Cyclin-dependent kinase 4 drives cystic kidney disease in the absence of mTORC1 signaling activity.

Progression of cystic kidney disease has been linked to activation of the mTORC1 signaling pathway. Yet the utility of mTORC1 inhibitors to treat patients with polycystic kidney disease remains controversial despite promising preclinical data. To define the cell intrinsic role of mTORC1 for cyst development, the mTORC1 subunit gene Raptor was selectively inactivated in kidney tubular cells lacking cilia due to simultaneous deletion of the kinesin family member gene Kif3A. In contrast to a rapid onset of cyst formation and kidney failure in mice with defective ciliogenesis, both kidney function, cyst formation discerned by magnetic resonance imaging and overall survival were strikingly improved in mice additionally lacking Raptor. However, these mice eventually succumbed to cystic kidney disease despite mTORC1 inactivation. In-depth transcriptome analysis revealed the rapid activation of other growth-promoting signaling pathways, overriding the effects of mTORC1 deletion and identified cyclin-dependent kinase (CDK) 4 as an alternate driver of cyst growth. Additional inhibition of CDK4-dependent signaling by the CDK4/6 inhibitor Palbociclib markedly slowed disease progression in mice and human organoid models of polycystic kidney disease and potentiated the effects of mTORC1 deletion/inhibition. Our findings indicate that cystic kidneys rapidly adopt bypass mechanisms typically observed in drug resistant cancers. Thus, future clinical trials need to consider combinatorial or sequential therapies to improve therapeutic efficacy in patients with cystic kidney disease.

TNF reduces osteogenic cell fate in PDL cells at transcriptional and functional levels without alteration of periodontal proliferative capacity.

AIMS: To investigate the effect of tumor necrosis factor (TNF) on the growth of human periodontal ligament (PDL) cells, their osteogenic differentiation and modulation of their matrix secretion in vitro. METHODS: The influence of 10 ng/ml TNF on proliferation and metabolic activity of PDL cells was analyzed by cell counting (DAPI [4',6-diamidino-2-phenylindole] staining) and the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. In addition, cells were cultured under control conditions and osteogenic conditions (media containing 10 mM ß-glycerophosphate). Quantitative expression analysis of genes encoding the osteogenic markers alkaline phosphatase (ALP), collagen type I alpha 1 chain (COL1A1), osteoprotegerin (OPG), and osteopontin (OPN) was performed after 7 and 14 days of cultivation. Calcium deposits were stained with alizarin red. RESULTS: Our studies showed that 10 ng/ml TNF did not affect the survival and metabolic activity of PDL cells. Quantitative expression analysis revealed that long-term cultures with TNF impaired osteogenic cell fate at early and late developmental stages. Furthermore, TNF significantly reduced matrix secretion in PDL cells. CONCLUSION: The present data confirm TNF as a regulatory factor of proinflammatory remodeling that influences the differentiation behavior but not the metabolism and cell proliferation of the periodontium. Therefore, TNF represents an interesting target for the regulation of orthodontic remodeling processes in the periodontium.

Long-term outcomes of patients with IgA nephropathy in the German CKD cohort.

