Rationale & Objective: Sodium-glucose cotransporter-2 inhibitors (SGLT2I) are a standard of care treatment for chronic kidney disease (CKD). However, the effects of SGLT2I on tubular function and stress are rare. Study Design: A monocentric, prospective, observational study. Setting & Participants: Patients with CKD who were referred to the outpatient clinic of the Department of Nephrology at Göttingen University Hospital and for whom SGLT2I therapy was indicated as part of standard treatment were prospectively included and treated with SGLT2I in accordance with current guidelines. Analytical Approach: Urine samples were collected, and the levels of urinary α1-microglobulin (uα-1-MG), urinary Dickkopf-3 (uDKK3), and urinary albumin creatinine ratio (UACR), each normalized to urinary creatinine, were measured at baseline and after 6 months of therapy. Results: Total of 57 patients were included. The mean age was 66.4 ± 12.4 years; 42.1% were female. At baseline, mean estimated glomerular filtration rate was 42.0 ± 15.3 mL/min/1.73m2. Within 6 months, the mean estimated glomerular filtration rate decreased by −2.1 ± 7.6 mL/min/1.73 m2 (P = 0.04). While overall patients, UACR (P = 0.23) and uDKK3 (P = 0.87) remained unchanged, there was a significant increase in median uα-1-MG within 6-months (+9.7 mg/g creatinine [IQR 1.0-22.2]; P < 0.001). Although patients with A3 albuminuria (n = 14) showed only a numerical increase of median uα-1-MG level (+4.6 mg/g creatinine [IQR −7.1 to 20.8]; P = 0.30), there was a significant increase of median uα-1-MG in stage A1 (n=22, +11.5 mg/g creatinine [IQR 3.8-23.2]; P < 0.001) and A2 patients (n = 21, +7.2 mg/g creatinine [IQR 0.7-30.8]; P = 0.006). Limitations: The small sample size, lack of a control group, and the short follow-up duration may restrict the findings. Conclusions: This study demonstrates an increase in uα-1-MG during SGLT2I treatment in patients with CKD, particularly in UACR stage A1, in which reduction of hyperfiltration is less pronounced. Because uDKK3 as a parameter of tubular damage remained stable, the increase in uα-1-MG may reflect a functional reduction of protein reabsorption and thus reduced intratubular protein overload as a potential nephroprotective effect. Plain-Language Summary: This study analyzed urine samples from patients receiving sodium-glucose cotransporter inhibitors (SGLT2I) as part of their treatment for chronic kidney disease. The urinary concentrations of the proteins α-1-microglobulin, Dickkopf-3, and albumin were measured before the start of treatment and after 6 months of therapy. Although the concentrations of albumin and Dickkopf-3 remained unchanged, the urinary concentration of α-1-microglobulin increased significantly during the 6-month SGLT2-I therapy. Because Dickkopf-3 levels as a marker of injury remained constant, the increase in α-1-microglobulin in urine could be an expression of functionally reduced protein reabsorption from the urine and thus reduced protein overload in the kidney cells, possibly representing a kidney-protective effect of SGLT2-I.

Aims Cardiac fibrosis remains a significant and currently untreatable contributor to mortality in chronic heart disease. This study aimed to comprehensively map cell-specific pathological changes in fibrotic heart tissue from aortic stenosis patients using single-nucleus RNA sequencing (snRNA-seq). We specifically sought to identify novel cell-specific candidate genes involved in cardiac fibrosis and validate their functional impact using both in vitro and in vivo models. Our ultimate goal was to enhance the understanding of fibrogenesis in aortic stenosis, thereby guiding the development of targeted antifibrotic therapies.Methods and results We conducted single nucleus RNA sequencing (snRNA-seq) on human fibrotic aortic stenosis and non-failing donor heart samples. The final dataset consisted of 41 330 nuclei distributed across 28 cell clusters representing 6 major cell types. SnRNA-seq revealed a disease-specific subpopulation of fibroblasts, characterized by the presence of both anti-fibrotic and pro-fibrotic markers. Interestingly, TCF21, a basic helix-loop-helix transcription factor, was identified as a potential key regulator within this distinct fibroblast population, and we performed functional studies to identify its role in disease progression accordingly. Loss of TCF21 in cardiac fibroblasts caused increased myofibroblast-like gene expression and differentiation, whereas upregulation of TCF21 in cardiac fibroblasts exerted an anti-fibrotic effect. Adeno-associated virus-mediated cardiac fibroblast-specific knockdown and overexpression in vivo further demonstrate that TCF21 improves cardiac remodelling.Conclusion This investigation highlights the role of TCF21 as a protective regulator in cardiac fibrosis. Moreover, the upregulation of TCF21 holds promise as a strategy for treating cardiac fibrosis.

