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Division of Pediatric Nephrology and Gastroenterology, Department of Pediatrics and Adolescent Medicine, Comprehensive Center for Pediatrics, Medical University of Vienna, Vienna, Austria
Division of Pediatric Nephrology and Gastroenterology, Department of Pediatrics and Adolescent Medicine, Comprehensive Center for Pediatrics, Medical University of Vienna, Vienna, Austria
Division of Nephrology and Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
Division of Pediatric Nephrology and Gastroenterology, Department of Pediatrics and Adolescent Medicine, Comprehensive Center for Pediatrics, Medical University of Vienna, Vienna, Austria
Delta 4 GmbH, Vienna, AustriaDivision of Pediatric Nephrology and Gastroenterology, Department of Pediatrics and Adolescent Medicine, Comprehensive Center for Pediatrics, Medical University of Vienna, Vienna, Austria
Focal segmental glomerulosclerosis (FSGS) is a glomerular lesion often associated with nephrotic syndrome. It is also associated with a high risk of progression to end-stage kidney disease. Current treatment of FSGS is limited to systemic corticosteroids or calcineurin inhibition, along with inhibitors of the renin-angiotensin-aldosterone system. FSGS is heterogeneous in etiology, and novel therapies targeting specific, dysregulated molecular pathways represent a major unmet medical need. We have generated a network-based molecular model of FSGS pathophysiology using previously established systems biology workflows to allow computational evaluation of compounds for their predicted interference with molecular processes contributing to FSGS. We identified the anti-platelet drug clopidogrel as a therapeutic option to counterbalance dysregulated FSGS pathways. This prediction of our computational screen was validated by testing clopidogrel in the adriamycin FSGS mouse model. Clopidogrel improved key FSGS outcome parameters and significantly reduced urinary albumin to creatinine ratio (P < 0.01) and weight loss (P < 0.01), and ameliorated histopathological damage (P < 0.05). Clopidogrel is used to treat several cardiovascular diseases linked to chronic kidney disease. Clopidogrel's favorable safety profile and its efficacy in the adriamycin mouse FSGS model thus recommend it as an attractive drug repositioning candidate for clinical trial in FSGS.
There is no specific therapy approved for treatment of focal segmental glomerulosclerosis (FSGS). Patients are currently treated with systemic corticosteroids, calcineurin inhibitors and inhibitors of the renin-angiotensin-aldosterone system. Novel approaches are needed to reduce the high proportion of FSGS patients progressing to end stage kidney disease despite treatment.
Translational Significance
We applied network-based bioinformatics to identify a mechanistic overlap between the pathophysiology of FSGS and the mechanism of action of clopidogrel. Clopidogrel significantly attenuated disease severity in the adriamycin mouse model of FSGS. The favorable safety profile of clopidogrel repositions it as an attractive candidate for testing in human clinical trials for FSGS.
Introduction
Focal segmental glomerulosclerosis (FSGS) is a glomerular lesion suggestive of injury to podocytes, especially if associated with nephrotic syndrome.
The kidney glomerulus filters blood through a fine sieve consisting of a fenestrated endothelium, the glomerular basement membrane and the slit diaphragm built by interdigitating podocyte foot processes.
Disruption of the slit diaphragm may lead to podocyte injury and a histopathologic lesion characterized by sclerosis in some areas (“segmental”) of some kidney glomeruli (“focal”). Without adequate treatment FSGS frequently progresses to end-stage kidney disease.
Currently, there is no specific therapy available for FSGS with the majority of patients with nephrotic range proteinuria being treated with systemic corticosteroids or calcineurin inhibitors. Both come with severe side effects and a high number of patients are unresponsive or refractory, ultimately leading to end-stage kidney disease.
Next to the huge burden for patients, renal replacement therapy generates high costs for health care systems emphasizing the importance to develop novel FSGS therapies to slow down progression of the disease.
Despite several recent FSGS trials, no single specific drug has yet emerged to significantly improve the situation for patients not responding to conventional therapy.
FSGS, however, is the consequence of a variety of etiologies including genetic mutations in key molecular pathways, viruses, drugs, hypertension, or circulating factors.
These causes lead to the disruption of a complex network of molecular processes especially relevant for podocyte differentiation, homeostasis and survival making FSGS a particular challenge for the development of novel therapeutic options.