Background: The importance of albuminuria as opposed to proteinuria in predicting kidney outcomes in primary immunoglobulin A nephropathy (IgAN) is not well established. Methods: From 2010 to 2012, 421 patients with biopsy-proven IgAN have been enrolled into the German Chronic Kidney Disease (GCKD) cohort, a prospective observational cohort study (N = 5217). Adjudicated endpoints include a composite kidney endpoint (CKE) consisting of eGFR decline >40%, eGFR <15 ml/min/1.73 m2 and initiation of kidney replacement therapy; the individual components of the CKE; and combined major adverse cardiac events (MACE), including non-fatal myocardial infarction, non-fatal stroke and all-cause mortality. The associations between the incidence of CKE and baseline factors, including demographics, laboratory values and comorbidities were analysed using the Cox proportional hazards regression model. Results: The mean age of IgAN patients at baseline was 51.6 years (± 13.6) and 67% were male. The patient-reported duration of disease at baseline was 5.9 ± 8.1 years. Baseline median urine albumin:creatinine ratio (UACR) was 0.4 g/g [interquartile range (IQR) 0.1-0.8] and mean eGFR was 52.5 ± 22.4 ml/min/1.73 m2. Over a follow-up of 6.5 years, 64 (15.2%) patients experienced a >40% eGFR decline, 3 (0.7%) reached eGFR <15 ml/min/1.73 m2, 53 (12.6%) initiated kidney replacement therapy and 28% of the patients experienced the CKE. Albuminuria, with reference to <0.1 g/g, was most associated with CKE. Hazard ratios (HRs) at UACRs of 0.1-0.6 g/g, 0.6-1.4 g/g, 1.4-2.2 g/g and >2.2 g/g were 2.03 [95% confidence interval (CI) 1.02-4.05], 3.8 (95% CI 1.92-7.5), 5.64 (95% CI 2.58-12.33) and 5.02 (95% CI 2.29-11-03), respectively. Regarding MACE, the presence of diabetes [HR 2.53 (95% CI 1.11-5.78)] was the most strongly associated factor, whereas UACR and eGFR did not show significant associations. Conclusion: In the GCKD IgAN subcohort, more than every fourth patient experienced a CKE event within 6.5 years. Our findings support the use of albuminuria as a surrogate to assess the risk of poor kidney outcomes.

Generation of a conditional cellular senescence model using proximal tubule cells and fibroblasts from human kidneys.

Emerging evidence highlights cellular senescence's pivotal role in chronic kidney disease (CKD). Proximal tubule epithelial cells (PTECs) and fibroblasts are major players in CKD and serve as cellular sources of senescence. The generation of a conditionally immortalized human kidney cell model would allow to better understand the specific mechanisms and factors associated with cellular senescence in a controlled setting, devoid of potential confounding factors such as age and comorbidities. In addition, the availability of human kidney cell lines for preclinical research is sparse and most cell lines do not reflect their in vivo counterparts due to their altered behavior as immortalized cancer-like cells. In this study, PTECs and fibroblasts from human kidneys were isolated and transduced with doxycycline-inducible simian virus 40 large T antigen (SV40LT) vector. By comparing their gene expression with single-cell RNA sequencing data from human kidneys, the newly produced human kidney cell lines demonstrated significant resemblances to their in vivo counterparts. As predicted, PTECs showed functional activity and fibroblasts responded to injury with fibrosis. Withdrawal of the immortalizing factor doxycycline led to p21+ cell-cycle arrest and the key hallmarks of senescence. The obtained senescence gene set largely overlapped between both cell lines and with the previously published SenMayo set of senescence-associated genes. Furthermore, crosstalk experiments showed that senescent PTECs can cause a profibrotic response in fibroblasts by paracrine actions. In 76 human kidney sections, the number of p21+ cells correlated with the degree of fibrosis, age and reduced glomerular filtration, validating the role of senescence in CKD. In conclusion, we provide a novel cellular ex vivo model to study kidney senescence which can serve as a platform for large scale compounds testing.

Long-term outcomes of adults with FSGS in the German Chronic Kidney Disease cohort.