Dear Editor, Aortic stenosis (AS) is the most prevalent valvular disorder in the western world imposing significant morbidity and mortality. Progressive AS primarily affects the left ventricle, leading to adaptations of the myocardium, resulting in cardiac hypertrophy and myocardial fibrosis (MF). We and others recently demonstrated a link between the amount of MF and impaired outcomes following aortic valve replacement in patients with severe AS,1 highlighting its clinical relevance. Although cardiac biopsy remains the gold standard for MF quantification, this method is complex, invasive, and may be associated with severe complications. Thus, easier ways of determining MF levels are direly needed. Different peptides of collagen metabolism have recently been suggested as serum biomarkers for MF and/or associated risk prediction in patients with heart failure (HF).2 For instance, low serum levels of procollagen type I C-terminal propeptide (PICP), formed during the extracellular conversion of procollagen type I into mature fibril-forming collagen type I, were shown to be linked to reduced risk of cardiovascular death or HF-associated hospitalization in HF patients with reduced ejection fraction (EF). Levels of the amino-terminal propeptide of procollagen type III (PIIINP), formed during the conversion of procollagen type III into mature collagen type III, correlated with the collagen type III volume fraction in HF patients with ischemic heart disease or idiopathic dilated cardiomyopathy. The level of MF is not only a matter of quantity, that is, collagen deposition, but also quality as reflected by the degree of myocardial collagen cross-linking. This can be indirectly assessed by the serum collagen type I C-terminal telopeptide to matrix metalloproteinase-1 ratio (CITP:MMP1), a negative index of myocardial collagen cross-linking, and can be used for HF-associated hospitalization risk prediction in hypertensive HF.3 Investigations on serum biomarker identification for MF assessment in AS patients with different hemodynamic subtypes have not yet been performed. Thus, our study was designed to evaluate the association of PICP, PIIINP, and CITP:MMP1 with histological MF (measured as collagen volume fraction) in cardiac biopsies, obtained during transcatheter aortic valve replacement (TAVR), in our well-defined cohort of patients with severe AS.1 Biomarkers were measured in serum samples from 95 AS patients prior to TAVR. This study focused on the four different hemodynamic AS subtypes: (I) normal EF and high transvalvular pressure gradient (NEF-HG AS), (II) low/reduced EF and high gradient (LEF-HG AS), (III) low/reduced EF and low gradient referred to as classical low-flow low-gradient AS (classical LF-LG AS), and (IV) paradoxical low-flow low-gradient despite normal EF (PLF-LG AS). Details regarding hemodynamics (Table S1) and a detailed description of material and methods can be found in the Supporting Information. Of the 95 study participants, 39 belonged to the NEF-HG AS group, 14 to LEF-HG AS, 26 to classical LF-LG AS, and 16 to PLF-LG-AS. The mean age of the study cohort was 78.4 years (±6.8), with males constituting 67% of the participants. Cardiovascular risk factors and comorbidities were comparably distributed across the four groups (Table S1). Regarding the analyzed serum biomarker levels, no statistically significant differences across the four groups could be observed (Figure 1A). Upon histological examination of fibrosis and as previously reported,1 LEF-HG AS and classical LF-LG AS patients displayed a markedly higher MF burden compared to NEF-HG AS patients (p < 0.05) (Figure 1B). Importantly, we discovered a trend for a negative association between the level of histological MF and serum CITP:MMP1 ratios in the cohort as a whole (r = −0.18; p = 0.08), which could predominantly be attributed to the highly significant association in the classical LF-LG AS group (r = −0.62; p = 0.002) (Figure 1C,D). None of the associations with the collagen deposition markers PICP and PIIINP in any of the groups were significant (Figure 1C). Of note, all analyses including serum biomarkers were adjusted for renal function (i.e., estimated glomerular filtration rate), age, and sex. To our knowledge, this is the first study assessing the three well-characterized serum biomarkers of collagen metabolism, that is, PICP, PIIINP, and