Application of spatial-omics to the classification of kidney biopsy samples in transplantation.

Improvement of long-term outcomes through targeted treatment is a primary concern in kidney transplant medicine. Currently, the validation of a rejection diagnosis and subsequent treatment depends on the histological assessment of allograft biopsy samples, according to the Banff classification system. However, the lack of (early) disease-specific tissue markers hinders accurate diagnosis and thus timely intervention. This challenge mainly results from an incomplete understanding of the pathophysiological processes underlying late allograft failure. Integration of large-scale multimodal approaches for investigating allograft biopsy samples might offer new insights into this pathophysiology, which are necessary for the identification of novel therapeutic targets and the development of tailored immunotherapeutic interventions. Several omics technologies - including transcriptomic, proteomic, lipidomic and metabolomic tools (and multimodal data analysis strategies) - can be applied to allograft biopsy investigation. However, despite their successful application in research settings and their potential clinical value, several barriers limit the broad implementation of many of these tools into clinical practice. Among spatial-omics technologies, mass spectrometry imaging, which is under-represented in the transplant field, has the potential to enable multi-omics investigations that might expand the insights gained with current clinical analysis technologies.

Spatial metabolomics in tissue injury and regeneration.

Tissue homeostasis is intricately linked to cellular metabolism and metabolite exchange within the tissue microenvironment. The orchestration of adaptive cellular responses during injury and repair depends critically upon metabolic adaptation. This adaptation, in turn, shapes cell fate decisions required for the restoration of tissue homeostasis. Understanding the nuances of metabolic processes within the tissue context and comprehending the intricate communication between cells is therefore imperative for unraveling the complexity of tissue homeostasis and the processes of injury and repair. In this review, we focus on mass spectrometry imaging as an advanced platform with the potential to provide such comprehensive insights into the metabolic instruction governing tissue function. Recent advances in this technology allow to decipher the intricate metabolic networks that determine cellular behavior in the context of tissue resilience, injury, and repair. These insights not only advance our fundamental understanding of tissue biology but also hold implications for therapeutic interventions by targeting metabolic pathways critical for maintaining tissue homeostasis.

Validating human induced pluripotent stem cell-specific quality control tests for the release of an intermediate drug product in a Good Manufacturing Practice quality system.

One of the challenges in Good Manufacturing Practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) production is the validation of quality control (QC) tests specific for hiPSCs, which are required for GMP batch release. This study presents a comprehensive description of the validation process for hiPSC-specific GMP-compliant QC assays; more specifically, the validation of assays to assess the potential presence of residual episomal vectors (REVs), the expression of markers of the undifferentiated state and the directed differentiation potential of hiPSCs. Critical aspects and specific acceptance criteria were formulated in a validation plan prior to assay validation. Assay specificity, sensitivity and reproducibility were tested, and the equipment used for each assay was subjected to performance qualification. A minimum input of 20 000 cells (120 ng of genomic DNA) was defined for accurate determination of the presence of REVs. Furthermore, since vector loss in hiPSC lines is a passage-dependent process, we advocate screening for REVs between passages eight and 10, as testing at earlier passages might lead to unnecessary rejection of hiPSC lines. The cutoff value for assessment of markers of the undifferentiated state was set to the expression of at least three individual markers on at least 75% of the cells. When multi-color flow cytometry panels are used, a fluorescence minus one control is advised to ensure the control for fluorescent spread. For the assay to assess the directed differentiation potential, the detection limit was set to two of three positive lineage-specific markers for each of the three individual germ layers. All of our assays proved to be reproducible and specific. Our data demonstrate that our implemented analytical procedures are suitable as QC assays for the batch release of GMP-compliant hiPSCs.

Long-term renal and cardiovascular risks of tacrolimus in patients with lupus nephritis.

