Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Basement membrane turnover and circulating matrix biomarkers in kidney disease

ABSTRACT: Chronic kidney disease (CKD) is characterised by kidney fibrosis and represents a major public health concern. Alport syndrome, a common monogenic cause of kidney failure caused by genetic variants in the type IV collagen genes COL4A3, COL4A4or COL4A5, leads to abnormal basement membrane remodelling and kidney fibrosis. Understanding the dynamics and mechanisms of kidney matrix remodelling is crucial for directing emerging gene therapies and developing non-invasive biomarkers to detect and stage kidney fibrosis earlier, ultimately enabling risk stratification and timely intervention. To define the temporal and spatial dynamics of kidney matrix remodelling, we applied a multi-omics strategy in a Col4a5-/-mousemodel of X-linked Alport syndrome, integrating stable isotope metabolic labelling, deep proteomicprofiling, bulk RNA sequencing, super-resolution imaging, and bioinformatic prediction.Transcriptomic profiling identified enrichment of extracellular matrix degradation pathways and upregulation of matrix proteases. This being the RNA-seq component.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Leo Zeef

PROVIDER: E-MTAB-15467 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Chronic kidney disease (CKD) is characterised by kidney fibrosis and represents a major public health concern. Alport syndrome, a common monogenic cause of kidney failure caused by genetic variants in the type IV collagen genes COL4A3, COL4A4 or COL4A5, leads to abnormal basement membrane remodelling and kidney fibrosis. Understanding the dynamics and mechanisms of kidney matrix remodelling is crucial for directing emerging gene therapies and developing non-invasive biomarkers to detect and stage kidney fibrosis earlier, ultimately enabling risk stratification and timely intervention. To define the temporal and spatial dynamics of kidney matrix remodelling, we applied a multi-omics strategy in a Col4a5-/- mouse model of X-linked Alport syndrome, integrating stable isotope metabolic labelling, deep proteomic profiling, bulk RNA sequencing, super-resolution imaging, and bioinformatic prediction. Transcriptomic profiling identified enrichment of extracellular matrix degradation pathways and upregulation of matrix proteases. 13C-Lysine labelling coupled with mass spectrometry revealed altered abundance and accelerated turnover of basement membrane components in Alport kidneys. Super-resolution imaging confirmed spatial disorganisation of matrix proteins, supporting a loss of structural integrity. Using peptide location fingerprinting, we mapped damage modifications and predicted fragmentation in ~40 kidney matrix proteins, including collagens, laminins and nidogens. Targeted assays validated the presence of these predicted matrix fragments in serum from children with COL4A3–5 variants, demonstrating detectable alterations in circulating basement membrane peptides compared to healthy controls. These data define an uncoupling of matrix synthesis and degradation as a hallmark of Alport-associated kidney fibrosis. Our integrative approach establishes a framework for linking matrix proteolysis and basement membrane fragmentation with clinically accessible biomarkers of kidney matrix remodelling. The detection of circulating matrix fragments offers a promising non-invasive strategy to monitor basement membrane integrity and stage kidney fibrosis, with potential utility in both diagnosis and therapeutic response assessment in Alport syndrome and broader CKD contexts.

Project description:Fibrosis is defined as an abnormal matrix remodeling and loss of tissue homeostasis due to excessive synthesis and accumulation of extracellular matrix proteins in tissues. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1(PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs including kidney. Therefore, the PAI-1 knockout model of cardiac fibrosis provides an excellent opportunity to find the igniter(s) of cardiac fibrosis and its status in unaffected organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase- and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways were upregulated or downregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in several biological processes including fibrogenesis. Total RNA was extracted from hearts and kidneys derived from three PAI-1 knockout (12- month old) and three wild-type mice (12-month old) using RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The quality of RNA (RNA Integrity, RIN) in all 12 samples (3 wildtype hearts; 3 PAI-1 KO hearts; 3 wildtype kidneys; and 3 PAI-1 KO kidneys) was checked using the bioanalyzer. We have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney.

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Basement membrane turnover and circulating matrix biomarkers in kidney disease

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