A way to increase chances of success might be to identify compounds being capable to interfere with several dysregulated FSGS molecular mechanisms and processes at different levels. In this translational study we modeled the complex pathophysiology of FSGS on a molecular level using established computational workflows and datasets. This network-based FSGS molecular model was used to evaluate whether clopidogrel, a platelet inhibitor that has previously shown potential to ameliorate Thy-1 glomerulonephritis in a model of chronic kidney injury,
also significantly interferes with core processes of FSGS pathophysiology. The therapeutic potential of clopidogrel for FSGS was confirmed in vivo in the adriamycin nephropathy mouse model.
Material and methods
Modeling FSGS pathophysiology
We constructed a network-based molecular model of FSGS pathophysiology following our previously described workflows.
Canagliflozin reduces inflammation and fibrosis biomarkers: a potential mechanism of action for beneficial effects of SGLT2 inhibitors in diabetic kidney disease.
A systems pharmacology workflow with experimental validation to assess the potential of anakinra for treatment of focal and segmental glomerulosclerosis.
FSGS associated molecular features were extracted from scientific publications annotated with the MeSH term “Glomerulosclerosis, Focal Segmental.” Data from NCBI's gene2pubmed mapping file were used to link proteins to FSGS covering human genes as well as genes from animal models. Genes and proteins were also automatically extracted using predefined catalogs from the set of MeSH-annotated FSGS publications by named entity recognition. Automatically extracted proteins were complemented by manually curated gene sets extracted from review articles on FSGS. FSGS-associated proteins were mapped onto a protein-protein dependency network of experimentally determined protein-protein interaction data from three independent databases, complemented by computationally inferred protein-protein dependencies based on a set of pre-defined data sources.
From this network we retrieved the connected component subgraph of proteins sharing one or more edges with at least one other protein from the initial FSGS set. The final FSGS molecular model was generated after subgraph segmentation using the MCODE algorithm with the following parameters: Degree cutoff: 2; Include Loops: Off; Haircut: Off; Fluff: Off; Node Score Cutoff: 0.5; K-Core: 2; Max. Depth: 4.
Nodes initially unassigned to a specific cluster were subsequently added to nearest neighbor clusters. In case of ambiguity, nodes were left unassigned.
Functional enrichment analysis
The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used for gene set enrichment analysis.
BT Sherman, M Hao, J Qiu, et al., DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update), Nucleic Acids Res, 2022,2022 Jul 5;50(W1):W216-W221. doi: 10.1093/nar/gkac194.
to identify and rank by false-discovery rate (FDR) the most highly enriched molecular pathways based on the protein set of the FSGS pathophysiology molecular model. Disease-specific pathways unrelated to nephrology were excluded from the final ranking. Top enriched gene ontology (GO) biological process terms were also identified for individual clusters of the FSGS pathophysiology molecular model using DAVID.
Modeling clopidogrel mechanism of action (MoA) in the context of FSGS pathophysiology
The clopidogrel-associated gene set was generated from clopidogrel-associated publications as described above for the FSGS gene set. Areas of the FSGS molecular model affected by clopidogrel treatment (proteins and protein-protein interactions shared between the FSGS molecular model and the clopidogrel MoA molecular model) were identified by network interference analysis. Due to the “guilt-by-association” principle, this method can discover unknown or hidden drug-disease relationships that have not been previously reported. Proteins of the resulting interference signature were assessed for regulation during development and/or progression of FSGS and for regulation by clopidogrel based on information in those publications that formed the basis for including the proteins into the respective molecular models in the first place.
Induction of FSGS in the mouse model
Female Balb/c mice (experiment 1: n = 17; experiment 2: n = 17) aged 12–16 weeks weighing >20 g were used for the experiment. Doxorubicin (adriamycin; Sigma-Aldrich St. Louis, MO) was diluted in sterile ddH2O and injected into the tail vein (27G needles) at a dose of 10.5 mg/kg (200 µL/25 g body weight). Mice were anaesthetized by intra peritoneal injection prior to tail vein injection with a mix of esketamin (Ketanest 12.5 mg/mL; Pfizer, NY) and xylazine hydrochloride (Rompun 0.25%; Bayer AG, Leverkusen, Germany) in 0.9% NaCl (200 µL/25 g body weight). All animal procedures were approved by the animal ethics committee of the Medical University of Vienna and Federal Ministry of Education, Science and Research (BMBWF 2020-0.200.379) and conducted in accordance with institutional guidelines that comply with the Directive of the European Parliament and of the Council on the Protection of Animals Used for Scientific Purposes. To monitor disease course, weekly values of urine albumin-creatinine ratio (UACR) were determined in spot urine. Results were normalized to body weight and controlled for age.