BACKGROUND AND HYPOTHESIS: Despite continuous advancement, treatment of lupus nephritis (LN) remains challenging. Recent guidelines now include a regimen incorporating tacrolimus as a first-line treatment option. Even though tacrolimus is effective in combination with mycophenolate and corticosteroids, concerns remain regarding long-term use, given its association with increased cardiovascular risks including nephrotoxicity, hypertension, dyslipidemia and hyperglycemia in kidney transplant recipients. However, in LN, long-term evaluations and head-to-head comparisons are lacking and thus the safety profile remains ill-defined. We hypothesized that chronic toxicity also occurs in LN patients. Therefore, this study aimed to assess long-term cardiovascular and renal outcomes of tacrolimus in LN patients. METHODS: This observational cohort study examined adult LN patients treated with tacrolimus, assessing renal outcomes, hypertension, diabetes, dyslipidemia, cardiovascular events and the Framingham risk score. The results were compared to a control group of CNI-naïve LN patients. RESULTS: Of the 219 LN patients in this study, 43 (19.6%) had tacrolimus exposure. Over a median follow-up of 7.1 years, tacrolimus use was associated with significant kidney function decline (6.8 ml/min/1.73m2, versus 0.8 in the control group). The incidence of end-stage kidney disease was similar. Cardiovascular event incidence was equally low in both groups. The 10-year risk of coronary heart disease was lower in the tacrolimus group, primarily due to age differences. HbA1c levels were higher in the tacrolimus group (37.4 mmol/mol) than in controls (33.6 mmol/mol), although the incidence of diabetes was similar. There were no differences in the occurrence of hypertension or dyslipidemia. CONCLUSIONS: Our study demonstrated that tacrolimus exposure was associated with long-term kidney function loss in LN patients. Although cardiovascular risk factors and events were similar to patients never exposed to tacrolimus, there may be an increased risk of developing diabetes. Therefore, our study supports vigilance towards renal adverse effects in LN patients treated with tacrolimus.

Disruption of memory B-cell trafficking by belimumab in patients with systemic lupus erythematosus.

OBJECTIVES: Autoreactive memory B cells contribute to chronic and progressive courses in autoimmune diseases like systemic lupus erythematosus (SLE). The efficacy of belimumab (BEL), the first approved biologic treatment for SLE and lupus nephritis (LN), is generally attributed to depletion of activated naïve B cells and inhibition of B cell activation. BEL's effect on memory B cells (MBCs) is currently unexplained. We performed an in-depth cellular and transcriptomic analysis of BEL's impact on the blood MBC compartment in patients with SLE. METHODS: A retrospective meta-analysis was conducted, pooling flow cytometry data from four randomized trials involving 1245 patients with SLE treated with intravenous BEL or placebo. Then, extensive MBC phenotyping was performed using high-sensitivity flow cytometry in patients with mild/moderate SLE and severe SLE/LN treated with subcutaneous BEL. Finally, transcriptomic characterization of surging MBCs was performed by single-cell RNA sequencing. RESULTS: In BEL-treated patients, a significant increase in circulating MBCs, in a broad range of MBC subsets, was established at week 2, gradually returning to baseline by week 52. The increase was most prominent in patients with higher SLE disease activity, serologically active patients, and patients aged ≤18 years. MBCs had a non-proliferating phenotype with a prominent decrease in activation status and downregulation of numerous migration genes. CONCLUSION: Upon BEL initiation, an increase of MBCs was firmly established. In the small cohort investigated, circulating MBCs were de-activated, non-proliferative, and demonstrated characteristics of disrupted lymphocyte trafficking, expanding on our understanding of the therapeutic mechanism of B cell-activating factor inhibition by BEL. TRIAL REGISTRATION: ClinicalTrials.gov NCT00071487, NCT00410384, NCT01632241, NCT01649765, NCT03312907, NCT03747159.

A cell-free nutrient-supplemented perfusate allows four-day ex vivo metabolic preservation of human kidneys.

