Serelaxin as a potential treatment for renal dysfunction in cirrhosis: Preclinical evaluation and results of a randomized phase 2 trial

<div><p>Background</p><p>Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension.</p><p>Methods and findings</p><p>To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 μg/kg/d and then 60 min at 30 μg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow.</p><p>Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; <i>p <</i> 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study’s main limitations were the relatively small sample size and stable, well-compensated population.</p><p>Conclusions</p><p>Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01640964" target="_blank">NCT01640964</a></p></div

Modulating <i>E. coli</i> membrane content via co-expression of saturated and unsaturated acyl-ACP targeting thioesterases.

<p>a.) The percentage of unsaturated C₁₆–C₁₈ and cyclopropane (C_17Δ) fatty acids were calculated from FAMEs made by base-catalyzed methylation of fatty acids extracted from cultures expressing combinations of BTE and GeoTE at 8 h and 24 h post-inoculation. GeoTE-expressing cells (GeoTE+ BTE−) and GeoTE/BTE co-expressing cells (GeoTE+ BTE+) have a reduced unsaturated content relative to BTE-expressing cells (BTE+ GeoTE−). Error bars represent standard deviations about the mean of biological triplicate samples. * = P-value<0.05, ** = P-value<0.01 compared to BTE⁺ GeoTE⁻ cultures at the same sampling time. b.) Transcript levels of <i>fabA</i> and <i>fabB</i> determined by qPCR on samples harvested 1 hour hour post-induction were normalized to BTE− GeoTE− (RL08ara pBAD33-BTE-H204A pBAD18-GeoTE-H173A) samples. Levels of <i>fabA</i> and <i>fabB</i> were decreased in cells expressing only BTE. Conversely, levels of <i>fabA</i> or <i>fabB</i> were statistically the same or higher in cells expressing GeoTE or GeoTE and BTE. Error bars represent propagated standard errors about the mean of biological triplicate samples. * = P-value<0.05 for C_q values compared against <i>fabA</i> or <i>fabB</i> in cultures expressing only non-functional thioesterases (BTE⁻ GeoTE⁻). c.) The percentage of intact cells were calculated from histograms of cells stained with SYTOX Green 8 h post-inoculation. Cultures expressing only BTE were the least intact. Cultures expressing only GeoTE, and both GeoTE and BTE were over 50% intact. Error bars represent standard errors about the mean of biological triplicate samples. ** = P-value<0.01 compared to BTE⁺ GeoTE⁻. d.) FFA titers from strains expressing combinations of BTE and GeoTE. FFA titers were determined at 8 and 24 h post-inoculation. Cultures expressing only GeoTE exhibited the highest titer at 8 h, with a minor increase observed after 24 h. Nearly equivalent titers were reached after 24 h in cultures expressing only BTE, or co-expressing BTE and GeoTE. Error bars represent standard deviations about the mean of biological triplicate samples. * = P-value<0.05, ** = P-value<0.01 compared to BTE⁺ GeoTE⁻ cultures at same sampling time.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Connection between FabR and unsaturated membrane content in FFA producing <i>E. coli</i>.

<p>a.) Transcript levels of <i>fabA</i> and <i>fabB</i> determined by qPCR on samples harvested 4.6 hours after inoculation were normalized to RL08ara harboring pTrc99A-BTE-H204A and pBAD33*. In strains carrying <i>fabR</i> (RL08ara, pBAD33*, labeled RL08ara, and RL08ara Δ<i>fabR</i>, pBAD33*fabR, labeled <i>fabR+</i>), expression of BTE (+) reduced both <i>fabA</i> and <i>fabB</i> levels. In Δ<i>fabR</i> strains (RL08ara Δ<i>fabR</i>, pBAD33*) levels of <i>fabB</i> were increased in cells expressing both BTE-H204A (−) and BTE (+). Error bars represent propagated standard errors about the mean of biological triplicate samples. P-values were calculated by a t-test of the C_q values of <i>fabA</i> or <i>fabB</i> in strain RL08ara BTE-H204A Samples with P-values less <0.05 were marked with an asterisk. b.) The percentage of unsaturated C₁₆–C₁₈ and cyclopropane (C_17Δ) fatty acids were calculated from fatty acid samples extracted from cultures 8 hours post-inoculation. Expression of BTE dramatically increased unsaturated content in strains harboring chromosomal <i>fabR</i> (RL08ara), and further increased unsaturated content in the Δ<i>fabR</i> strain. Overexpression of <i>fabR</i> on a plasmid in the Δ<i>fabR</i> strain restored unsaturated content to a lower level than present in RL08ara. Error bars represent standard errors about the mean of biological triplicate samples. * = P-value<0.05, ** = P-value<0.01 compared against RL08ara BTE⁻ for BTE⁻ cultures or RL08ara BTE⁺ for BTE⁺ cultures at the same sampling time. c.) The percentage of intact cells were calculated from histograms of cells stained with SYTOX Green. In BTE-H204A-expressing cultures, deletion of <i>fabR</i> had little effect on percent intact cells. In BTE-expressing cultures, a dramatic decrease in the number of intact cells was observed as a result of deletion of <i>fabR</i>. Samples from <i>fabR</i>+ cells exhibited altered histograms and were not quantified (#). Error bars represent standard errors about the mean of biological triplicate samples. ** = P-value<0.01 compared against RL08ara BTE⁻ for BTE⁻ cultures or RL08ara BTE⁺ for BTE⁺ cultures. d.) Effect of <i>fabR</i> deletion on C₈–C₁₄ (predominantly free) fatty acid titer produced in BTE-expressing cultures. Reduced titers were observed at both 8 h and 24 h growth in the Δ<i>fabR</i> strain. Error bars represent standard deviations about the mean of biological triplicate samples.</p