Diet and FSGS therapy
48 hours postadriamycin injection, clopidogrel therapy was initiated and maintained for 5 weeks. Standard commercial chow (ssniff Spezialdiäten GmbH, Soest, Germany) contained crude protein (19%), crude fat (3.3%), crude fiber (4.9%), crude ash (6.4%), starch (36.5%) and sugar (4.7%). Control group animals (n = 9) were fed standard chow. Clopidogrel-treated animals (n = 8) were fed standard chow with 200 mg clopidogrel added per kg of chow (Sigma-Aldrich) produced by ssniff. All animals were allowed to eat ad libitum assuming a daily average uptake of ∼4 g of chow per mouse (=0.8 mg Clopidogrel per mouse), yielding a daily oral clopidogrel intake of ∼30 mg/kg, as has been recently used in other mouse models.
One animal in each group was found dead and thus excluded from further analysis.
Urine collection and measurements
Spot urine was collected every week and creatinine levels were measured with standard protocols in the Clinical Institute for Laboratory Medicine of the General Hospital of Vienna. Urinary albumin was measured by enzyme-linked immunosorbent assay (ELISA) using antimouse serum albumin antibody (ab34807; diluted 1:2,000, Abcam, Cambridge, UK) and horseradish peroxidase (HRP)-coupled anti mouse serum albumin antibody (ab19195; diluted 1:100,000, Abcam). Reactions were developed with 3,3´,5,5´-tetramethylbenzidine (TMB) (Promega, Madison, Wis). Standard curves were generated with mouse albumin (Sigma-Aldrich). Color reaction was stopped with 2M H2SO4 and absorbance was measured at 450 nm (Epoch, BioTek, Winooski, VT) using the Gen5 software (BioTek).
Histological analysis
Kidneys harvested at the end of the experiment were formalin-fixed, paraffin-embedded and stained with Periodic acid-Schiff (PAS) in the Department of Clinical Pathology of the Medical University of Vienna according to standard protocols. Samples were scanned using Pannoramic Flash 250 and Pannoramic Scanner 2.1.1-250 software and analyzed using CaseCenter v2.9 (all from 3Dhistech, Budapest, Hungary). The presence of segmentally and/or globally sclerotic glomeruli and tubular ectasia were quantitively evaluated by an expert renal pathologist masked to treatment.
Statistical analysis
R v4.0.2 was used for statistical analysis. Repeated measures ANOVA was performed to analyze clopidogrel's impact on urinary albumin-to-creatinine ratio (UACR) and weight loss over time using the rstatix package (v0.6.0). Graphics were generated with ggplot2 (v3.3.2) and ggpubr (v0.4.0). Chi-square tests were used to assess statistical significance in network interference analysis comparing the molecular models for FSGS pathophysiology and clopidogrel MoA.
Results
Computational network interference analysis predicts beneficial modulation of FSGS pathophysiology by clopidogrel
Our workflow resulted in a molecular model of FSGS pathophysiology that encompassed 376 proteins (Supplementary Table I). The 2 most highly enriched molecular pathways were “Nephrotic syndrome” and “Primary focal segmental glomerulosclerosis.” Additional enriched molecular pathways included those related to signaling, immune-relation, hemostasis, cell adhesion and extracellular matrix (Fig 1, A). The full list of significantly enriched molecular pathways based on the set of FSGS model proteins is provided in Supplementary Table II. The FSGS molecular model consisted of 12 clusters ranging in size from 3 to 182 proteins. The largest cluster consisted of proteins involved in intracellular signaling cascades and in regulation of gene expression (Fig 1, B). Clusters 2 and 3 were linked to hemostasis, with enrichment in cluster 2 of proteins involved in platelet activation and enrichment in cluster 3 of proteins linked to plasminogen activation and regulation of fibrinolysis. Cluster 4 consisted of proteins involved in glomerular filtration and cell-cell adhesion. These proteins also formed links to proteins of clusters 5 and 6 involved in cell adhesion, cell migration, extracellular matrix, and glomerular basement membrane. Clusters 7 and 8 included proteins associated with immune response, chemokine signaling and inflammatory processes. Cluster 9 consisted of proteins involved in regulation of apoptosis and/or cell proliferation. Cluster 10 linked to calcineurin-nuclear factor of activated T cells (NFAT) signaling and Ca2+ transport; calcineurin inhibitors are standard therapeutic options for FSGS patients. Cluster 11 included proteins of the renin–angiotensin–aldosterone system (RAAS) cascade; RAAS inhibitors are also routinely used as part of FSGS management. Cluster 12 consisted of nuclear pore complex proteins mediating nucleocytoplasmic transport. The full list of enriched GO biological process terms for each cluster is available in Supplementary Table III.