The growing disparity between the demand for transplants and the available donor supply, coupled with an aging donor population and increasing prevalence of chronic diseases, highlights the urgent need for the development of platforms enabling reconditioning, repair, and regeneration of deceased donor organs. This necessitates the ability to preserve metabolically active kidneys ex vivo for days. However, current kidney normothermic machine perfusion (NMP) approaches allow metabolic preservation only for hours. Here we show that human kidneys discarded for transplantation can be preserved in a metabolically active state up to 4 days when perfused with a cell-free perfusate supplemented with TCA cycle intermediates at subnormothermia (25 °C). Using spatially resolved isotope tracing we demonstrate preserved metabolic fluxes in the kidney microenvironment up to Day 4 of perfusion. Beyond Day 4, significant changes were observed in renal cell populations through spatial lipidomics, and increases in injury markers such as LDH, NGAL and oxidized lipids. Finally, we demonstrate that perfused kidneys maintain functional parameters up to Day 4. Collectively, these findings provide evidence that this approach enables metabolic and functional preservation of human kidneys over multiple days, establishing a solid foundation for future clinical investigations.

Hypertension and diabetes, but not leptin and adiponectin, mediate the relationship between body fat and chronic kidney disease.

PURPOSE: Obesity may promote kidney damage through hemodynamic and hormonal effects. We investigated the association between body mass index (BMI), total body fat (TBF) and chronic kidney disease (CKD) and whether hypertension, diabetes, leptin and adiponectin mediated these associations. METHODS: In this cross-sectional analysis of the Netherlands Epidemiology of Obesity study, 6671 participants (45-65 y) were included. We defined CKD as eGFR <60 ml/min/1.73 m2 and/or moderately increased albuminuria. The percentage of mediation was calculated using general structural equation modeling, adjusted for potential confounding factors age, sex, smoking, ethnicity, physical activity and Dutch healthy diet index. RESULTS: At baseline mean (SD) age was 56 (6), BMI 26.3 (4.4), 44% men, and 4% had CKD. Higher BMI and TBF were associated with 1.08 (95%CI 1.05; 1.11) and 1.05-fold (95%CI 1.02; 1.08) increased odds of CKD, respectively. As adiponectin was not associated with any of the outcomes, it was not studied further as a mediating factor. The association between BMI and CKD was 8.5% (95%CI 0.5; 16.5) mediated by diabetes and 22.3% (95%CI 7.5; 37.2) by hypertension. In addition, the association between TBF and CKD was 9.6% (95%CI -0.4; 19.6) mediated by diabetes and 22.4% (95%CI 4.2; 40.6) by hypertension. We could not confirm mediation by leptin in the association between BMI and CKD (35.6% [95%CI -18.8; 90.3]), nor between TBF and CKD (59.7% [95%CI -7.1; 126.6]). CONCLUSION: Our results suggest that the relations between BMI, TBF and CKD are in part mediated by diabetes and hypertension.

Role of dietary interventions on microvascular health in South-Asian Surinamese people with type 2 diabetes in the Netherlands: A randomized controlled trial.

BACKGROUND/OBJECTIVES: We investigated whether dietary interventions, i.e. a fasting mimicking diet (FMD, Prolon®) or glycocalyx mimetic supplementation (EndocalyxTM) could stabilize microvascular function in Surinamese South-Asian patients with type 2 diabetes (SA-T2DM) in the Netherlands, a patient population more prone to develop vascular complications. SUBJECTS/METHODS: A randomized, placebo controlled, 3-arm intervention study was conducted in 56 SA-T2DM patients between 18 and 75 years old, for 3 consecutive months, with one additional follow up measurement 3 months after the last intervention. Sublingual microcirculation was assessed with SDF-imaging coupled to the GlycoCheckTM software, detecting red blood cell velocity, capillary density, static and dynamic perfused boundary region (PBR), and the overall microvascular health score (MVHS). Linear mixed models and interaction analysis were used to investigate the effects the interventions had on microvascular function. RESULTS: Despite a temporal improvement in BMI and HbA1c after FMD the major treatment effect on microvascular health was worsening for RBC-velocity independent PBRdynamic, especially at follow-up. Glycocalyx supplementation, however, reduced urinary MCP-1 presence and improved both PBRdynamic and MVHSdynamic, which persisted at follow-up. CONCLUSIONS: We showed that despite temporal beneficial changes in BMI and HbA1c after FMD, this intervention is not able to preserve microvascular endothelial health in Dutch South-Asian patients with T2DM. In contrast, glycocalyx mimetics preserves the microvascular endothelial health and reduces the inflammatory cytokine MCP-1. CLINICAL STUDY REGISTRATION: NCT03889236.

Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products.

BACKGROUND AIMS: Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. METHODS: Our standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). RESULTS: Two MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). CONCLUSIONS: We successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

Ethnic Disparities in Lipid Metabolism and Clinical Outcomes between Dutch South Asians and Dutch White Caucasians with Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) poses a higher risk for complications in South Asian individuals compared to other ethnic groups. To shed light on potential mediating factors, we investigated lipidomic changes in plasma of Dutch South Asians (DSA) and Dutch white Caucasians (DwC) with and without T2DM and explore their associations with clinical features. Using a targeted quantitative lipidomics platform, monitoring over 1000 lipids across 17 classes, along with 1H NMR based lipoprotein analysis, we studied 51 healthy participants (21 DSA, 30 DwC) and 92 T2DM patients (47 DSA, 45 DwC) from the MAGNetic resonance Assessment of VICTOza efficacy in the Regression of cardiovascular dysfunction in type 2 dIAbetes mellitus (MAGNA VICTORIA) study. This comprehensive mapping of the circulating lipidome allowed us to identify relevant lipid modules through unbiased weighted correlation network analysis, as well as disease and ethnicity related key mediatory lipids. Significant differences in lipidomic profiles, encompassing various lipid classes and species, were observed between T2DM patients and healthy controls in both the DSA and DwC populations. Our analyses revealed that healthy DSA, but not DwC, controls already exhibited a lipid profile prone to develop T2DM. Particularly, in DSA-T2DM patients, specific lipid changes correlated with clinical features, particularly diacylglycerols (DGs), showing significant associations with glycemic control and renal function. Our findings highlight an ethnic distinction in lipid modules influencing clinical outcomes in renal health. We discover distinctive ethnic disparities of the circulating lipidome and identify ethnicity-specific lipid markers. Jointly, our discoveries show great potential as personalized biomarkers for the assessment of glycemic control and renal function in DSA-T2DM individuals.

Fasting mimicking diet in diabetic mice partially preserves glomerular endothelial glycocalyx coverage, without changing the diabetic metabolic environment.

Intermittent fasting has become of interest for its possible metabolic benefits and reduction of inflammation and oxidative damage, all of which play a role in the pathophysiology of diabetic nephropathy. We tested in a streptozotocin (60 mg/kg)-induced diabetic apolipoprotein E knockout mouse model whether repeated fasting mimicking diet (FMD) prevents glomerular damage. Diabetic mice received 5 FMD cycles in 10 wk, and during cycles 1 and 5 caloric measurements were performed. After 10 wk, glomerular endothelial morphology was determined together with albuminuria, urinary heparanase-1 activity, and spatial mass spectrometry imaging to identify specific glomerular metabolic dysregulation. During FMD cycles, blood glucose levels dropped while a temporal metabolic switch was observed to increase fatty acid oxidation. Overall body weight at the end of the study was reduced together with albuminuria, although urine production was dramatically increased without affecting urinary heparanase-1 activity. Weight loss was found to be due to lean mass and water, not fat mass. Although capillary loop morphology and endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced together with the presence of UDP-glucuronic acid. Mass spectrometry imaging further revealed reduced protein catabolic breakdown products and increased oxidative stress, not different from diabetic mice. In conclusion, although FMD preserves partially glomerular endothelial glycocalyx, loss of lean mass and increased glomerular oxidative stress argue whether such diet regimes are safe in patients with diabetes.NEW & NOTEWORTHY Repeated fasting mimicking diet (FMD) partially prevents glomerular damage in a diabetic mouse model; however, although endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced in the presence of UDP-glucuronic acid. The weight loss observed was of lean mass, not fat mass, and increased glomerular oxidative stress argue whether such a diet is safe in patients with diabetes.