Background: Focal segmental glomerulosclerosis (FSGS) can lead to kidney failure in adults. This study examines the progression of FSGS in the German Chronic Kidney Disease (GCKD) cohort. Methods: The GCKD study (N = 5217), a prospective cohort, included 159 patients with biopsy-confirmed FSGS recruited from 2010 to 2012. Baseline was defined as the first study visit. Adjudicated endpoints included a composite kidney endpoint (CKE), including an estimated glomerular filtration rate (eGFR) decrease >40%, eGFR <15 ml/min/1.73 m2 or initiation of kidney replacement therapy and combined major adverse cardiovascular events (MACE), including non-fatal myocardial infarction or stroke and all-cause mortality. Associations between baseline demographics, laboratory data, comorbidity and CKE and MACE were analysed using the Cox proportional hazards regression model. Results: The mean age at baseline was 52.1 ± 13.6 years, with a disease duration of 4.72 years (quartile 1: 1; quartile 3: 6) before joining the study. The median urinary albumin:creatinine ratio (UACR) at baseline was 0.7 g/g (IQR 0.1;1.8), while mean eGFR was 55.8 ± 23 ml/min/1.73 m2. Based on clinical and pathological features, 69 (43.4%) patients were categorized as primary FSGS, 55 (34.6%) as secondary FSGS and 35 (22%) as indeterminate. Over a follow-up of 6.5 years, 44 patients reached the composite kidney endpoint and 16 individuals had at least one MACE. UACR ≥0.7 g/g was strongly associated with both the composite kidney endpoint {hazard ratio [HR] 5.27 [95% confidence interval (CI) 2.4-11.5]} and MACE [HR 3.37 (95% CI 1.05-10.82)] compared with <0.7 g/g, whereas a higher eGFR at baseline (per 10 ml/min) was protective for both endpoints [HR 0.8 (95% CI 0.68-0.95) and HR 0.63 (95% CI 0.46-0.88), respectively]. Patients with secondary FSGS experienced a greater rate of eGFR decline than patients with primary FSGS. Conclusions: Lower eGFR and higher albuminuria are key risk factors for kidney disease progression and cardiovascular events in patients with FSGS.

Senescence of endothelial cells promotes phenotypic changes in adventitial fibroblasts: possible implications for vascular aging.

Aging is the most important risk factor for the development of cardiovascular diseases. Senescent cells release plethora of factors commonly known as the senescence-associated secretory phenotype, which can modulate the normal function of the vascular wall. It is currently not well understood if and how endothelial cell senescence can affect adventitial niche. The aim of this study was to characterize oxidative stress-induced endothelial cells senescence and identify their paracrine effects on the primary cell type of the adventitia, the fibroblasts. Human aortic endothelial cells (HAEC) were treated with hydrogen peroxide to induce premature senescence. Mass spectrometry analysis identified several proteomic changes in senescent HAEC with top upregulated secretory protein growth differentiation factor 15 (GDF-15). Treatment of the human adventitial fibroblast cell line (hAdv cells) with conditioned medium (CM) from senescent HAEC resulted in alterations in the proteome of hAdv cells identified in mass spectrometry analysis. Majority of differentially expressed proteins in hAdv cells treated with CM from senescent HAEC were involved in the uptake and metabolism of lipoproteins, mitophagy and ferroptosis. We next analyzed if some of these changes and pathways might be regulated by GDF-15. We found that recombinant GDF-15 affected some ferroptosis-related factors (e.g. ferritin) and decreased oxidative stress in the analyzed adventitial fibroblast cell line, but it had no effect on erastin-induced cell death. Contrary, silencing of GDF-15 in hAdv cells was protective against this ferroptotic stimuli. Our findings can be of importance for potential therapeutic strategies targeting cell senescence or ferroptosis to alleviate vascular diseases.

Coronary Microvascular Dysfunction in Acute Cholestasis-Induced Liver Injury.