CITP:MMP1, in AS patients with different hemodynamic subtypes. We uncovered a hitherto unrecognized role for CITP:MMP1 as promising serum biomarker for estimating MF levels specifically in classical LF-LG AS patients. From a pathophysiological perspective, the results point toward myocardial collagen cross-linking as relevant mechanism specifically in this hemodynamic entity. Collagen cross-linking plays a critical role in determining the resistance of collagen fibers to MMP-mediated degradation; thus, enhanced cross-linking of collagen type I fibers specifically reduces the cleavage of CITP by MMP1. Consequently, the negative association of the CITP:MMP1 ratio with histological MF in classical LF-LG AS patients is suggestive of more collagen cross-linking and resistance to degradation, causing reduced cleavage of CITP by MMP1. This results in increased levels of MF and consequently lower CITP:MMP1 serum ratios. It is likely that this mechanism contributes to the adverse myocardial remodeling and worst prognosis of classical LF-LG AS among all four AS subtypes even after TAVR.1 This association could not be observed in any of the other groups, emphasizing the importance to distinguish between them as pathophysiological mechanisms may be fundamentally different. Moreover, our results on PICP and PIIINP suggest that collagen deposition, as compared to cross-linking, may not be as relevant in our cohort of AS patients, irrespective of the hemodynamic subtype. This could potentially be attributed to the timing of collagen deposition, which may have occurred before the biopsy was taken with no further relevant active collagen production. However—due to the rather small sample size—a type II error, that is, the failure to reject a false null hypothesis, can also not be ruled out at this point. The relevance to distinguish between hemodynamic AS subgroups is not only important for the identification of novel biomarkers, but also with respect to a more personalized therapeutic strategy. In this regard, assessing CITP:MMP1 serum levels in patients with classical LF-LG AS may help to identify individuals who particularly benefit from a medication with antifibrotic properties. In line with this consideration, Ravassa et al. already demonstrated the potential of CITP:MMP1 assessment regarding the favorable effects through spironolactone treatment in (i) HF patients with preserved EF (HFpEF) (Aldo-DHF trial) and (ii) patients at risk of HF (HOMAGE trial).4, 5 In both studies, patients with higher CITP:MMP1 ratios, that is, a lower degree of collagen cross-linking, showed various clinical benefits as compared to patients with lower ratios following spironolactone treatment. Considering the exploratory nature of our study—with its relatively small sample size and primary focus on the correlation between collagen biomarkers and histological fibrosis—larger studies are needed to expand these observations and to evaluate the relationship between CITP:MMP1 and cardiovascular outcome parameters. It will therefore be interesting to assess serum CITP:MMP1 in the ongoing, prospective REDUCE-MFA trial (NCT05230901). In this cohort, patients with severe AS are treated with low-dose dihydralazine and spironolactone, aiming to reduce MF. Assessing CITP:MMP1 with subsequent analysis of a potential MF reduction and altered outcome will allow to get a much clearer understanding of how this biomarker could be implemented into clinical practice. In summary, we identified CITP:MMP1 as a novel serum biomarker for estimating MF in classical low-flow low-gradient AS. This is the first study to demonstrate a significant correlation between the serum CITP:MMP1 ratio and MF in a clinically relevant disease context. S.G. conceived the study, performed data acquisition, performed statistical analyses, interpreted data, wrote the manuscript draft, and revised it. P.B. conceived the study, performed data acquisition, performed statistical analyses, interpreted data, wrote the manuscript draft, and revised it. N.B.P. performed statistical analyses and reviewed the manuscript draft. S.R. performed collagen metabolism serum biomarker measurements, discussed results and strategy, interpreted data, and revised the manuscript. S.v.H. interpreted data, discussed results and strategy, and revised the manuscript. A.F. discussed results and strategy, interpreted data, and