T-Cell Mediated Immune Rejection of Beta-2-Microglobulin Knockout Induced Pluripotent Stem Cell-Derived Kidney Organoids.

Immune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as "stealth" organoids, hold promise for clinical transplantation. To address immune rejection, we investigated the impact of genetically modifying human leukocyte antigen (HLA) class I in kidney organoids prior to transplantation. By using CRISPR-Cas9, we successfully knocked out beta-2-microglobulin (B2M), resulting in iPSCs devoid of HLA class I surface expression. In vitro, the B2M knockout protected kidney organoids derived from these iPSCs against T-cell rejection. To assess in vivo protection, unmodified (control) and B2M-/- kidney organoids were transplanted into humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs). Successful engraftment of human PBMCs was confirmed, and after 4 weeks, we observed no discernible difference in the infiltration rate, proliferation, or cytotoxicity of CD4+ and CD8+ T cells between control and B2M-/- organoids. Both groups of organoids showed compromised tissue integrity, displaying tubulitis and loss of tubule integrity. Notably, while B2M-/- organoids failed to express HLA class I on their cell surface, there was preexisting expression of HLA class II in both control and B2M-/- organoids transplanted into mice with human PBMCs. HLA class II expression was not limited to antigen-presenting cells but also evident in epithelial cells of the kidney organoid, posing an additional immunological challenge to its transplantation. Consequently, we conclude that B2M knockout alone is insufficient to protect iPSC-derived kidney organoids from T-cell-mediated immune rejection. Additionally, our findings suggest that modulating HLA class II signaling will be necessary to prevent rejection following transplantation.

Using an artificial intelligence tool incorporating natural language processing to identify patients with a diagnosis of ANCA-associated vasculitis in electronic health records.

BACKGROUND: Because anti-neutrophil cytoplasmatic antibody (ANCA)-associated vasculitis (AAV) is a rare, life-threatening, auto-immune disease, conducting research is difficult but essential. A long-lasting challenge is to identify rare AAV patients within the electronic-health-record (EHR)-system to facilitate real-world research. Artificial intelligence (AI)-search tools using natural language processing (NLP) for text-mining are increasingly postulated as a solution. METHODS: We employed an AI-tool that combined text-mining with NLP-based exclusion, to accurately identify rare AAV patients within large EHR-systems (>2.000.000 records). We developed an identification method in an academic center with an established AAV-training set (n = 203) and validated the method in a non-academic center with an AAV-validation set (n = 84). To assess accuracy anonymized patient records were manually reviewed. RESULTS: Based on an iterative process, a text-mining search was developed on disease description, laboratory measurements, medication and specialisms. In the training center, 608 patients were identified with a sensitivity of 97.0 % (95%CI [93.7, 98.9]) and positive predictive value (PPV) of 56.9 % (95%CI [52.9, 60.1]). NLP-based exclusion resulted in 444 patients increasing PPV to 77.9 % (95%CI [73.7, 81.7]) while sensitivity remained 96.3 % (95%CI [93.8, 98.0]). In the validation center, text-mining identified 333 patients (sensitivity 97.6 % (95%CI [91.6, 99.7]), PPV 58.2 % (95%CI [52.8, 63.6])) and NLP-based exclusion resulted in 223 patients, increasing PPV to 86.1 % (95%CI [80.9, 90.4]) with 98.0 % (95%CI [94.9, 99.4]) sensitivity. Our identification method outperformed ICD-10-coding predominantly in identifying MPO+ and organ-limited AAV patients. CONCLUSIONS: Our study highlights the advantages of implementing AI, notably NLP, to accurately identify rare AAV patients within large EHR-systems and demonstrates the applicability and transportability. Therefore, this method can reduce efforts to identify AAV patients and accelerate real-world research, while avoiding bias by ICD-10-coding.