BACKGROUND: Previous studies have shown cardiac abnormalities in acute liver injury, suggesting a potential role in the associated high mortality. METHODS: We designed an experimental study exploring the short-term effects of acute cholestasis-induced liver injury on cardiac function and structure in a rodent bile duct ligation (BDL) model to elucidate the potential interplay. Thirty-seven male Sprague-Dawley rats were subjected to BDL surgery (n = 28) or served as sham-operated (n = 9) controls. Transthoracic echocardiography, Doppler evaluation of the left anterior descending coronary artery, and myocardial contrast echocardiography were performed at rest and during adenosine and dobutamine stress 5 days after BDL. Immunohistochemical staining of myocardial tissue samples for hypoxia and inflammation as well as serum analysis were performed. RESULTS: BDL animals exhibited acute liver injury with elevated transaminases, bilirubin, and total circulating bile acids (TBA) 5 days after BDL (TBA control: 0.81 ± 2.54 µmol/L vs. BDL: 127.52 ± 57.03 µmol/L; p < 0.001). Concurrently, cardiac function was significantly impaired, characterized by reduced cardiac output (CO) and global longitudinal strain (GLS) in the echocardiography at rest and under pharmacological stress (CO rest control: 120.6 ± 24.3 mL/min vs. BDL 102.5 ± 16.6 mL/min, p = 0.041; GLS rest control: -24.05 ± 3.8% vs. BDL: -18.5 ± 5.1%, p = 0.01). Myocardial perfusion analysis revealed a reduced myocardial blood flow at rest and a decreased coronary flow velocity reserve (CFVR) under dobutamine stress in the BDL animals (CFVR control: 2.1 ± 0.6 vs. BDL: 1.7 ± 0.5 p = 0.047). Immunofluorescence staining indicated myocardial hypoxia and increased neutrophil infiltration. CONCLUSIONS: In summary, acute cholestasis-induced liver injury can lead to impaired cardiac function mediated by coronary microvascular dysfunction, suggesting that major adverse cardiac events may contribute to the mortality of acute liver failure. This may be due to endothelial dysfunction and direct bile acid signaling.

Harnessing Artificial Intelligence for Enhanced Renal Analysis: Automated Detection of Hydronephrosis and Precise Kidney Segmentation.

Background and objective: Hydronephrosis is essential in the diagnosis of renal colic. We automated the detection of hydronephrosis from ultrasound images to standardize the therapy and reduce the misdiagnosis of renal colic. Methods: Anonymously collected ultrasound images of human kidneys, both normal and hydronephrotic, were preprocessed for neural networks. Six "state of the art" models were trained and cross-validated for the detection of hydronephrosis, and two convolutional networks were used for kidney segmentation. In the testing phase, performance metrics included true positives, true negatives, false positives, false negatives, accuracy, and F1 score, while the evaluation of the segmentation task involved accuracy, precision, dice, jaccard, recall, and ASSD. Key findings and limitations: A total of 523 sonographic kidney images (423 nonhydronephrotic and 100 hydronephrotic) were collected from three different ultrasound devices. After training on this dataset, all models were used to evaluate 200 new ultrasound kidney images (142 nonhydronephrotic and 58 hydronephrotic kidneys). The highest validation accuracy (98.5%) was achieved by the AlexNet model (GoogLeNet 97%, AlexNet_v2 96%, ResNet50 96%, ResNet101 97.5%, and ResNet152 95%). The deeplabv3_resnet50 and deeplabv3_resnet101 reached a dice coefficient of 94.74% and 94.48%, respectively, on the task of automated kidney segmentation. The study is limited by analyzing only hydronephrosis, but this specific focus enabled high detection accuracy. Conclusions and clinical implications: We show that our automated ultrasound deep learning model can be trained and used to interpret and segmentate ultrasound images from different sources with high accuracy. This method will serve as an automated tool in the diagnostic algorithm of acute renal failure in the future. Patient summary: Hydronephrosis is crucial in the diagnosis of renal colic. Recent advances in artificial intelligence allow automated detection of hydronephrosis in ultrasound images with high accuracy. These methods will help standardize the diagnosis and treatment renal colic.

Lineage tracing reveals transient phenotypic adaptation of tubular cells during acute kidney injury.

Tubular injury is the hallmark of acute kidney injury (AKI) with a tremendous impact on patients and health-care systems. During injury, any differentiated proximal tubular cell (PT) may transition into a specific injured phenotype, so-called "scattered tubular cell" (STC)-phenotype. To understand the fate of this specific phenotype, we generated transgenic mice allowing inducible, reversible, and irreversible tagging of these cells in a murine AKI model, the unilateral ischemia-reperfusion injury (IRI). For lineage tracing, we analyzed the kidneys using single-cell profiling during disease development at various time points. Labeled cells, which we defined by established endogenous markers, already appeared 8 h after injury and showed a distinct expression set of genes. We show that STCs re-differentiate back into fully differentiated PTs upon the resolution of the injury. In summary, we show the dynamics of the phenotypic transition of PTs during injury, revealing a reversible transcriptional program as an adaptive response during disease.