revised the manuscript. E.M.Z. performed histological measurements and revised the manuscript. M.P. performed echocardiography and TAVR, organized the clinical trial, and revised the manuscript. G.H. is the trial's principal investigator, provided serum samples for biomarker measurements, discussed results and strategy, interpreted data, and revised the manuscript. M.S. conceived, designed, and directed the study (lead), was involved in all analyses, and wrote the manuscript. All authors have read and approved the final manuscript. We thank Swetlana Hartmann and Kristina Schröder (both Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany) for technical support and Prof. Dr. Karl Toischer, Dr. Bo E. Beuthner (both Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany), Prof. Dr. Tim Beissbarth (Department of Bioinformatics, University Medical Center Göttingen, Göttingen, Germany), and Prof. Dr. Javier Diez (Program of Cardiovascular Disease, Centro de Investigacion Medica Aplicada Universidad de Navarra [CIMA], Pamplona, Spain) for excellent scientific advise. This work was supported by the German Research Foundation (DFG; CRC 1002, TP D01 to G.H.); S.G. was funded by the University Medical Center Göttingen—UMG Clinician Scientist Program. The authors declare no conflicts of interest. The study was approved by the Ethics Committee of the University Medical Center Göttingen (number: 10/5/16) and complied with the Declaration of Helsinki. Written informed consent for study participation including collection of myocardial biopsies was obtained from all study participants. Data are available from the corresponding author upon reasonable request. 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.

Abstract Background and Aims Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by immune system dysfunction, leading to inflammation and damage in various organs and tissues. Renal involvement known as lupus nephritis is one of the most severe complications occurring in 60% of patients with SLE, significantly impacting patient morbidity and mortality. Understanding the intricate relationship between SLE and renal manifestations is crucial for effective management and improved patient outcomes. Recently, the cell surface urokinase plasminogen activator receptor (uPAR) has emerged as a significant player in the pathogenesis of lupus nephritis. However, the molecular mechanisms through which uPAR influences lupus nephritis remain an active area of investigation. Therefore, we here pursued a transcriptome array-based approach to identify molecular patterns in association with uPAR in lupus nephritis. Method PLAUR mRNA expression levels (encoding uPAR, reporter ID: 210845_s_at, platform: Affymetrix Human Genome U133 Plus 2.0 Array, altCDF v10) were extracted from microdissected control kidneys (glomerular compartment: n = 14) and lupus nephritis (glomerular: n = 32, tubulointerstitial: n = 32). Pathway analysis was performed for gene enrichment associated with PLAUR mRNA expression with a correlation threshold of ≥0.5 by using reactome (https://reactome.org), pathways with a predefined entities value of P ≤ .001 were included. Data was validated in cultured podocytes stressed with Adriamycin, protein expression profiling was performed by mass spectrometry in combination with stable isotope labelling by amino acids in cell culture (SILAC). Results We here identified a predominant glomerular expression of uPAR in lupus nephritis that was increased as compared to healthy controls. While glomerular uPAR expression was predominantly associated with interferon signaling, none of type I or type II interferons were induced. Interestingly, we identified distinct interferon hub genes that were associated with uPAR in lupus nephritis and enriched in stressed podocytes (GBP2: P < .0001, IFI30: P = .0006, IFIT1: P < .0001, IFITM3: P = .0308). Finally, uPAR and associated interferon hub genes correlated with proteinuria in lupus nephritis. Conclusion The therapeutic implications of targeting uPAR in lupus nephritis are promising, and ongoing research endeavors are aimed at unraveling the complexities of uPAR as a potential diagnostic marker and therapeutic target for this challenging renal pathology. We here expand our current knowledge and link uPAR upregulation in lupus nephritis to distinct interferon hub genes in stressed podocytes.

