Differential prognostic utility of adiposity measures in chronic kidney disease

Objective Adipose tissue contributes to adverse outcomes in chronic kidney disease (CKD), but there is uncertainty regarding the prognostic relevance of different adiposity measures. We analyzed the associations of neck circumference (NC), waist circumference (WC), and body mass index (BMI) with clinical outcomes in patients with mild to severe CKD. Methods The German Chronic Kidney Disease (GCKD) study is a prospective cohort study, which enrolled Caucasian adults with mild to severe CKD, defined as estimated glomerular filtration rate (eGFR): 30–60 mL/min/1.73 m2, or >60 mL/min/1.73 m2 in the presence of overt proteinuria. Associations of NC, WC and BMI with all-cause death, major cardiovascular events (MACE: a composite of non-fatal stroke, non-fatal myocardial infarction, peripheral artery disease intervention, and cardiovascular death), kidney failure (a composite of dialysis or transplantation) were analyzed using multivariable Cox proportional hazards regression models adjusted for confounders and the Akaike information criteria (AIC) were calculated. Models included sex interactions with adiposity measures. Results A total of 4537 participants (59% male) were included in the analysis. During a 6.5-year follow-up, 339 participants died, 510 experienced MACE, and 341 developed kidney failure. In fully adjusted models, NC was associated with all-cause death in women (HR 1.080 per cm; 95% CI 1.009–1.155), but not in men. Irrespective of sex, WC was associated with all-cause death (HR 1.014 per cm; 95% CI 1.005–1.038). NC and WC showed no association with MACE or kidney failure. BMI was not associated with any of the analyzed outcomes. Models of all-cause death including WC offered the best (lowest) AIC. Conclusion In Caucasian patients with mild to severe CKD, higher NC (in women) and WC were significantly associated with increased risk of death from any cause, but BMI was not

E. coli HMS174(DE3) is a sustainable alternative to BL21(DE3)

Abstract Background Escherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG. We recently published a few studies regarding lactose induction in BL21(DE3) strains. BL21(DE3) can only take up the glucose-part of the disaccharide when fed with lactose. However, initially additional glucose has to be supplied as otherwise the ATP-related lactose uptake barely happens. Yet, as lactose is an inexpensive compound compared to glucose and IPTG, a new induction strategy by a lactose-only feed during induction seems attractive. Thus, we investigated this idea in the galactose metabolizing strain HMS174(DE3). Results We show that strain HMS174(DE3) can be cultivated on lactose as sole carbon source during induction. We demonstrate that strain HMS174(DE3) exhibits higher product and biomass yields compared to BL21(DE3) when cultivated in a lactose fed-batch. More importantly, HMS174(DE3) cultivated on lactose even expresses more product than BL21(DE3) in a standard IPTG induced glucose fed-batch at the same growth rate. Finally, we demonstrate that productivity in HMS174(DE3) lactose-fed batch cultivations can easily be influenced by the specific lactose uptake rate (qs,lac). This is shown for two model proteins, one expressed in soluble form and one as inclusion body. Conclusions As strain HMS174(DE3) expresses even slightly higher amounts of target protein in a lactose fed-batch than BL21(DE3) in a standard cultivation, it seems a striking alternative for recombinant protein production. Especially for large scale production of industrial enzymes cheap substrates are essential. Besides cost factors, the strategy allows straight forward adjustment of specific product titers by variation of the lactose feed rate

Valorisation of cheese whey as substrate and inducer for recombinant protein production in E. coli HMS174(DE3)

Every year worldwide around 190 million tons of cheese whey are generated resulting in a huge environmental burden. We recently published a study where we showed that E. coli strain HMS174(DE3) can be cultivated using only lactose as C-source and inducer. Motivated by the results we investigated using a concentrated whey feed instead of the lactose feed. Spray drying whey and dissolving the powder allowed preparation of a 40-fold concentrated whey containing 91% lactose and 81% protein of the original whey. Cultivations using the concentrated whey feed instead of a defined lactose feed revealed 39% higher growth rates, 24% higher biomass yields and even higher specific product titers for the model enzymes, flavanone 3-hydroxylase and chalcone 3- hydroxylase. Our strategy simultaneously provides a cheap substrate for large-scale production of technical enzymes and an excellent opportunity for cheese whey valorization, reducing the biological burden resulting from whey wastewaters.Fonds zur Förderung der Wissenschaftlichen Forschung