Unsaturated fatty acid biosynthesis and regulation.

<p>Unsaturated fatty acid biosynthesis begins with the isomerization of <i>trans</i>-2-decenoyl-ACP to <i>cis</i>-3-decenoyl-ACP by FabA. Instead of being reduced, this intermediate is condensed with malonyl-ACP by FabB. The resulting unsaturated β-ketoacyl-ACP is processed analogous to its saturated counterpart until unsaturated C₁₆ and C₁₈ acyl-ACPs are made and incorporated into phospholipids. Unsaturated fatty acid biosynthesis is feedback-inhibited at the transcriptional level by FabR, which exhibits increased repression of transcription of <i>fabA</i> and <i>fabB</i> when bound to enoyl-ACP species <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054031#pone.0054031-Zhu1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054031#pone.0054031-Feng2" target="_blank">[17]</a>. Expression of a thioesterase cleaves acyl-ACPs to generate FFA. BTE expression preferentially cleaves saturated C₁₂-acyl-ACPs (solid blue arrow) and minorly cleaves unsaturated C₁₂- (dashed blue arrow) and saturated and unsaturated C₁₄-acyl-ACPs, thereby depleting saturated, long chain acyl-ACPs, the key regulatory signal for controlling fatty acid biosynthesis. As a result, flux through the saturated (prior to C₁₀) and unsaturated pathway increases. Inset: the four arrows represent the elongation (FabB/FabF) (dashed arrow) in which the acyl chain represented by R grows by 2 carbons, ketoreduction (FabG), dehydration (FabZ), and enoyl reduction (FabI) reactions that comprise one round of fatty acid elongation and reduction.</p

Viability analysis of strains expressing combinations of BTE and GeoTE.

<p>Viability analysis of strains expressing combinations of BTE and GeoTE.</p

Generation of a platform strain for ionic liquid tolerance using adaptive laboratory evolution.

BackgroundThere is a need to replace petroleum-derived with sustainable feedstocks for chemical production. Certain biomass feedstocks can meet this need as abundant, diverse, and renewable resources. Specific ionic liquids (ILs) can play a role in this process as promising candidates for chemical pretreatment and deconstruction of plant-based biomass feedstocks as they efficiently release carbohydrates which can be fermented. However, the most efficient pretreatment ILs are highly toxic to biological systems, such as microbial fermentations, and hinder subsequent bioprocessing of fermentative sugars obtained from IL-treated biomass.MethodsTo generate strains capable of tolerating residual ILs present in treated feedstocks, a tolerance adaptive laboratory evolution (TALE) approach was developed and utilized to improve growth of two different Escherichia coli strains, DH1 and K-12 MG1655, in the presence of two different ionic liquids, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3-methylimidazolium chloride ([C4C1Im]Cl). For multiple parallel replicate populations of E. coli, cells were repeatedly passed to select for improved fitness over the course of approximately 40&nbsp;days. Clonal isolates were screened and the best performing isolates were subjected to whole genome sequencing.ResultsThe most prevalent mutations in tolerant clones occurred in transport processes related to the functions of mdtJI, a multidrug efflux pump, and yhdP, an uncharacterized transporter. Additional mutations were enriched in processes such as transcriptional regulation and nucleotide biosynthesis. Finally, the best-performing strains were compared to previously characterized tolerant strains and showed superior performance in tolerance of different IL and media combinations (i.e., cross tolerance) with robust growth at 8.5% (w/v) and detectable growth up to 11.9% (w/v) [C2C1Im][OAc].ConclusionThe generated strains thus represent the best performing platform strains available for bioproduction utilizing IL-treated renewable substrates, and the TALE method was highly successful in overcoming the general issue of substrate toxicity and has great promise for use in tolerance engineering