Proteins and signalling pathways targeted by dapagliflozin and finerenone: Insights from <scp>DAPA</scp> ‐ <scp>CKD</scp> and <scp>FIGARO</scp> ‐ <scp>DKD</scp>

Sodium glucose co-transporter 2 (SGLT2) inhibitors and the non-steroidal mineralocorticoid receptor antagonist (nsMRA) finerenone reduce the risk of cardiorenal events in various clinical settings.1-3 Post hoc analyses demonstrated that the cardio-kidney protective effects of SGLT2 inhibitors were consistent in patients who were or were not treated with conventional MRAs.4 Similarly, post hoc analyses from clinical trials with finerenone reported that the cardio-kidney treatment benefits of finerenone were observed irrespective of concomitant use of SGLT2 inhibitors.5 These data suggest that the clinical benefits of both drug classes are achieved through independent and likely complementary mechanisms. The CONFIDENCE trial was designed to assess whether combined initiation of the SGLT2 inhibitor empagliflozin with finerenone would further reduce albuminuria in patients with chronic kidney disease (CKD) and type 2 diabetes compared to either treatment alone. The trial demonstrated that the combined initiation of empagliflozin and finerenone led to a statistically significant greater reduction in albuminuria compared to either treatment alone and was well tolerated.6 These data provide further support for the complementary mechanisms of both drug classes to preserve long-term kidney function. Large-scale proteomics followed by pathway analysis may help understand the molecular mechanisms and signalling pathways by which SGLT2 inhibitors and finerenone exert their beneficial effects. To test the hypothesis that the pharmacological mechanisms of dapagliflozin and finerenone involve different yet complementary molecular effects and pathways in patients with type 2 diabetes and CKD, we performed a proteomic analysis of the individual patient data from clinical outcome trials with the SGLT2 inhibitor dapagliflozin and the nsMRA finerenone. We did a post hoc analysis on available proteomic data from 1616 and 929 patients with type 2 diabetes and CKD and available proteomic data from the Dapagliflozin and Prevention of Adverse outcomes in Chronic Kidney Disease (DAPA-CKD) and the Finerenone in Reducing Cardiovascular Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trials. Both were multicentre, double-blind, placebo-controlled, randomised trials. DAPA-CKD enrolled 4304 patients with CKD, estimated glomerular filtration rate (eGFR) of 25–75 mL/min/1.73 m2 and a urinary albumin-to-creatinine ratio (UACR) of 200–5000 mg/g, with or without type 2 diabetes to evaluate the efficacy and safety of dapagliflozin. The FIGARO-DKD trial included 7352 patients with type 2 diabetes with a UACR of 30–300 mg/g and an eGFR of 25–90 mL/min/1.73 m2 or a UACR of 300–5000 mg/g and an eGFR of at least 60 mL/min/1.73 m2, to evaluate the efficacy and safety of finerenone. Plasma proteomics was performed as a substudy in both trials using the Olink Explore 3072 panel. Samples were analysed in the central Olink laboratory according to the manufacturer's standard operating procedure. In the DAPA-CKD trial, plasma proteomic data was available at baseline and at 1-year post-randomisation; for the FIGARO-DKD substudy (referred to as FIGARO-BM, ClinicalTrials.gov number: NCT05013008), samples from the first post-randomisation visit (Month 4) until Month 48 post-randomisation were analysed. Samples from the baseline visit were not available. Analysis of covariance (ANCOVA) was performed to assess the effect of dapagliflozin versus placebo on changes in plasma protein levels from baseline to year 1 in DAPA-CKD; proteins were deemed to be significant if the adjusted p-value was below 0.05. In FIGARO-BM, linear mixed models for repeated measurements were used, including treatment, visit, treatment × visit interaction, and sex as fixed effects to determine the effect of finerenone relative to placebo on plasma proteins.7 At Year 1, proteins were deemed to be significant if the adjusted p-value was below 0.01. Proteins affected by finerenone or dapagliflozin were subsequently subjected to Ingenuity Pathway Analysis with the Ingenuity Knowledge Base as reference set. The characteristics of the two study populations are shown in Table 1. The median age was 64 and 65 years; 75% and 66% were male in FIGARO-BM and DAPA-CKD, respectively. At baseline, the median eGFR was higher (65.3 vs. 43.0 mL/min/1.73 m2) and median UACR (203 vs. 990 mg/g) was lower in FIGARO-BM compared to DAPA-CKD. Renin Angiotensin System inhibitors were used by >98% of participants in both trials. Analysis of the individual proteins demonstrated very little overlap in proteins affected by finerenone and dapagliflozin treatment (Figure S1). Accordingly, pathway enrichment analysis showed that both finerenone and dapagliflozin target different molecular pathways (Figure 1). Enriched pathways associated with finerenone treatment included coagulation, inflammatory, and immune signalling-related pathways. In contrast, enriched pathways linked to dapagliflozin treatment response included pathways involved in lipid metabolism, gas exchange of erythrocytes, as well as leukocyte migration (Figure 1). Notable pathways affected by finerenone and dapagliflozin included the insulin-like growth factor pathway, the endothelin-1 pathway, atherosclerosis signalling, and pathways related to extracellular matrix remodelling, such as hepatic fibrosis and signalling by bone morphogenetic proteins. This proteomic analysis of two phase 3 clinical trials indicates that the response to nsMRA finerenone and the SGLT2 inhibitor dapagliflozin is partially mediated by divergent pathways. These findings provide further mechanistic support that the molecular effects of both drug classes are independent and possibly complimentary. Finerenone