.

Endoplasmic reticulum stress inhibition attenuates hypertensive chronic kidney disease through reduction in proteinuria.

Endoplasmic reticulum (ER) stress is implicated in chronic kidney disease (CKD) development in patients and in animal models. Here we show that ER stress inhibition through 4-phenylbutyric acid (4-PBA) administration decreases blood pressure, albuminuria, and tubular casts in an angiotensin II/deoxycorticosterone acetate/salt murine model of CKD. Lower albuminuria in 4-PBA-treated mice was associated with higher levels of cubilin protein in renal tissue membrane fractions. 4-PBA decreased renal interstitial fibrosis, renal CD3+ T-cell and macrophage infiltration, mRNA expression of TGFß1, Wnt signaling molecules, and ER stress-induced pro-inflammatory genes. CHOP deficient mice that underwent this model of CKD developed hypertension comparable to wild type mice, but had less albuminuria and tubular casts. CHOP deficiency resulted in higher nephrin levels and decreased glomerulosclerosis compared to wild type mice; this effect was accompanied by lower macrophage infiltration and fibrosis. Our findings portray ER stress inhibition as a means to alleviate hypertensive CKD by preserving glomerular barrier integrity and tubular function. These results demonstrate ER stress modulation as a novel target for preserving renal function in hypertensive CKD.

GRP78 and CHOP modulate macrophage apoptosis and the development of bleomycin-induced pulmonary fibrosis.

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78(+/-) , Chop(-/-) and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78(+/-) mice were strongly protected from bleomycin-induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL-positive cells were observed in Grp78(+/-) mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78(+/-) mice was also strongly positive for cleaved caspase-3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop(-/-) mice resulted in increased quasi-static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase-1-positive macrophages that were negative for cleaved caspase-3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78- and CHOP-mediated macrophage apoptosis was found to protect against bleomycin-induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78-mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Development of a Model of Chronic Kidney Disease in the C57BL/6 Mouse with Properties of Progressive Human CKD.

Chronic kidney disease (CKD) is a major healthcare problem with increasing prevalence in the population. CKD leads to end stage renal disease and increases the risk of cardiovascular disease. As such, it is important to study the mechanisms underlying CKD progression. To this end, an animal model was developed to allow the testing of new treatment strategies or molecular targets for CKD prevention. Many underlying risk factors result in CKD but the disease itself has common features, including renal interstitial fibrosis, tubular epithelial cell loss through apoptosis, glomerular damage, and renal inflammation. Further, CKD shows differences in prevalence between the genders with premenopausal women being relatively resistant to CKD. We sought to develop and characterize an animal model with these common features of human CKD in the C57BL/6 mouse. Mice of this genetic background have been used to produce transgenic strains that are commercially available. Thus, a CKD model in this strain would allow the testing of the effects of numerous genes on the severity or progression of CKD with minimal cost. This paper describes such a mouse model of CKD utilizing angiotensin II and deoxycorticosterone acetate as inducers.

Crosstalk between the unfolded protein response and NF-κB-mediated inflammation in the progression of chronic kidney disease.

The chronic inflammatory response is emerging as an important therapeutic target in progressive chronic kidney disease. A key transcription factor in the induction of chronic inflammation is NF-κB. Recent studies have demonstrated that sustained activation of the unfolded protein response (UPR) can initiate this NF-κB signaling phenomenon and thereby induce chronic kidney disease progression. A key factor influencing chronic kidney disease progression is proteinuria and this condition has now been demonstrated to induce sustained UPR activation. This review details the crosstalk between the UPR and NF-κB pathways as pertinent to chronic kidney disease. We present potential tools to study this phenomenon as well as potential therapeutics that are emerging to regulate the UPR. These therapeutics may prevent inflammation specifically induced in the kidney due to proteinuria-induced sustained UPR activation.

SREBP-1 Mediates Angiotensin II-Induced TGF-ß1 Upregulation and Glomerular Fibrosis.