Comparison of three creatinine-based equations to predict adverse outcome in a cardiovascular high-risk cohort: an investigation using the SPRINT research materials.

Background: Novel creatinine-based equations have recently been proposed but their predictive performance for cardiovascular outcomes in participants at high cardiovascular risk in comparison to the established CKD-EPI 2009 equation is unknown. Method: In 9361 participants from the United States included in the randomized controlled SPRINT trial, we calculated baseline estimated glomerular filtration rate (eGFR) using the CKD-EPI 2009, CKD-EPI 2021, and EKFC equations and compared their predictive value of cardiovascular events. The statistical metric used is the net reclassification improvement (NRI) presented separately for those with and those without events. Results: During a mean follow-up of 3.1 ± 0.9 years, the primary endpoint occurred in 559 participants (6.0%). When using the CKD-EPI 2009, the CKD-EPI 2021, and the EKFC equations, the prevalence of CKD (eGFR <60 ml/min/1.73 m2 or >60 ml/min/1.73 m2 with an ACR ≥30 mg/g) was 37% vs. 35.3% (P = 0.02) vs. 46.4% (P < 0.001), respectively. The corresponding mean eGFR was 72.5 ± 20.1 ml/min/1.73 m2 vs. 73.2 ± 19.4 ml/min/1.73 m2 (P < 0.001) vs. 64.6 ± 17.4 ml/min/1.73 m2 (P < 0.001). Neither reclassification according to the CKD-EPI 2021 equation [CKD-EPI 2021 vs. CKD-EPI 2009: NRIevents: -9.5% (95% confidence interval (CI) -13.0% to -5.9%); NRInonevents: 4.8% (95% CI 3.9% to 5.7%)], nor reclassification according to the EKFC equation allowed better prediction of cardiovascular events compared to the CKD-EPI 2009 equation (EKFC vs. CKD-EPI 2009: NRIevents: 31.2% (95% CI 27.5% to 35.0%); NRInonevents: -31.1% (95% CI -32.1% to -30.1%)). Conclusion: Substituting the CKD-EPI 2009 with the CKD-EPI 2021 or the EKFC equation for calculation of eGFR in participants with high cardiovascular risk without diabetes changed the prevalence of CKD but was not associated with improved risk prediction of cardiovascular events for both those with and without the event.

The role of desmoglein-2 in kidney disease.

Desmosomes are multi-protein cell-cell adhesion structures supporting cell stability and mechanical stress resilience of tissues, best described in skin and heart. The kidney is exposed to various mechanical stimuli and stress, yet little is known about kidney desmosomes. In healthy kidneys, we found desmosomal proteins located at the apical-junctional complex in tubular epithelial cells. In four different animal models and patient biopsies with various kidney diseases, desmosomal components were significantly upregulated and partly miss-localized outside of the apical-junctional complexes along the whole lateral tubular epithelial cell membrane. The most upregulated component was desmoglein-2 (Dsg2). Mice with constitutive tubular epithelial cell-specific deletion of Dsg2 developed normally, and other desmosomal components were not altered in these mice. When challenged with different types of tubular epithelial cell injury (unilateral ureteral obstruction, ischemia-reperfusion, and 2,8-dihydroxyadenine crystal nephropathy), we found increased tubular epithelial cell apoptosis, proliferation, tubular atrophy, and inflammation compared to wild-type mice in all models and time points. In vitro, silencing DSG2 via siRNA weakened cell-cell adhesion in HK-2 cells and increased cell death. Thus, our data show a prominent upregulation of desmosomal components in tubular cells across species and diseases and suggest a protective role of Dsg2 against various injurious stimuli.

Detection of PatIent-Level distances from single cell genomics and pathomics data with Optimal Transport (PILOT).