Journal Article Accepted manuscript Single-nucleus transcriptomics reveals adrenergic and STAT3 signaling in paradoxical low-flow low-gradient -specific cardiomyocyte subclusters: implications for aortic stenosis pathogenesis and treatment Get access Xingbo Xu, Xingbo Xu Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany Correspondence: Ph.D. Xingbo Xu, Heart Research Building (DZHK), Department of Cardiology and Pneumology, University Medical Center of Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany, Telephone: +49-(0)551-3963633, Email: xingbo.xu@med.uni-goettingen.de; Dr. Gerd Hasenfuß, MD, Department of Cardiology and Pneumology, University Medical Center of Göttingen, Georg-August-University, Robert-Koch-Str. 40, 37075 Göttingen, Germany, Telephone: +49-(0)551-3967601, Fax: +49-(0)551-3967601, Email: hasenfus@med.uni-goettingen.de https://orcid.org/0000-0002-8249-5507 Search for other works by this author on: Oxford Academic PubMed Google Scholar Xiaoying Tan, Xiaoying Tan German Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, GermanyDepartment of Nephrology and Rheumatology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Baolong Cui, Baolong Cui Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Niels B Paul, Niels B Paul Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyDepartment of Medical Bioinformatics, University Medical Center Göttingen, Goldschmidtstraße 1, 37077 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Manar Elkenani, Manar Elkenani Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Bo E Beuthner, Bo E Beuthner Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Weichao Li, Weichao Li Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Belal A Mohamed, Belal A Mohamed Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Moritz Schnelle, Moritz Schnelle German Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, GermanyDepartment of Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Miriam Puls, Miriam Puls Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany https://orcid.org/0000-0001-5540-1264 Search for other works by this author on: Oxford Academic PubMed Google Scholar ... Show more Elisabeth M Zeisberg, Elisabeth M Zeisberg Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Michael Zeisberg, Michael Zeisberg Department of Nephrology and Rheumatology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Tim Beißbarth, Tim Beißbarth Department of Medical Bioinformatics, University Medical Center Göttingen, Goldschmidtstraße 1, 37077 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Karl Toischer, Karl Toischer Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Gerd Hasenfuß Gerd Hasenfuß Clinic for Cardiology and Pulmonology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, GermanyGerman Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany Correspondence: Ph.D. Xingbo Xu, Heart Research Building (DZHK), Department of Cardiology and Pneumology, University Medical Center of Göttingen, Robert-Koch-Str. 42a, 37075 Göttingen, Germany, Telephone: +49-(0)551-3963633, Email: xingbo.xu@med.uni-goettingen.de; Dr. Gerd Hasenfuß, MD, Department of Cardiology and Pneumology, University Medical Center of Göttingen, Georg-August-University, Robert-Koch-Str. 40, 37075 Göttingen, Germany, Telephone: +49-(0)551-3967601, Fax: +49-(0)551-3967601, Email: hasenfus@med.uni-goettingen.de Search for other works by this author on: Oxford Academic PubMed Google Scholar Cardiovascular Research, cvae137, https://doi.org/10.1093/cvr/cvae137 Published: 29 June 2024 Article history Received: 15 February 2024 Revision received: 22 April 2024 Accepted: 17 June 2024 Published: 29 June 2024

Oncogenic KRASG12D (KRAS*) is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC) and is a known repressor of tumor immunity. Conditional elimination of KRAS* in genetic mouse models of PDAC leads to the reactivation of FAS, CD8+ T cell-mediated apoptosis, and complete eradication of tumors. KRAS* elimination recruits activated CD4+ and CD8+ T cells and promotes the activation of antigen-presenting cells. Mechanistically, KRAS*-mediated immune evasion involves the epigenetic regulation of Fas death receptor in cancer cells, via methylation of its promoter region. Furthermore, analysis of human RNA sequencing identifies that high KRAS expression in PDAC tumors shows a lower proportion of CD8+ T cells and demonstrates shorter survival compared with tumors with low KRAS expression. This study highlights the role of CD8+ T cells in the eradication of PDAC following KRAS* elimination and provides a rationale for the combination of KRAS* targeting with immunotherapy to control PDAC.