Fig 1Network-based mechanistic model of FSGS pathophysiology. Panel A: Barplot of the 20 most highly enriched molecular pathways based on the full protein set of the FSGS molecular model. (R) = Reactome pathway; (W) = Wikipathway; ECM = extracellular matrix. Panel B: Graphical representation of the FSGS molecular model. Each cluster is represented by 2 top enriched GO biological process terms.
The MoA molecular model constructed for clopidogrel consisted of 102 proteins (Supplementary Table IV). Network alignment analysis identified 39 proteins of the FSGS molecular model also found in the clopidogrel MoA model, yielding the highly significant network interference signature depicted in Fig 2 (P<0.0001 based on a chi-square test, vs a reference set of proteins randomly selected from any molecular disease or drug model in the employed bioinformatics workflow).
Fig 2Modeled impact of clopidogrel on FSGS pathophysiology. Proteins of the interference signatures of the molecular model of FSGS disease pathophysiology and of the clopidogrel MoA molecular model are depicted in the context of FSGS pathophysiological mechanisms and annotated by their gene symbols. Green nodes represent proteins predicted to be beneficially impacted by clopidogrel treatment. The red node represents a protein predicted to be detrimentally affected by clopidogrel. Cluster colors are in line with those given in Fig 1.
Proteins involved in FSGS development and progression beneficially impacted by clopidogrel included, among others, tumor necrosis factor (TNF), nuclear factor erythroid 2–related factor 2 (NRF2), transforming growth factor beta 1 (TGF-β1), plasminogen activator inhibitor (PAI-1), CD40 ligand (CD40LG), Ras homolog family member A (RHOA), adenosine monophosphate-activated protein kinase (AMPK), and the principal protein target of clopidogrel, the P2RY12 purinergic receptor of platelets and other tissues. Table I presents the network interference signature, showing proposed roles of these proteins in FSGS and predicted impacts of clopidogrel. Supplementary Table V holds additional information on these proteins along with references to key publications.
Table 1Proteins involved in FSGS development and progression and the proposed effect of clopidogrel.
Symbol
FSGS involvement
Clopidogrel effect
P2RY12
reduction of platelets/sclerosis/inflammation
direct target, ADP receptor inhibition
TNF
promotes podocyte damage and FSGS progression
systemic reduction
NFE2L2 (NRF2)
protective factor
increased in HL60 cells
TGFB1
promotes fibrosis
reduction in CKD, GN, and DN
FN1
promotes fibrosis
reduction in GN and DN
COL1A1
promotes fibrosis
reduction in DN
ICAM1
related to sclerosis/inflammation in mesangial cells in patients
reduction by P2Y12 inhibitor in CKD and DN
SERPINE1 (PAI-1)
promotes fibrosis
reduction in GN, plasma
FGA/B/G
promotes fibrosis
reduction in GN
EDN1
promotes fibrosis
reduction in CKD
CCL2 (MCP-1)
promotes FSGS
reduction in CKD
NLRP3
promotes inflammation and fibrosis
inhibition in sepsis-induced kidney injury
PRKAA1 (AMPK)
promotes podocyte survival
activated in HL60 cells
RHOA
promotes FSGS; important cytoskeleton/foot process regulator
Clopidogrel attenuates FSGS-associated weight loss and UACR in-vivo
FSGS was induced in Balb/c mice by intravenous administration of adriamycin. 48 hours postadriamycin treatment, clopidogrel therapy was initiated. To monitor disease course, weekly values of UACR were determined in spot urine samples. In line with our bioinformatics prediction, clopidogrel-treated mice showed reduced albuminuria (Fig 3). The beneficial effect of clopidogrel became more pronounced over time as evidenced by decreasing confidence intervals (P < 0.001, repeated-measures ANOVA). In a second independent experiment in a different cohort of mice these results were confirmed. Taking into account all time points, clopidogrel reduced UACR on average by 61% (P < 0.001, repeated-measures ANOVA). Body weight as an indicator for disease severity and drug tolerability was measured weekly. Supplementary Fig 1 shows that clopidogrel-treated animals experienced less weight loss than control animals (P < 0.01, repeated-measures ANOVA).
