Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5–2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62–0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16–1·59), representing a 50% (42–58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all >0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council

Investigating mass transfer limitations during biodegradation of micropollutants with compound-specific isotope analysis.  

Worldwide, thousands of xenobiotics are discharged into the environment either by accident, e.g. in spills, or on purpose, e.g. when pesticides like atrazine or glyphosate are applied on agricultural fields. Even though most of these chemicals are initially degraded by bacteria, this degradation seems to stall at low concentrations in ground water and surface water. As a consequence, humans are exposed to a large number of these persistent chemical pollutants in drinking water. Two competing paradigms claim that biodegradation is either mass transfer limited or cell physiology limited. While multiple methods, e.g. proteomics, are available to study physiological adaptation, pinpointing mass transfer limitations is challenging, as simple concentration measurements are not sufficient. Therefore, we employed isotope fractionation during biodegradation, a promising concentration independent tool to identify and distinguish different rate determining steps of reactions, to unravel underlying mass transfer limitations during pollutant biodegradation. To mimic oligotrophic conditions where biodegradation seems to stall, we cultivated the atrazine degrader Arthrobacter aurescens TC1 in chemostat with atrazine as the sole carbon and nitrogen source. The dilution rate was varied and we observed a decreasing isotope fractionation factor from ε13C = -5.4 ‰ at 85 µg∙L-1 atrazine down to ε13C = -2.3 ‰ at 33 µg∙L-1 with decreasing residual atrazine concentrations. Thus, we were able to pinpoint a rapid onset of rate limiting mass transfer across the cell envelope when bacteria adapt to oligotrophic conditions and transition to stationary phase at slow growth rates. To further elucidate the role of the cell envelope as barrier to biodegradation, we (i) compared atrazine uptake in Gram-positive Arthrobacter aurescens TC1 and Gram-negative Polaromonas sp. NeaC and (ii) studied glyphosate permeation in liposome models systems and during biodegradation. The intrinsic enzymatic fractionation factor of atrazine hydrolysis by TrzN ε13C = -5.3 ‰ was masked in whole cells of Polaromonas sp. NeaC ε13C = -3.5 ‰, but not in Gram-positive Arthrobacter aurescens TC1. As the atrazine degradation rates were not reduced after inhibition of active transporter, we identified the outer membrane in Gram-negative Polaromonas sp. NeaC as the barrier to atrazine influx. High glyphosate permeation rates in the liposome model system indicate that passive membrane permeation is also an underestimated uptake pathway for charged pollutants like glyphosate. Additionally, that this glyphosate uptake is not rate determining for glyphosate biodegradation was confirmed by strong isotope fractionation during glyphosate biodegradation by a newly isolated degrader strain Ochrobactrum sp. FrEM. To sum up, this thesis not only unravels the role of passive membrane permeation for pollutant degradation but also addresses the environmental implications of rate limiting mass transfer at low concentrations

Isotope fractionation pinpoints membrane permeability as a barrier to atrazine biodegradation in gram-negative <em>Polaromonas sp. Nea-C</em>.

Biodegradation of persistent pesticides like atrazine often stalls at low concentrations in the environment. While mass transfer does not limit atrazine degradation by the Gram-positive Arthrobacter aurescens TC1 at high concentrations (&gt;1 mg/L), evidence of bioavailability limitations is emerging at trace concentrations (&lt;0.1 mg/L). To assess the bioavailability constraints on biodegradation, the roles of cell wall physiology and transporters remain imperfectly understood. Here, compound-specific isotope analysis (CSIA) demonstrates that cell wall physiology (i.e., the difference between Gram-negative and Gram-positive bacteria) imposes mass transfer limitations in atrazine biodegradation even at high concentrations. Atrazine biodegradation by Gram-negative Polaromonas sp. Nea-C caused significantly less isotope fractionation (ϵ(C) = -3.5 &permil;) than expected for hydrolysis by the enzyme TrzN (ϵ(C) = -5.0 &permil;) and observed in Gram-positive Arthrobacter aurescens TC1 (ϵ(C) = -5.4 &permil;). Isotope fractionation was recovered in cell-free extracts (ϵ(C) = -5.3 &permil;) where no cell envelope restricted pollutant uptake. When active transport was inhibited with cyanide, atrazine degradation rates remained constant demonstrating that atrazine mass transfer across the cell envelope does not depend on active transport but is a consequence of passive cell wall permeation. Taken together, our results identify the cell envelope of the Gram-negative bacterium Polaromonas sp. Nea-C as a relevant barrier for atrazine biodegradation

Modeling of contaminant biodegradation and compound-specific isotope fractionation in chemostats at low dilution rates.

We present a framework to model microbial transformations in chemostats and retentostats under transient or quasi-steady state conditions. The model accounts for transformation-induced isotope fractionation and mass transfer across the cell membrane. It also verifies that the isotope fractionation epsilon can be evaluated as the difference of substrate-specific isotope ratios between inflow and outflow. We explicitly considered that the dropwise feeding of substrate into the reactor at very low dilution rates leads to transient behavior of concentrations and transformation rates and use this information to validate conditions under which a quasi-steady state treatment is justified. We demonstrate the practicality of the code by modeling a chemostat experiment of atrazine degradation at low dilution/growth rates by the strain Arthrobacter aurescens TCl. Our results shed light on the interplay of processes that control biodegradation and isotope fractionation of contaminants at low (mu g/L) concentration levels. With the help of the model, an estimate of the mass-transfer coefficient of atrazine through the cell membrane was achieved (0.0025s(-1))

Rate-limiting mass transfer in micropollutant degradation revealed by isotope fractionation in chemostat.