Background Patients with chronic heart failure (HF) show an increased risk for the occurrence of chronic kidney disease and cardiorenal syndrome. Urinary Dickkopf‐3 (uDKK3), a stress‐induced, tubular profibrotic glycoprotein, may be elevated in HF as early as in New York Heart Association class I HF and may indicate subsequent decline in estimated glomerular filtration rate (eGFR). Methods and Results uDKK3 levels in patients with HF and controls were measured by enzyme‐linked immunosorbent assay. eGFR was determined up to 5 years in HF. Change in eGFR was assessed with respect to baseline uDKK3 using (mixed‐effect) linear and logistic regression models. A total of 488 patients with chronic HF and 45 control patients were included. Patients with HF showed higher median uDKK3 levels than controls (259.6 pg/mg creatinine [interquartile range (IQR), 119.2–509.4 pg/mg creatinine] versus 107.5 pg/mg creatinine [IQR, 60.5–181.2 pg/mg creatinine], P <0.001). Regression models demonstrated a significant association between log uDKK3 and the decline in eGFR during a median of 13 months (IQR, 12–59 months) (estimated higher eGFR loss, 0.8039 mL/min per 1.73 m 2 /year [95% CI, 0.002–1.606 mL/min per 1.73 m 2 /year], P =0.049; odds ratio, 1.345 [95% CI, 1.049–1.741], P =0.021). uDKK3 levels ≥354 pg/mg creatinine were associated with a significantly higher risk for eGFR decline at 1‐year follow‐up (estimated higher eGFR loss, 4.538 mL/min per 1.73 m 2 [95% CI, 1.482–9.593 mL/min per 1.73 m 2 ]), P =0.004). Even patients with HF without chronic kidney disease (n=334) had higher uDKK3 levels compared with controls (233.4 [IQR, 109.0–436.9 pg/mg creatinine] versus 107.5 [IQR, 60.5–181.2 pg/mg creatinine], P <0.001). Conclusions The present findings indicate that uDKK3 is a promising prognostic biomarker for subsequent eGFR decline in patients with HF, irrespective of the presence of chronic kidney disease and even in the early stages of HF. This potential allows for early intervention to mitigate the deterioration of kidney function. Further investigation is warranted to validate its clinical utility.

Cardiac fibrosis is the hallmark of all forms of chronic heart disease. Activation and proliferation of cardiac fibroblasts are the prime mediators of cardiac fibrosis. Existing studies show that ROS and inflammatory cytokines produced during fibrosis not only signal proliferative stimuli but also contribute to DNA damage. Therefore, as a prerequisite to maintain sustained proliferation in fibroblasts, activation of distinct DNA repair mechanism is essential. In this study, we report that TET3, a DNA demethylating enzyme, which has been shown to be reduced in cardiac fibrosis and to exert antifibrotic effects does so not only through its demethylating activity but also through maintaining genomic integrity by facilitating error-free homologous recombination (HR) repair of DNA damage. Using both in vitro and in vivo models of cardiac fibrosis as well as data from human heart tissue, we demonstrate that the loss of TET3 in cardiac fibroblasts leads to spontaneous DNA damage and in the presence of TGF-β to a shift from HR to the fast but more error-prone non-homologous end joining repair pathway. This shift contributes to increased fibroblast proliferation in a fibrotic environment. In vitro experiments showed TET3’s recruitment to H2O2-induced DNA double-strand breaks (DSBs) in mouse cardiac fibroblasts, promoting HR repair. Overexpressing TET3 counteracted TGF-β-induced fibroblast proliferation and restored HR repair efficiency. Extending these findings to human cardiac fibrosis, we confirmed TET3 expression loss in fibrotic hearts and identified a negative correlation between TET3 levels, fibrosis markers, and DNA repair pathway alteration. Collectively, our findings demonstrate TET3’s pivotal role in modulating DDR and fibroblast proliferation in cardiac fibrosis and further highlight TET3 as a potential therapeutic target.