Fig 3Clopidogrel therapy reduces albuminuria in mice with adriamycin nephropathy. Urinary albumin-to-creatinine ratio (UACR) values of control (n=8) and clopidogrel-treated mice (n = 7) postadriamycin injection. Significance was assessed via repeated-measures ANOVA. Error bars represent mean values and standard deviations..
Clopidogrel significantly affects sclerosis and tubular atrophy in vivo
Mice were euthanized five weeks post induction of FSGS. Kidneys were collected and processed for PAS staining to determine the impact of clopidogrel on the histopathological features of FSGS. Sclerotic glomeruli and protein casts of the highest severity are only visible in adriamycin mice and not in the clopidogrel-treated animals (Fig 4, A). The histological damage score, defined as the sum of the glomerular sclerosis severity score, and the tubular ectasia severity score, assessed by expert nephro-pathologists, was significantly lower (P < 0.05, Student's t-test) in clopidogrel-treated animals (0.86) than in adriamycin controls (2.67) (Fig 4, B). In both independent experiments clopidogrel reduced the damage score on average by 67.9% (95% confidence interval -11.7 to -98.1%; P < 0.05).
Fig 4Clopidogrel therapy ameliorates the histopathology of adriamycin nephropathy in mice. Panel A: Representative PAS staining of kidney sections from mice with adriamycin nephropathy treated without (control) or with clopidogrel. Black arrows indicate selected sclerotic areas. Black asterisks indicate protein casts representing tubular ectasia. Panel B: 100%-stacked column chart depicting segmental glomerular sclerosis scores (left) or of tubular ectasia severity scores (right). Numbers in the bar stacked charts represent (number of individual mice). Segmental sclerosis scores: 0: n.d., 1: 1%–4%, 2: 5%–9%, 3: 10%–19%, 4: >=20%. Tubular ectasia scores: 0: none, 1: mild, 2: moderate, 3: strong, 4: very strong.
The data together demonstrate that clopidogrel administration beginning twodays after adriamycin administration significantly attenuated weight loss, albuminuria, and histopathological damage in the kidneys in the adriamycin FSGS mouse model.
Discussion
We have identified clopidogrel as promising potential therapeutic option for FSGS through a network-based drug repositioning approach. We found a significant in silico overlap between the molecular models constructed for FSGS pathophysiology and the clopidogrel MoA, with clopidogrel exerting a strongly beneficial impact on key FSGS proteins and mechanisms. In vivo, clopidogrel significantly improved adriamycin nephropathy, an established and widely used mouse model of FSGS.
Clopidogrel treatment significantly reduced albuminuria, weight loss, glomerulosclerosis and tubular ectasia. This is to our knowledge the first demonstration of a beneficial impact of clopidogrel treatment on an in vivo model of FSGS.
Clopidogrel is a P2Y12 receptor inhibitor and anti-platelet drug approved for prophylactic use in patients at elevated risk for myocardial infarction, unstable angina, stroke and peripheral arterial disease.
CJ Beavers and IA Naqvi, Clopidogrel, StatPearls [Internet], 2022, StatPearls Publishing; Treasure Island (FL), [cited 2022 May 18]. Available at: http://www.ncbi.nlm.nih.gov/books/NBK470539/. September 2022.
are additional gene products modulated by clopidogrel that are centrally involved in fibrotic processes. Fibronectin 1 was also identified as a top hub gene for FSGS with diagnostic potential.
Anti-inflammatory effects of clopidogrel intake in renal transplant patients: effects on platelet-leukocyte interactions, platelet CD40 ligand expression, and proinflammatory biomarkers.
Clopidogrel thus exerts anti-coagulant, anti-inflammatory, anti-oxidative, immune-modulating and anti-apoptotic effects influencing several genes and processes relevant to FSGS. Of note, in the context of gastric cancer, clopidogrel upregulates toll-like receptor 4 (TLR4),
our translational study is the first to demonstrate beneficial impact of clopidogrel on FSGS. Another P2Y12 inhibitor, ticagrelor, and recently also clopidogrel have been shown to improve diabetic nephropathy in vivo.
. From a clinical point of view, clopidogrel appears particularly attractive as many of its FSGS-relevant molecular targets are complementary to those of the current standard of care, namely corticosteroids, calcineurin inhibitors, and inhibitors of RAAS. Addition of clopidogrel to such regimens might thus benefit FSGS patients unresponsive or refractory to conventional therapies.
We performed our experiments in the most commonly used model of FSGS, the adriamycin mouse model of FSGS. Adriamycin intercalates into DNA pairs and inhibits topoisomerases and other nucleotide metabolic enzymes.