Although clinical applications represent the next challenge in single-cell genomics and digital pathology, we still lack computational methods to analyze single-cell or pathomics data to find sample-level trajectories or clusters associated with diseases. This remains challenging as single-cell/pathomics data are multi-scale, i.e., a sample is represented by clusters of cells/structures, and samples cannot be easily compared with each other. Here we propose PatIent Level analysis with Optimal Transport (PILOT). PILOT uses optimal transport to compute the Wasserstein distance between two individual single-cell samples. This allows us to perform unsupervised analysis at the sample level and uncover trajectories or cellular clusters associated with disease progression. We evaluate PILOT and competing approaches in single-cell genomics or pathomics studies involving various human diseases with up to 600 samples/patients and millions of cells or tissue structures. Our results demonstrate that PILOT detects disease-associated samples from large and complex single-cell or pathomics data. Moreover, PILOT provides a statistical approach to find changes in cell populations, gene expression, and tissue structures related to the trajectories or clusters supporting interpretation of predictions.

Mechanisms of kidney fibrosis and routes towards therapy.

Kidney fibrosis is the final common pathway of virtually all chronic kidney diseases (CKDs) and is therefore considered to be a promising therapeutic target for these conditions. However, despite great progress in recent years, no targeted antifibrotic therapies for the kidney have been approved, likely because the complex mechanisms that initiate and drive fibrosis are not yet completely understood. Recent single-cell genomic approaches have allowed novel insights into kidney fibrosis mechanisms in mouse and human, particularly the heterogeneity and differentiation processes of myofibroblasts, the role of injured epithelial cells and immune cells, and their crosstalk mechanisms. In this review we summarize the key mechanisms that drive kidney fibrosis, including recent advances in understanding the mechanisms, as well as potential routes for developing novel targeted antifibrotic therapeutics.

The challenges of research data management in cardiovascular science: a DGK and DZHK position paper-executive summary.

The sharing and documentation of cardiovascular research data are essential for efficient use and reuse of data, thereby aiding scientific transparency, accelerating the progress of cardiovascular research and healthcare, and contributing to the reproducibility of research results. However, challenges remain. This position paper, written on behalf of and approved by the German Cardiac Society and German Centre for Cardiovascular Research, summarizes our current understanding of the challenges in cardiovascular research data management (RDM). These challenges include lack of time, awareness, incentives, and funding for implementing effective RDM; lack of standardization in RDM processes; a need to better identify meaningful and actionable data among the increasing volume and complexity of data being acquired; and a lack of understanding of the legal aspects of data sharing. While several tools exist to increase the degree to which data are findable, accessible, interoperable, and reusable (FAIR), more work is needed to lower the threshold for effective RDM not just in cardiovascular research but in all biomedical research, with data sharing and reuse being factored in at every stage of the scientific process. A culture of open science with FAIR research data should be fostered through education and training of early-career and established research professionals. Ultimately, FAIR RDM requires permanent, long-term effort at all levels. If outcomes can be shown to be superior and to promote better (and better value) science, modern RDM will make a positive difference to cardiovascular science and practice. The full position paper is available in the supplementary materials.

Matrix Metalloproteinase-7 in Urinary Extracellular Vesicles Identifies Rapid Disease Progression in Autosomal Dominant Polycystic Kidney Disease.