Abstract Oncogenic KrasG12D (Kras*) is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC), and a known repressor of tumor immunity. Conditional extinction of Kras* in genetic mouse models of PDAC leads to reactivation of Fas, CD8+ T cell mediated apoptosis, and complete eradication of tumors. Kras* extinction recruits CD4+ and CD8+ T cells, promotes the activation of putative antigen presenting CD11b−CD11c+ dendritic cells, and suppresses CD11b+ myeloid cell infiltration. Our findings shows that Kras* drives T cell suppression in PDAC. Mechanistically, Kras* mediated immune evasion involves epigenetic regulation of the death receptor Fas in cancer cells, via repression of its promoter region mediated by functional recruitment of DNA methyltransferase 1 (DNMT1), trimethylation of histone 3 at lysine 27 (H3K27me3), the histone methyltransferase EZH2, and suppression of acetylation of histone 3 at lysine 27 (H3K27ac). Further, analysis of human RNA sequencing reveals that Kras* expression levels inversely correlate with FAS expression and PDAC tumors with high KRAS demonstrate low CD8+ T cell infiltration, which is associated with shorter overall survival in PDAC patients. Given that specific and effective targeting of Kras* results in the CD8+ T cell mediated, Fas dependent apoptosis of cancer cells and eradication of PDAC in mice, this study highlights the direct role of Kras* in suppression of tumor immunity and supports a role for immunotherapy in combination with Kras* targeting strategies to control PDAC. Citation Format: Valerie S. LeBleu, Krishnan K. Mahadevan, Elena V. Ramirez, Yang Chen, Bingrui Li, Amari M. Sockwell, Mihai Gagea, Hikaru Sugimoto, Desiree Tampe, Michael Zeisberg, Haoqiang Ying, Abhinav K. Jain, Ronald A. DePinho, Aniban Maitra, Kathleen M. McAndrews, Raghu Kalluri. Extinction of oncogenic Kras in genetic mouse models eradicates pancreatic cancer by inducing Fas-dependent apoptosis by CD8+ T cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B082.

BACKGROUND:Cardiac fibrosis plays a major pathophysiological role in any form of chronic heart disease, and high levels are associated with poor outcome. Diffuse and focal cardiac fibrosis are different subtypes, which have different pathomechanisms and prognostic implications. The total fibrosis burden in endomyocardial biopsy tissue was recently proved to play an independent prognostic role in aortic stenosis patients after transcatheter aortic valve implantation (TAVI).AIMS:Here, for the first time, we aim to assess the specific impact of different fibrosis subtypes on sudden cardiac death (SCD) as a primary reason for cardiovascular mortality after TAVI.METHODS:The fibrosis pattern was assessed histologically in the left ventricular biopsies obtained during TAVI interventions in 161 patients, who received a structured follow-up thereafter.RESULTS:Receiver operating characteristic analyses, performed 6, 12, 24 and 48 months after TAVI, showed diffuse, but not focal, fibrosis as a significant predictor for SCD at all timepoints, with the highest area under the curve at the first time point and a decrease in its SCD predictivity over time. In both multivariate Cox proportional hazards and Fine-Gray competing risk models, including both fibrosis subtypes, as well as age, sex and ejection fraction, high diffuse fibrosis remained statistically significant. Accordingly, it represents an independent SCD predictor, most importantly for the occurrence of early events.CONCLUSIONS:The burden of diffuse cardiac fibrosis plays an important and independent prognostic role regarding SCD early after TAVI. Therefore, the histological evaluation of fibrosis topography has value as a prognostic tool for TAVI patients and may help to tailor individualised approaches to optimise their postinterventional management.

Abstract Background and Aims Avacopan is a novel promising treatment for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) targeting the complement system by blocking C5a receptor C5aR1. We here pursued a transcriptome array-based approach to dissect molecular signatures associated with C5aR1 in renal vasculitis. Method C5aR1 mRNA expression levels (reporter ID: 210845_s_at, platform: Affymetrix Human Genome U133 Plus 2.0 Array, altCDF v10) were extracted from microdissected samples of renal vasculitis (glomerular: n = 23, tubulointerstitial: n = 21). Pathway analysis was performed for gene enrichment associated with PLAUR mRNA expression with a correlation threshold of ≥0.5 by using reactome (http://reactome.org), pathways with a predefined entities value of p≤0.001 were included. Results We here identified a predominant glomerular expression of C5aR1 in renal vasculitis as compared to the tubulointerstitial compartment (p < 0.0001). Furthermore, glomerular C5aR1 induction correlated with impaired kidney function (eGFR: 0.0193). Gene set enrichment identified various inflammatory signatures including neutrophil degranulation and innate immunity, and particularly enrichment of urokinase plasminogen activator receptor (uPAR). uPAR itself also correlated with impairment of kidney function in renal vasculitis and particularly active glomerular lesions (cellular crescents and necrosis, p < 0.0001). Conclusion The therapeutic implications of targeting uPAR in renal vasculitis are promising, and ongoing research endeavors are aimed at unraveling the complexities of uPAR as a potential diagnostic marker and therapeutic target for this challenging renal pathology. We here expand our current knowledge and link C5aR1 to inflammatory responses driven by glomerular uPAR upregulation in renal vasculitis.