Adriamycin at the applied dose and route of administration causes podocyte injury and foot process effacement leading to glomerulosclerosis, tubulointerstitial inflammation, fibrosis, albuminuria and kidney injury in Balb/c mice.
While adriamycin nephropathy replicates all relevant histopathological features of FSGS, it cannot (as also true for other animal models of FSGS) serve as a model for all the multiple etiologies of human FSGS and resembles particularly primary FSGS.
Therefore, our data suggest that clopidogrel might be most beneficial in patients with primary FSGS. However, the beneficial effect of clopidogrel reported in other glomerular disease contexts encourages the speculation that it might benefit a substantial proportion of FSGS patients. Our positive preclinical data for clopidogrel suggests a phase II trial with selected patients might be a productive next step in the evaluation of the therapeutic potential of clopidogrel in human FSGS.
In contrast to clinical development of new chemical entity drugs for FSGS, the repositioning approach allows focus on proof of efficacy, since the drug candidates have undergone previous safety profile testing. As mouse data cannot be directly extrapolated to human data, dose-finding studies will be needed in clinical trial settings. Clopidogrel has been positively reviewed in CKD patients treated for clopidogrel's original indications.
This could increase acceptability among investigators and patients and facilitate recruitment, often the greatest barrier to new drug development for rare diseases.
In addition to identification of clopidogrel as a potential therapeutic option in FSGS, our successful bioinformatics approach for evaluation of a specific drug MoA in the context of a particular disease pathophysiology represents a promising and efficient general strategy for accelerated therapeutics development.
Conclusion
Our translational study has identified clopidogrel as a promising therapeutic option for FSGS via network-based bioinformatics analysis. Clopidogrel significantly improved key outcome parameters in the adriamycin FSGS mouse model, including albuminuria, weight loss, and histopathological lesions of the kidney. These data, along with a favorable drug safety profile, endorse clopidogrel as an attractive candidate for drug repositioning and subsequent clinical trial evaluation for patients suffering from FSGS.
Patents
PCT patent application entitled “CLOPIDOGREL FOR USE IN THE TREATMENT OF FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS)” was filed on December 3, 2021 (W02022/117862 A1).
Data Availability Statement
All data from this study are included within the manuscript, including the Supplemental Information.
Acknowledgments
Conflict of interest: C. A. Gebeshuber, M. Ley, and P. Perco are employees of Delta 4 GmbH. C. Aufricht is co-founder of Delta 4 GmbH and member of the scientific advisory board. K. Kratochwill is co-founder of Delta 4 GmbH and part of Delta 4’s management team. Delta 4 GmbH has filed the patent application given above. S. L. Alper is a consultant to the Medical University of Vienna as well as to Quest Diagnostics, Inc. All other authors declare no conflicts of interest.
Author contributions are as follows: C. A. Gebeshuber developed the experimental outline, conducted the animal experiments, interpreted the data and wrote the first version of the manuscript. P. Perco performed the bioinformatics and statistical analyses, interpreted the data, prepared the figures, and wrote the first version of the manuscript. H. Regele and C. Kornauth performed the histopathological diagnoses of the FSGS samples. H. Schachner conducted the histopathological staining. L. Daniel-Fischer performed the tail vein injections and performed manuscript revision. M. Ley supported data analysis and figure generation. S. L. Alper critiqued and revised the manuscript. R. Herzog provided experimental support. C. Aufricht conceptualized the study, supported statistical analyses, and supported manuscript preparation. K. Kratochwill supervised the project and supported manuscript preparation. All authors have read and approved the final version of the manuscript.
Canagliflozin reduces inflammation and fibrosis biomarkers: a potential mechanism of action for beneficial effects of SGLT2 inhibitors in diabetic kidney disease.
A systems pharmacology workflow with experimental validation to assess the potential of anakinra for treatment of focal and segmental glomerulosclerosis.
BT Sherman, M Hao, J Qiu, et al., DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update), Nucleic Acids Res, 2022,2022 Jul 5;50(W1):W216-W221. doi: 10.1093/nar/gkac194.
CJ Beavers and IA Naqvi, Clopidogrel, StatPearls [Internet], 2022, StatPearls Publishing; Treasure Island (FL), [cited 2022 May 18]. Available at: http://www.ncbi.nlm.nih.gov/books/NBK470539/. September 2022.
Anti-inflammatory effects of clopidogrel intake in renal transplant patients: effects on platelet-leukocyte interactions, platelet CD40 ligand expression, and proinflammatory biomarkers.
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