treatment affected a variety of molecular signalling pathways linked to inflammation, oxidative stress, and lipid metabolism, such as TNFR1 signalling, endothelin-1 signalling, and retinoid X receptor signalling pathways. Preclinical studies previously reported that finerenone reduces lipid uptake in cardiomyocytes and cellular inflammation through downregulation of peroxisome proliferator-activated receptor gamma (PPARƔ), a key receptor involved in fatty acid metabolism.8 In addition, finerenone reduced oxidative stress in various experimental models through downregulation of reactive oxygen species and improving endothelial function.9, 10 Observed signalling pathways previously associated with dapagliflozin treatment included mitogen-activated protein kinase pathways and pathways linked to oxygen transport by erythrocytes.11, 12 Both finerenone and dapagliflozin affected the endothelin-1 signalling pathway and pathways related to extracellular matrix remodelling. Previous in vitro studies have shown that SGLT2 inhibition with empagliflozin reduces endothelin-1 expression in human proximal tubular cells, whereas aldosterone has been shown to promote endothelin-1 production, providing mechanistic support for the link between both finerenone and dapagliflozin treatment and endothelin-1 signalling.13-15 Furthermore, our analyses revealed that both finerenone and dapagliflozin modulate pathways associated with extracellular matrix remodelling, including those involved in hepatic fibrosis and BMP signalling. These observations are consistent with previous studies highlighting the anti-fibrotic potential of both agents through regulation of fibrogenic signalling cascades.16, 17 Results of this study are hypothesis generating and are limited by the indirect cross-trial comparison of finerenone and dapagliflozin. Proteomic analyses from the CONFIDENCE trial with direct comparison of combination treatment of finerenone and SGLT2 inhibitors versus monotherapy with these agents are needed to confirm our findings. Moreover, due to the lack of baseline samples in the finerenone biomarker study, we were unable to adjust for interindividual differences in protein levels at baseline. Finally, the selection of the Olink circulating plasma proteins was not random, but based on existing knowledge about the relevance of selected proteins in multiple diseases. We cannot exclude the possibility that other relevant proteins and signalling pathways were omitted in our analyses. In conclusion, the molecular pathways targeted by finerenone and dapagliflozin seem to differ in patients with type 2 diabetes and CKD, supporting their independent and possibly additive effects to confer long-term kidney protection. The authors thank all patients and investigators of the DAPA-CKD and FIGARO-DKD trials for their support. This study was sponsored by AstraZeneca and Bayer, and conducted within the framework of the PRIME-CKD project, funded by the European Union's Horizon Research and Innovation Actions (HORIZON-HLTH-2022-TOOL-11-01: Tools and technologies for a healthy society) under grant agreement no. 101095146. Mark André de la Rambelje reports no conflicts. Adriaan Voors received consultancy fees and/or research support paid to his employer from Adrenomed, Anacardio, AstraZeneca, Bayer AG, BMS, Boehringer Ingelheim, Cardurion, Corteria, Eli Lilly, Merck, Moderna, Novartis, Novo Nordisk, Rycarma, Salubris Bio. Peter J. Greasley is an employee and shareholder in AstraZeneca. Mario Berger is a Bayer employee and holds Bayer AG shares. Hiddo J. L. Heerspink reports funding to conduct clinical trials from AstraZeneca, Bayer, Boehringer Ingelheim, Janssen, and Novo Nordisk (all to the University of Groningen); consulting fees from AstraZeneca, Alexion, Alnylum, Bayer, Boehringer Ingelheim, Biocity Pharmaceutics, Dimerix, Eli Lilly, Janssen, Novartis, Novo Nordisk, Roche, and Travere Therapeutics; lecture fees from AstraZeneca, Bayer, and Novo Nordisk. The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/dom.70193. Availability of the data from the FIGARO-DKD trial underlying this publication will be determined according to Bayer's commitment to the EFPIA/PhRMA ‘Principles for responsible clinical trial data sharing’. This pertains to scope, timepoint and process of data access. As such, Bayer commits to sharing upon request from qualified scientific and medical researchers patient-level clinical trial data, study-level clinical trial data, and protocols from clinical trials in patients for medicines and indications approved in the United States (US) and European Union (EU) as necessary for conducting legitimate research. This applies to data on new medicines and indications that have been approved by the EU and US regulatory agencies on or after 01 January 2014. Interested researchers can use https://vivli.org to request access to anonymized patient-level data and supporting documents from clinical studies to conduct further research that can help advance medical science or improve patient care. Information on the Bayer criteria for listing studies and other relevant information is provided in the member section of the portal. Data access will be granted to anonymized patient-level data, protocols and clinical study reports after approval by an independent scientific review panel. Bayer is not involved in the decisions made by the independent review panel. Bayer will take all necessary measures to ensure that patient privacy is safeguarded. Data from the DAPA-CKD trial underlying the findings described in this manuscript may be obtained in accordance with AstraZeneca's data sharing policy described at https://astrazenecagrouptrials.pharmacm.com/ST/Submission/Disclosure. Figure S1. Individual proteins changed by finerenone and dapagliflozin relative to placebo. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.