SIGNIFICANCE STATEMENT: There is an unmet need for biomarkers of disease progression in autosomal dominant polycystic kidney disease (ADPKD). This study investigated urinary extracellular vesicles (uEVs) as a source of such biomarkers. Proteomic analysis of uEVs identified matrix metalloproteinase 7 (MMP-7) as a biomarker predictive of rapid disease progression. In validation studies, MMP-7 was predictive in uEVs but not in whole urine, possibly because uEVs are primarily secreted by tubular epithelial cells. Indeed, single-nucleus RNA sequencing showed that MMP-7 was especially increased in proximal tubule and thick ascending limb cells, which were further characterized by a profibrotic phenotype. Together, these data suggest that MMP-7 is a biologically plausible and promising uEV biomarker for rapid disease progression in ADPKD. BACKGROUND: In ADPKD, there is an unmet need for early markers of rapid disease progression to facilitate counseling and selection for kidney-protective therapy. Our aim was to identify markers for rapid disease progression in uEVs. METHODS: Six paired case-control groups ( n =10-59/group) of cases with rapid disease progression and controls with stable disease were formed from two independent ADPKD cohorts, with matching by age, sex, total kidney volume, and genetic variant. Candidate uEV biomarkers were identified by mass spectrometry and further analyzed using immunoblotting and an ELISA. Single-nucleus RNA sequencing of healthy and ADPKD tissue was used to identify the cellular origin of the uEV biomarker. RESULTS: In the discovery proteomics experiments, the protein abundance of MMP-7 was significantly higher in uEVs of patients with rapid disease progression compared with stable disease. In the validation groups, a significant >2-fold increase in uEV-MMP-7 in patients with rapid disease progression was confirmed using immunoblotting. By contrast, no significant difference in MMP-7 was found in whole urine using ELISA. Compared with healthy kidney tissue, ADPKD tissue had significantly higher MMP-7 expression in proximal tubule and thick ascending limb cells with a profibrotic phenotype. CONCLUSIONS: Among patients with ADPKD, rapid disease progressors have higher uEV-associated MMP-7. Our findings also suggest that MMP-7 is a biologically plausible biomarker for more rapid disease progression.

Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms.

The role of hematopoietic Hedgehog signaling in myeloproliferative neoplasms (MPNs) remains incompletely understood despite data suggesting that Hedgehog (Hh) pathway inhibitors have therapeutic activity in patients. We aim to systematically interrogate the role of canonical vs. non-canonical Hh signaling in MPNs. We show that Gli1 protein levels in patient peripheral blood mononuclear cells (PBMCs) mark fibrotic progression and that, in murine MPN models, absence of hematopoietic Gli1, but not Gli2 or Smo, significantly reduces MPN phenotype and fibrosis, indicating that GLI1 in the MPN clone can be activated in a non-canonical fashion. Additionally, we establish that hematopoietic Gli1 has a significant effect on stromal cells, mediated through a druggable MIF-CD74 axis. These data highlight the complex interplay between alterations in the MPN clone and activation of stromal cells and indicate that Gli1 represents a promising therapeutic target in MPNs, particularly that Hh signaling is dispensable for normal hematopoiesis.

Multiomic profiling of transplant glomerulopathy reveals a novel T-cell dominant subclass.

Kidney transplant (KTx) biopsies showing transplant glomerulopathy (TG) (glomerular basement membrane double contours (cg) > 0) and microvascular inflammation (MVI) in the absence of C4d staining and donor-specific antibodies (DSAs) do not fulfill the criteria for chronic active antibody-mediated rejection (CA-AMR) diagnosis and do not fit into any other Banff category. To investigate this, we initiated a multicenter intercontinental study encompassing 36 cases, comparing the immunomic and transcriptomic profiles of 14 KTx biopsies classified as cg+MVI DSA-/C4d- with 22 classified as CA-AMR DSA+/C4d+ through novel transcriptomic analysis using the NanoString Banff-Human Organ Transplant (B-HOT) panel and subsequent orthogonal subset analysis using two innovative 5-marker multiplex immunofluorescent panels. Nineteen genes were differentially expressed between the two study groups. Samples diagnosed with CA-AMR DSA+/C4d+ showed a higher glomerular abundance of natural killer cells and higher transcriptomic cell type scores for macrophages in an environment characterized by increased expression of complement-related genes (i.e., C5AR1) and higher activity of angiogenesis, interstitial fibrosis tubular atrophy, CA-AMR, and DSA-related pathways when compared to samples diagnosed with cg+MVI DSA-/C4d-. Samples diagnosed with cg+MVI DSA-/C4d- displayed a higher glomerular abundance and activity of T cells (CD3+, CD3+CD8+, and CD3+CD8-). Thus, we show that using novel multiomic techniques, KTx biopsies with cg+MVI DSA-/C4d- have a prominent T-cell presence and activity, putting forward the possibility that these represent a more T-cell dominant phenotype.