Voclosporin and the Antiviral Effect Against SARS-CoV-2 in Immunocompromised Kidney Patients.

Introduction: Immunocompromised kidney patients are at increased risk of prolonged SARS-CoV-2 infection and related complications. Preclinical evidence demonstrates a more potent inhibitory effect of voclosporin on SARS-CoV-2 replication than tacrolimus in vitro. We investigated the potential antiviral effects of voclosporin on SARS-CoV-2 in immunocompromised patients. Methods: First, we conducted a prospective, randomized, open-label, proof-of-concept study in 20 kidney transplant recipients (KTRs) on tacrolimus-based immunosuppression who contracted mild to moderate SARS-CoV-2 infection. Patients were randomized to continue tacrolimus or switch to voclosporin. Second, we performed a post hoc analysis on SARS-CoV-2 infections in 216 patients with lupus nephritis (LN) on standard immunosuppression who were randomly exposed to voclosporin or placebo as part of a clinical trial that was conducted during the worldwide COVID-19 pandemic. Results: The primary end point was clearance of SARS-CoV-2 viral load and that did not differ between voclosporin-treated KTRs (median 12 days, interquartile range [IQR] 8-28) and tacrolimus-treated KTRs (median 12 days, IQR 4-16) nor was there a difference in clinical recovery. Pharmacokinetic analyses demonstrated that, when voclosporin trough levels were on-target, SARS-CoV-2 viral load dropped significantly more (ΔCt 7.7 [3.4-10.7]) compared to tacrolimus-treated KTRs (ΔCt 2.7 [2.0-4.3]; P = 0.035). In voclosporin-exposed patients with LN, SARS-CoV-2 infection was detected in 6% (7/116) compared to 12% (12/100) in placebo-exposed patients (relative risk [RR] 1.4 [0.97-2.06]). Notably, no voclosporin-exposed patients with LN died from severe SARS-CoV-2 infection compared to 3% (3/100) in placebo-exposed patients (RR 2.2 [1.90-2.54]). Conclusion: This proof-of-concept study shows a potential positive risk-benefit profile for voclosporin in immunocompromised patients with SARS-CoV-2 infection. These results warrant further investigations on voclosporin to establish an equipoise between infection and maintenance immunosuppression.

Endothelial to mesenchymal transition in kidney fibrosis.

Fibrotic disease are characterized by the uncontrolled accumulation of extracellular matrix (ECM) components leading to disruption of tissue homeostasis. Myofibroblasts as main ECM-producing cells can originate from various differentiated cell types after injury. Particularly, the process of endothelial-to-mesenchymal transition (endMT), describing phenotypic shifts of endothelial cells (ECs) to adopt a fully mesenchymal identity, may contribute to the pool of myofibroblasts in fibrosis, while leading to capillary rarefaction and exacerbation of tissue hypoxia. In renal disease, incomplete recovery from acute kidney injury (AKI) and the ensuing fibrotic reaction stand out as major contributors to chronic kidney disease (CKD) development. While the focus has largely been on impaired tubular epithelial repair as a potential fibrosis-driving mechanism, alterations in the renal microcirculation post-AKI, and in particular endMT as a maladaptive response, could hold equal significance. Dysfunctional interplays among various cell types in the kidney microenvironment can instigate endMT. Transforming growth factor beta (TGF-ß) signaling, with its downstream activation of canonical/Smad-mediated and non-canonical pathways, has been identified as primary driver of this process. However, non-TGF-ß-mediated pathways involving inflammatory agents and metabolic shifts in intercellular communication within the tissue microenvironment can also trigger endMT. These harmful, maladaptive cell-cell interactions and signaling pathways offer potential targets for therapeutic intervention to impede endMT and decelerate fibrogenesis such as in AKI-CKD progression. Presently, partial reduction of TGF-ß signaling using anti-diabetic drugs or statins may hold therapeutic potential in renal context. Nevertheless, further investigation is warranted to validate underlying mechanisms and assess positive effects within a clinical framework.