Angiotensin II is an important mediator of CKD of diverse etiology. A common pathologic feature of CKD is glomerular fibrosis, a central mediator of which is the profibrotic cytokine TGF-ß. The mechanisms underlying the induction of TGF-ß and matrix by angiotensin II are not completely understood. Recent studies showed that overexpression of the transcription factor SREBP-1 induces glomerular sclerosis and that angiotensin II can activate SREBP-1 in tubular cells. We thus studied whether SREBP-1 is activated by angiotensin II and mediates angiotensin II-induced profibrogenic responses in primary rat mesangial cells. Treatment of cells with angiotensin II induced the upregulation and activation of SREBP-1. Angiotensin II-induced activation of SREBP-1 required signaling through the angiotensin II type I receptor and activation of PI3K/Akt in addition to the chaperone SCAP and protease S1P. Notably, angiotensin II-induced endoplasmic reticulum stress was identified as a key mediator of Akt-SREBP-1 activation, and inhibition of endoplasmic reticulum stress or SREBP-1 prevented angiotensin II-induced SREBP-1 binding to the TGF-ß promoter, TGF-ß upregulation, and downstream fibronectin upregulation. Endoplasmic reticulum stress alone, however, did not induce TGF-ß upregulation despite activating SREBP-1. Although not required for SREBP-1 activation by angiotensin II, EGF receptor signaling was necessary for activation of the SREBP-1 cotranscription factor Sp1, which provided a required second signal for TGF-ß upregulation. In vivo, endoplasmic reticulum stress and SREBP-1-dependent effects were induced in glomeruli of angiotensin II-infused mice, and administration of the SREBP inhibitor fatostatin prevented angiotensin II-induced TGF-ß upregulation and matrix accumulation. SREBP-1 and endoplasmic reticulum stress thus provide potential novel therapeutic targets for the treatment of CKD.

TDAG51 mediates epithelial-to-mesenchymal transition in human proximal tubular epithelium.

Epithelial-to-mesenchymal transition (EMT) contributes to renal fibrosis in chronic kidney disease. Endoplasmic reticulum (ER) stress, a feature of many forms of kidney disease, results from the accumulation of misfolded proteins in the ER and leads to the unfolded protein response (UPR). We hypothesized that ER stress mediates EMT in human renal proximal tubules. ER stress is induced by a variety of stressors differing in their mechanism of action, including tunicamycin, thapsigargin, and the calcineurin inhibitor cyclosporine A. These ER stressors increased the UPR markers GRP78, GRP94, and phospho-eIF2α in human proximal tubular cells. Thapsigargin and cyclosporine A also increased cytosolic Ca(2+) concentration and T cell death-associated gene 51 (TDAG51) expression, whereas tunicamycin did not. Thapsigargin was also shown to increase levels of active transforming growth factor (TGF)-ß1 in the media of cultured human proximal tubular cells. Thapsigargin induced cytoskeletal rearrangement, ß-catenin nuclear translocation, and α-smooth muscle actin and vinculin expression in proximal tubular cells, indicating an EMT response. Subconfluent primary human proximal tubular cells were induced to undergo EMT by TGF-ß1 treatment. In contrast, tunicamycin treatment did not produce an EMT response. Plasmid-mediated overexpression of TDAG51 resulted in cell shape change and ß-catenin nuclear translocation. These results allowed us to develop a two-hit model of ER stress-induced EMT, where Ca(2+) dysregulation-mediated TDAG51 upregulation primes the cell for mesenchymal transformation via Wnt signaling and then TGF-ß1 activation leads to a complete EMT response. Thus the release of Ca(2+) from ER stores mediates EMT in human proximal tubular epithelium via the induction of TDAG51.

Integrated stress response modulates cellular redox state via induction of cystathionine γ-lyase: cross-talk between integrated stress response and thiol metabolism.

The integrated stress response mediated by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2α phosphorylation induces activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor that regulates the expression of genes responsible for amino acid metabolism, cellular redox state, and anti-stress responses. Cystathionine γ-lyase (CSE) and cystathionine ß-synthase are critical enzymes in the transsulfuration pathway, which also regulate cellular redox status by modulating glutathione (GSH) levels. To determine the link between the integrated stress response and the transsulfuration pathway, we used homocysteine (Hcy) as an inducer of eIF2α phosphorylation and ATF4 gene induction. Mouse embryonic fibroblasts (MEFs) lacking ATF4 (ATF4(-/-)) had reduced GSH levels and increased reactive oxygen species and were susceptible to apoptotic cell death under normal culture conditions. Further, ATF4(-/-) MEFs were more sensitive to Hcy-induced cytotoxicity and showed significantly reduced intracellular GSH levels associated with apoptosis. ATF4(-/-) MEFs could be rescued from l-Hcy-induced apoptosis by ß-mercaptoethanol medium supplementation that increases cysteine levels and restores GSH synthesis. ATF4(-/-) MEFs showed little or no CSE protein but did express cystathionine ß-synthase. Further, ER stress-inducing agents, including tunicamycin and thapsigargin, induced the expression of CSE in ATF4(+/+) MEFs. Consistent with ATF4(-/-) MEFs, CSE(-/-) MEFs showed significantly greater apoptosis when treated with tunicamycin, thapsigargin, and l-Hcy, compared with CSE(+/+) MEFs. Liver and kidney GSH levels were also reduced in CSE(-/-) mice, suggesting that CSE is a critical factor in GSH synthesis and may act to protect the liver and kidney from a variety of conditions that cause ER stress.