AimsPatients with severe aortic stenosis (AS), low transvalvular flow (LF) and low gradient (LG) with normal ejection fraction (EF)—are referred to as paradoxical LF-LG AS (PLF-LG). PLF-LG patients develop more advanced heart failure symptoms and have a worse prognosis than patients with normal EF and high-gradient AS (NEF-HG). Despite its clinical relevance, the mechanisms underlying PLF-LG are still poorly understood.MethodsLeft ventricular (LV) myocardial biopsies of PLF-LG (n = 5) and NEF-HG patients (n = 6), obtained during transcatheter aortic valve implantation, were analyzed by LC-MS/MS after sequential extraction of cellular and extracellular matrix (ECM) proteins using a three-step extraction method. Proteomic data are available via ProteomeXchange with identifier PXD055391.Results73 cellular proteins were differentially abundant between the 2 groups. Among these, a network of proteins related to muscle contraction and arrhythmogenic cardiomyopathy (e.g., cTnI, FKBP1A and CACNA2D1) was found in PLF-LG. Extracellularly, upregulated proteins in PLF-LG were related to ATP synthesis and oxidative phosphorylation (e.g., ATP5PF, COX5B and UQCRB). Interestingly, we observed a 1.3-fold increase in cyclophilin A (CyPA), proinflammatory cytokine, in the extracellular extracts of PLF-LG AS patients (p < 0.05). Consistently, immunohistochemical analysis confirmed its extracellular localization in PLF-LG AS LV sections along with an increase in its receptor, CD147, compared to the NEF-HG AS patients. Levels of core ECM proteins, namely collagens and proteoglycans, were comparable between groups.ConclusionOur study pinpointed novel candidates and processes with potential relevance in the pathophysiology of PLF-LG. The role of CyPA in particular warrants further investigation.

Legends to Supplementary Figures S1-S4 from Chronic Bile Duct Injury Associated with Fibrotic Matrix Microenvironment Provokes Cholangiocarcinoma in p53-Deficient Mice

Abstract Background and Aims Ischemia and following hypoxia are a common cause of acute kidney injury (AKI). Strategies for prevention and treatment of AKI are limited. In several animal models, ischemic preconditioning (IPC) has been established as an effective intervention to prevent ischemic organ injury. An IPC protocol of ischemia and reperfusion mediates organ protection for a few hours. This first phase of organ protection is followed by a second, protective phase occurring after 24–48 h. The second phase, named second window of protection (SWOP), persists for 3–4 d. Mechanisms and transcriptional mediators during SWOP are mostly unknown. The aim of the underlying study was to investigate effectors and target genes during SWOP and to identify a potential pharmacological therapeutic target. Method 6–8-week-old C57BL/6 mice were undergoing an IPC intervention; five minutes of renal sinistra artery ischemia was followed by five minutes of reperfusion. This cycle was performed five times. Mice were sacrificed 2, 4, 8, 12, 25, 48, 72 h after IPC. Two additional groups of mice were pharmacologically preconditioned for 48 h with low dose FK506 and small molecule GPI1046. Analysis of murine kidneys were performed by SDS-Page and Western blot. Results The first phase after IPC (2–4 h) was characterized by an increasing expression of hypoxia inducible factor-1α (HIF1α); 48 h after intervention a significant increase of expression of aryl hydrocarbon receptor nuclear translocator (ARNT) and consecutive activin receptor-like kinase 3 (ALK3) was observed. Comparable induction of ARNT and ALK3 expression was obtained by pharmacological preconditioning with FK506 or GPI1046. Conclusion Affected by hypoxia, HIF1α activates transcriptional upregulation of ARNT 48 h after IPC. The increasing expression of ARNT, follows an increased ARNT homodimerization. ARNT homodimer binds to the palindromic E-box 5′-CACGTG-3′ of the proximal ALK3 promoter. By this it induces the expression of the renoprotective ALK3 during SWOP. The organ-protective effects of constitutively expressed ARNT during SWOP can be mimicked pharmacologically by administration of FK506 or GPI1046 and may be used as a future therapeutic target.