Heart and kidney organoids maintain organ-specific function in a microfluidic system.

Heart and kidney communicate with one another in an interdependent relationship and they influence each other's behavior reciprocally, as pathological changes in one organ can damage the other. Although independent human in vitro models for heart and kidney exist, they do not capture their dynamic crosstalk. We have developed a microfluidic system which can be used to study heart and kidney interaction in vitro. Cardiac microtissues (cMTs) and kidney organoids (kOs) derived from human induced pluripotent stem cells (hiPSCs) were generated and loaded into two separated communicating chambers of a perfusion chip. Static culture conditions were compared with dynamic culture under unidirectional flow. Tissue viability was maintained for minimally 72 h under both conditions, as indicated by the presence of sarcomeric structures coupled with beating activity in cMTs and the presence of nephron structures and albumin uptake in kOs. We concluded that this system enables the study of human cardiac and kidney organoid interaction in vitro while controlling parameters like fluidic flow speed and direction. Together, this "cardiorenal-unit" provides a new in vitro model to study the cardiorenal axis and it may be further developed to investigate diseases involving both two organs and their potential treatments.

Glycosaminoglycans and fucoidan have a protective effect on experimental glomerulonephritis.

Background: The glomerular endothelial glycocalyx is degraded during inflammation. The glycocalyx plays a pivotal role in endothelial function and is involved in many processes including binding of chemokines and cytokines, leukocyte trafficking, and preventing proteinuria. HS-based therapeutics are a promising novel class of anti-inflammatory drugs to restore a compromised endothelial glycocalyx under inflammatory conditions. Recently, we demonstrated that treatment with HS extracted from unstimulated glomerular endothelial glycocalyx (unstimulated HSglx) reduced albuminuria during anti-GBM induced glomerulonephritis. Since endothelial HS domains are distinct in unstimulated versus inflammatory conditions, we hypothesized that 1) unstimulated HSglx, 2) LPS-stimulated HSglx, 3) the HS-mimetic fucoidan and 4) the glycosaminoglycan preparation sulodexide, which is a mixture of low molecular weight heparin and dermatan sulfate, might have different beneficial effects in experimental glomerulonephritis. Methods: The effect of unstimulated HSglx, LPS HSglx, Laminaria japonica fucoidan, or sulodexide on experimental glomerulonephritis was tested in LPS-induced glomerulonephritis in mice. Analyses included urinary albumin creatinine measurement, cytokine expression in plasma and renal cortex, and renal influx of immune cells determined by flow cytometry and immunofluorescence staining. Furthermore, the observed in vivo effects were evaluated in cultured glomerular endothelial cells and peripheral blood mononuclear cells by measuring cytokine and ICAM-1 expression levels. The ability of the compounds to inhibit heparanase activity was assessed in a heparanase activity assay. Results: Treatment of mice with LPS HSglx or sulodexide near-significantly attenuated LPS-induced proteinuria. All treatments reduced plasma MCP-1 levels, whereas only fucoidan reduced IL-6 and IL-10 plasma levels. Moreover, all treatments reversed cortical ICAM-1 mRNA expression and both fucoidan and sulodexide reversed cortical IL-6 and nephrin mRNA expression. Sulodexide decreased renal influx of CD45+ immune cells whereas renal influx of macrophages and granulocytes remained unaltered for all treatments. Although all compounds inhibited HPSE activity, fucoidan and sulodexide were the most potent inhibitors. Notably, fucoidan and sulodexide decreased LPS-induced mRNA expression of ICAM-1 and IL-6 by cultured glomerular endothelial cells. Conclusion: Our data show a potentially protective effect of glycosaminoglycans and fucoidan in experimental glomerulonephritis. Future research should be aimed at the further identification of defined HS structures that have therapeutic potential in the treatment of glomerular diseases.