Posttranslational Modification of Heme b in a Bacterial Peroxidase: The Role of Heme to Protein Ester Bonds in Ligand Binding and Catalysis

The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases including myeloperoxidase (MPO) and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. The present study focuses on the model wild-type peroxidase from the cyanobacterium Lyngbya sp. PCC 8106 (LspPOX) and the mutants D109A, E238A and D109A/E238A which could be recombinantly produced as apo-proteins in E. coli, fully reconstituted to the respective heme b proteins and posttranslationally modified by hydrogen peroxide. This for the first time allows not only to directly compare the catalytic properties of the heme b and PTM forms, but also to study the impact of D109 and E238 on PTM and catalysis including Compound I formation and the two-electron reduction of Compound I by bromide, iodide and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238 - in contrast to exchange of D109 - does not cause significant structural rearrangements and changes of the catalytic properties neither in the heme b nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases

Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica

Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3′-hydroxylase (F3′H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3′H cDNA clones from apple leaves and tested recombinant Malus F3′Hs for their substrate specificity. From the two isolated cDNA clones, only F3′HII encoded a functionally active enzyme. In the F3′HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3′HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely

Posttranslational Modification of Heme <i>b</i> in a Bacterial Peroxidase: The Role of Heme to Protein Ester Bonds in Ligand Binding and Catalysis

The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. This study focuses on the model wild-type peroxidase from the cyanobacterium <i>Lyngbya</i> sp. PCC 8106 (LspPOX) and the mutants D109A, E238A, and D109A/E238A that could be recombinantly produced as apoproteins in <i>Escherichia coli</i>, fully reconstituted to the respective heme <i>b</i> proteins, and posttranslationally modified by hydrogen peroxide. This for the first time allows not only a direct comparison of the catalytic properties of the heme <i>b</i> and PTM forms but also a study of the impact of D109 and E238 on PTM and catalysis, including Compound I formation and the two-electron reduction of Compound I by bromide, iodide, and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238, in contrast to exchange of D109, does not cause significant structural rearrangements or changes in the catalytic properties neither in heme <i>b</i> nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases

Results from the German Chronic Kidney Disease (GCKD) study support association of relative telomere length with mortality in a large cohort of patients with moderate chronic kidney disease

Telomere length is known to be inversely associated with aging and has been proposed as a marker for aging-related diseases. Telomere attrition can be accelerated by oxidative stress and inflammation, both commonly present in patients with chronic kidney disease. Here, we investigated whether relative telomere length is associated with mortality in a large cohort of patients with chronic kidney disease stage G3 and A1-3 or G1-2 with overt proteinuria (A3) at enrollment. Relative telomere length was quantified in peripheral blood by a quantitative PCR method in 4,955 patients from the GCKD study, an ongoing prospective observational cohort. Complete four-year follow-up was available from 4,926 patients in whom we recorded 354 deaths. Relative telomere length was a strong and independent predictor of all-cause mortality. Each decrease of 0.1 relative telomere length unit was highly associated with a 14% increased risk of death (hazard ratio1.14 [95% confidence interval 1.06-1.22]) in a model adjusted for age, sex, baseline eGFR, urine albumin/creatinine ratio, diabetes mellitus, prevalent cardiovascular disease, LDL-cholesterol, HDL-cholesterol, smoking, body mass index, systolic and diastolic blood pressure, C-reactive protein and serum albumin. This translated to a 75% higher risk for those in the lowest compared to the highest quartile of relative telomere length. The association was mainly driven by 117 cardiovascular deaths (1.20 [1.05-1.35]) as well as 67 deaths due to infections (1.27 [1.07-1.50]). Thus, our findings support an association of shorter telomere length with all-cause mortality, cardiovascular mortality and death due to infections in patients with moderate chronic kidney disease