Biodegradation of persistent micropollutants like pesticides often slows down at low concentrations (mu g/L) in the environment. Mass transfer limitations or physiological adaptation are debated to be responsible. Although promising, evidence from compound-specific isotope fractionation analysis (CSIA) remains unexplored for bacteria adapted to this low concentration regime. We accomplished CSIA for degradation of a persistent pesticide, atrazine, during cultivation of Arthrobacter aurescens TCl in chemostat under four different dilution rates leading to 82, 62, 45, and 32 mu g/L residual atrazine concentrations. Isotope analysis of atrazine in chemostat experiments with whole cells revealed a drastic decrease in isotope fractionation with declining residual substrate concentration from epsilon(C) = -5.36 +/- 0.20 parts per thousand at 82 mu g/L to epsilon(C) = -2.32 +/- 0.28 parts per thousand at 32 mu g/L. At 82 mu g/L epsilon(C) represented the full isotope effect of the enzyme reaction. At lower residual concentrations smaller epsilon(C) indicated that this isotope effect was masked indicating that mass transfer across the cell membrane became rate-limiting. This onset of mass transfer limitation appeared in a narrow concentration range corresponding to about 0.7 mu M assimilable carbon. Concomitant changes in cell morphology highlight the opportunity to study the role of this onset of mass transfer limitation on the physiological level in cells adapted to low concentrations

High permeation rates in liposome systems explain rapid glyphosate biodegradation associated with strong isotope fractionation.

Bacterial uptake of charged organic pollutants such as the widely used herbicide glyphosate is typically attributed to active transporters, whereas passive membrane permeation as an uptake pathway is usually neglected. For 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) liposomes, the pH-dependent apparent membrane permeation coefficients (P-app) of glyphosate, determined by nuclear magnetic resonance (NMR) spectroscopy, varied from P-app (pH 7.0) = 3.7 (+/- 0.3) x 10(-7) m.s(-1) to P-app (pH 4.1) = 4.2 (+/- 0.1) x 10(-6) m.s(-1). The magnitude of this surprisingly rapid membrane permeation depended on glyphosate speciation and was, at circumneutral pH, in the range of polar, noncharged molecules. These findings point to passive membrane permeation as a potential uptake pathway during glyphosate biodegradation. To test this hypothesis, a Gram-negative glyphosate degrader, Ochrobactrum sp. FrEM, was isolated from glyphosate-treated soil and glyphosate permeation rates inferred from the liposome model system were compared to bacterial degradation rates. Estimated maximum permeation rates were, indeed, 2 orders of magnitude higher than degradation rates of glyphosate. In addition, biodegradation of millimolar glyphosate concentrations gave rise to pronounced carbon isotope fractionation with an apparent kinetic isotope effect, AKIE(carbon), of 1.014 +/- 0.003. This value lies in the range typical of non-masked enzymatic isotope fractionation demonstrating that glyphosate biodegradation was not subject to mass transfer limitations and glyphosate exchange across the cell membrane was rapid relative to enzymatic turnover

Defining lower limits of biodegradation: atrazine degradation regulated by mass transfer and maintenance demand in Arthrobacter aurescens TC1.

( )Exploring adaptive strategies by which microorganisms function and survive in low-energy natural environments remains a grand goal of microbiology, and may help address a prime challenge of the 21st century: degradation of man-made chemicals at low concentrations ("micropollutants"). Here we explore physiological adaptation and maintenance energy requirements of a herbicide (atrazine)-degrading microorganism (Arthrobacter aurescens TC1) while concomitantly observing mass transfer limitations directly by compound-specific isotope fractionation analysis. Chemostat-based growth triggered the onset of mass transfer limitation at residual concentrations of 30 mu g L-1 of atrazine with a bacterial population doubling time (t(d)) of 14 days, whereas exacerbated energy limitation was induced by retentostat-based near-zero growth (t(d) = 265 days) at 12 +/- 3 mu g L-1 residual concentration. Retentostat cultivation resulted in (i) complete mass transfer limitation evidenced by the disappearance of isotope fractionation (epsilon C-13 = -0.45%o +/- 0.36 parts per thousand) and (ii) a twofold decrease in maintenance energy requirement compared with chemostat cultivation. Proteomics revealed that retentostat and chemostat cultivation under mass transfer limitation share low protein turnover and expression of stress-related proteins. Mass transfer limitation effectuated slow-down of metabolism in retentostats and a transition from growth phase to maintenance phase indicating a limit of similar or equal to 10 mu g L-1 for long-term atrazine degradation. Further studies on other ecosystem-relevant microorganisms will substantiate the general applicability of our finding that mass transfer limitation serves as a trigger for physiological adaptation, which subsequently defines a lower limit of biodegradation

Defining lower limits of biodegradation: Atrazine degradation regulated by mass transfer and maintenance demand in Arthrobacter aurescens TC1 (vol 13, pg 2236, 2019).

Since publication of the original article the authors noticed that co-author Christian Griebler’s second affiliation was not included. This has now been added to the HTML and PDF versions of the paper. Furthermore, Supplementary Table 3 was not uploaded with the rest of the Supplementary Information files. This is now available to view on the HTML of the original article. The authors would like to apologies for any inconvenience caused