Biology subjects

Kers, J.

Publications and source records attributed to Kers, J..

4 recordsLinked to original sources

bioRxiv2024

Highly Repeatable Tissue Proteomics for Kidney Transplant Pathology: Technical and Biological Validation of Protein Analysis using LC-MS/MS

Accurate pathological assessment of tissue samples is key for diagnosis and optimal treatment decisions. Traditional pathology techniques suffer from subjectivity resulting in inter-observer variability, and limitations in identifying subtle molecular changes. Omics approaches provide both molecular evidence and unbiased classification, which increases the quality and reliability of final tissue assessment. Here, we focus on mass spectrometry (MS)-based proteomics as a method to reveal biopsy tissue differences. For MS data to be useful, molecular information collected from formalin fixed paraffin embedding (FFPE) biopsy tissues needs to be consistent and quantitatively accurate and contain sufficient clinically relevant molecular information. Therefore, we developed an MS-based workflow and assessed the analytical repeatability on 36 kidney biopsies, ultimately analysing molecular differences and similarities of over 5000 proteins per biopsy. Additional 301 transplant biopsies were analysed to understand other physical parameters including effects of tissue size, standing time in autosampler, and the effect on clinical validation. MS data were acquired using Data-Independent Acquisition (DIA) which provides gigabytes of data per sample in the form of high proteome (and genome) representation, at exquisitely high quantitative accuracy. The FFPE-based method optimised here provides a coefficient of variation below 20%, analysing more than 5000 proteins per sample in parallel. We also observed that tissue thickness does affect the outcome of the data quality: 5 m sections show more variation in the same sample than 10 m sections. Notably, our data reveals an excellent agreement for the relative abundance of known protein biomarkers with kidney transplantation lesion scores used in clinical pathological diagnostics. The findings presented here demonstrate the ease, speed, and robustness of the MS-based method, where a wealth of molecular data from minute tissue sections can be used to assist and expand pathology, and possibly reduce the inter-observer variability.

pathology↗

bioRxiv2024

Infiltrative classical monocyte-derived and SPP1 lipid-associated macrophages mediate inflammation and fibrosis in ANCA-associated glomerulonephritis

BackgroundKidney macrophage infiltration is a histological hallmark of vasculitic lesions and is strongly linked to disease activity in anti-neutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis (AGN). The precise mechanisms by which kidney macrophages influence local inflammation and long-term damage remain largely unknown. MethodsHere, we investigate kidney macrophage diversity using single-cell transcriptome analysis of 25,485 freshly retrieved unfrozen, high-quality kidney CD45+ immune cells from five AGN patients, a lupus nephritis and nephrectomy control. Detailed subclustering of myeloid cells was performed to identify disease-specific macrophage subtypes. Next, transcriptome differences between macrophage subsets and disease serotypes were assessed. Findings were validated by immunostainings of an extended cohort of kidney biopsies and flow cytometric analysis of peripheral blood monocytes. ResultsFour main macrophage subsets were identified, including a classical monocyte-derived macrophage (MDM) subset expressing a chemotactic (CXCL2, CXCL3, CXCL8, CCL3) and pro-inflammatory (IL1{beta}, TNF) set of markers and a osteopontin/SPP1+ lipid-associated macrophage (SPP1 LAMs) subtype exhibiting distinctive upregulation of fibrotic genesets. AGN samples revealed a markedly increased proportion of CD163+ macrophages, predominantly composed of classical MDMs, accompanied by resident-like C1Q macrophages, and SPP1 LAMs. An analogous trend was observed in the expansion of peripheral blood classical monocytes during active disease. The proteinase 3 (PR3)-AGN subtype exhibited heightened classical MDM infiltration and markers of acute inflammation, while interferon signaling and markers of chronicity were reduced compared to myeloperoxidase (MPO)-AGN. ConclusionsOur findings highlight the expression of inflammatory and fibrotic genes by kidney macrophage subsets in AGN. Classical monocyte dysregulation might contribute to inflammation in the pathogenesis of AGN. Targeting these specific monocyte/macrophage subsets may potentially control the inflammatory cascade and attenuate resulting fibrosis in AGN and kidney disease in general. Key points- Classical monocyte-derived macrophages are predominant in ANCA-associated glomerulonephritis and exhibit chemotactic and pro-inflammatory markers - Osteopontin/SPP1+ lipid-associated macrophages (SPP1 LAMs) show distinctive upregulation of fibrotic genesets - Understanding of the macrophage immune response supports exploration of macrophage-directed therapies for the treatment of autoimmune kidney diseases

immunology↗

bioRxiv2023

Air Pollution Aggravates Renal Ischemia-Reperfusion-Induced Acute Kidney Injury

Chronic kidney disease (CKD) has emerged as a significant global public health concern. Recent epidemiological studies have highlighted the link between exposure to fine particulate matter (PM2.5) and declined renal function. PM2.5 exerts its harmful effects on various organs through oxidative stress and inflammation. Acute kidney injury (AKI) resulting from ischemia reperfusion injury (IRI) involves similar biological processes involved in PM2.5 toxicity and is a known risk factor for CKD. The objective of this study was to investigate the impact of PM2.5 exposure on IRI-induced AKI. Mice were exposed to PM2.5 or filtered air for 12 weeks before IRI, and were euthanized 48h after IRI. Animals exposed to PM2.5 and IRI exhibited reduced glomerular filtration and impaired urine concentration ability. Moreover, they showed elevated tubular damage markers NGAL and KIM-1, along with significant tubular necrosis. PM2.5 exposure exacerbated local innate immune activation, leading to an increased infiltration of Ly6G+ granulocytes and F480+ macrophages in the kidney. This, in turn, contributed to heightened renal senescence markers and myofibroblast infiltration. Collectively, our findings suggest that AKI-induced hampered tubular function is worsened by PM2.5, leading to reduced resilience to stress, activation of aging mechanisms and early hallmarks of fibrosis. Decreasing PM2.5 and implementing preventive strategies can improve AKI patients outcome and prevent AKI progression.

pathology↗

bioRxiv2023

Advancing multi-day ex vivo kidney perfusion using spatially resolved metabolomics

The ability to preserve metabolically active kidneys ex vivo for multiple days may permit reconditioning, repair and regeneration of deceased donor kidneys. However, the kidneys high metabolic demand limits its functional preservation. Current approaches focus on normothermic machine perfusion (NMP) at 37{degrees}C or hypothermic machine perfusion (HMP) at 4-8{degrees}C. At normothermia, kidneys are metabolically active but ex vivo preservation is limited to hours. During hypothermia kidneys can be preserved up to 24 hours but are metabolically inactive and suffer cold-induced injury. Therefore, we revisited sub normothermic perfusion (at 25{degrees}C) as an alternative approach to preserve human kidneys in a metabolically active state for extended periods of time. In a custom-made platform that includes a cell-free perfusate enriched with TCA cycle fuels, urine recirculation, and continuous hemofiltration we perfused discarded human kidneys up to 8 days. Using spatially resolved single cell resolution isotope tracing we demonstrate active metabolism in all the different renal cell types over this period. However, beyond 4 days cell composition of nephron segments assessed with spatial lipidomics changed substantially and injury markers such as NGAL and LDH increased in the perfusate. Up to 4 days, perfused human discarded donor kidneys maintained metabolic fluxes, functional parameters and allow for reperfusion using a porcine auto transplantation model. These data underpin that extended multi-day metabolic preservation of human kidneys is achievable using a sub normothermic perfusion platform.

bioengineering↗