Heart-Type Fatty Acid Binding Protein, Cardiovascular Outcomes, and Death: Findings From the German CKD Cohort Study

Rationale & objective: Heart-type fatty acid binding protein (H-FABP) is a biomarker that has been shown to provide long-term prognostic information in patients with coronary artery disease independently of high-sensitivity troponin T (hs-TNT). We examined the independent associations of H-FABP with cardiovascular outcomes in patients with chronic kidney disease (CKD). Study design: Prospective cohort study. Setting & participants: 4,951 patients enrolled in the German Chronic Kidney Disease (GCKD) study with an estimated glomerular filtration rate of 30-60 mL/min/1.73 m2 or overt proteinuria (urinary albumin-creatinine ratio > 300 mg/g or equivalent). Exposure: Serum levels of H-FABP and hs-TNT were measured at study entry. Outcome: Noncardiovascular (non-CV) death, CV death, combined major adverse CV events (MACE), and hospitalization for congestive heart failure (CHF). Analytical approach: Hazard ratios (HRs) for associations of H-FABP and hs-TNT with outcomes were estimated using Cox regression analyses adjusted for established risk factors. Results: During a maximum follow-up of 6.5 years, 579 non-CV deaths, 190 CV deaths, 522 MACE, and 381 CHF hospitalizations were observed. In Cox regression analyses adjusted for established risk factors, H-FABP was associated with all 4 outcomes, albeit with lower HRs than those found for hs-TNT. After further adjustment for hs-TNT levels, H-FABP was found to be associated with non-CV death (HR, 1.57 [95% CI, 1.14-2.18]) and MACE (HR, 1.40 [95% CI, 1.02-1.92]) but with neither CV death (HR, 1.64 [95% CI, 0.90-2.99]) nor CHF hospitalizations (HR, 1.02 [95% CI, 0.70-1.49]). Limitations: Single-point measurements of H-FABP and hs-TNT. Uncertain generalizability to non-European populations. Conclusions: In this large cohort of patients with CKD, H-FABP was associated with non-CV death and MACE, even after adjustment for hs-TNT. Whether measurement of H-FABP improves cardiovascular disease risk prediction in these patients warrants further studies

Association of osteopontin with kidney function and kidney failure in chronic kidney disease patients: the GCKD study

Background: Osteopontin (OPN), synthesized in the thick ascending limb of Henle's loop and in the distal tubule, is involved in the pathogenesis of kidney fibrosis, a hallmark of kidney failure (KF). In a cohort of chronic kidney disease (CKD) patients, we evaluated OPN's association with kidney markers and KF. Methods: OPN was measured from baseline serum samples of German Chronic Kidney Disease study participants. Cross-sectional regression models for estimated glomerular filtration rate (eGFR) and urinary albumin-to-creatinine ratio (UACR) as well as Cox regression models for all-cause mortality and KF were evaluated to estimate the OPN effect. Additionally, predictive ability, of OPN and time-dependent population-attributable fraction were evaluated. Results: Over a median follow-up of 6.5 years, 471 KF events and 629 deaths occurred among 4,950 CKD patients. One-unit higher log(OPN) was associated with 5.5 mL/min/1.73m2 lower eGFR (95%CI: [-6.4,-4.6]) and 1% change in OPN with 0.7% higher UACR (estimated effect 0.7, 95%CI: [0.6,0.8]). Moreover, higher OPN levels were associated with a higher risk of KF (hazard ratio [HR] 1.4, 95%CI: [1.2,1.7]) and all-cause mortality (HR 1.5, 95%CI: [1.3,1.8]). After 6 years, 31% of the KF events could be attributed to higher OPN levels (95%CI: [3%,56%]). Conclusions: In this study, higher OPN levels were associated with kidney function markers worsening, and a higher risk for adverse outcomes. A larger proportion of KF could be attributed to higher OPN levels warranting further research on OPN with regards to its role in CKD progression and possible treatment options