Identification of Key Genes and Pathways in Nephritis Progression through Bulk-seq Data Analysis
In the initial phase of our study, we embarked on an extensive analysis of bulk-seq data derived from public databases to decipher the tissue-level changes occurring during the progression of Nephritis. Through differential gene expression analysis of bulk transcriptome data from IgA nephropathy patients and healthy kidney samples, our study has uncovered significant alterations in several key biological pathways (Fig. 1A&B). The upregulated genes predominantly fall within pathways such as Toll-like receptor signaling, inflammatory response, collagen formation, epithelial to mesenchymal transition, cytokine signaling in the immune system, TGF-beta signaling, and angiogenesis (Fig. 1B&C). These findings point towards an enhanced inflammatory response and fibrotic processes in IgA nephropathy. Specifically, the activation of Toll-like receptor signaling and cytokine signaling pathways suggests an amplified innate immune response, which could be driving the chronic inflammation and subsequent tissue damage in the kidneys16. The increase in collagen formation and TGF-beta signaling genes indicates progressive fibrosis, a critical factor in the deterioration of kidney function in IgA nephropathy17.
Conversely, the downregulated genes are involved in pathways such as renal system development, tube formation, response to hormone, oxidative phosphorylation, Hippo pathway, xenobiotic export from cells, and transport of small molecules. This downregulation reflects a decline in renal function and a disruption in metabolic processes (Fig. 1B&D). Notably, the decrease in genes associated with renal system development and tube formation could be indicative of impaired nephron structure and function, a hallmark of advancing IgA nephropathy. The reduced expression of oxidative phosphorylation genes points to an altered energy metabolism in the kidney cells, potentially impacting their ability to repair and maintain tissue integrity18.
Our analysis revealed interplay between heightened inflammation, fibrotic changes, and a decline in renal functionality and metabolism. These insights into the transcriptomic landscape of IgA nephropathy offer a deeper understanding of its pathophysiology and potential avenues for therapeutic intervention.
Establishment of Kidney Cell Atlas during the Pathogenesis of IgA Nephropathy
In our investigation of IgA nephropathy pathogenesis, we utilized single-cell data to construct a detailed kidney cell atlas. The cell types identified within IgA nephropathy and healthy kidney tissues included Proximal tubular cells (PTC), Distal tubular cells (DTC), Thick ascending limb cells (Thick-ALC), Thin ascending limb cells (Thin-ALC), Distal convoluted tubule cells (DCTC), Connecting tubule cells (CTC), Collecting duct principal cells (CDPC), Collecting duct intercalated cells (CDIC), Podocytes (Pod), Endothelial cells (EC), Mesangium cells (Mes), Macrophages (Mac), Fibroblasts (Fib), T cells (TC), and B cells (BC) (Fig. 2A&B). The cell type identification was corroborated by the expression profiles of classic markers and the functional annotation of cell-type-specific marker genes (Fig. 2C).
Subsequent cell proportion analysis showed an increase in the proportion of immune cells, including macrophages, T cells, and B cells, throughout the disease process, suggesting a heightened immune response. Additionally, there was an observed increase in the proportion of fibroblasts, which aligns with the fibrotic remodeling indicated by our transcriptomic data (Fig. 2D).
Crucially, the analysis revealed a decrease in the proportion of podocytes during the disease course (Fig. 2D). The reduction of these non-proliferative, filtration-critical cells underscores their potential loss or dysfunction in IgA nephropathy, which could contribute significantly to the pathological proteinuria and glomerular damage observed in affected patients.
This findings from the establishment of a kidney cell atlas in IgA nephropathy highlight the critical cellular dynamics that characterize the disease's progression. The increase in immune and fibroblast cell populations, together with the reduction in podocytes, illustrates a pathophysiological trajectory of heightened immune response, fibrotic remodeling, and compromised filtration capacity.
Differential Gene Expression in Renal Cell Types During IgA Nephropathy
Our analysis of the differential gene expression across renal cell types during IgA nephropathy revealed that podocytes and fibroblasts have a high number of differentially expressed genes (Fig. 3A). This suggests significant transcriptomic alterations in these cell types. Podocytes, alongside other renal parenchymal cells, showed numerous downregulated genes, while fibroblasts and immune cells had an abundance of upregulated genes (Fig. 3B).
Further investigation into the functions of these differentially expressed genes uncovered that upregulated genes were mainly involved in pathways related to response to hypoxia, cytokine stimulus, cytokine signaling in the immune system, extracellular matrix organization, oxidative stress, inflammatory response, apoptotic signaling, platelet aggregation, tight junctions, interferon signaling, complement activation, cell cycle, angiogenesis, leukocyte proliferation, cell-cell recognition, PPAR signaling, regulation of endothelial cell migration, and T cell activation. These pathways are intimately linked with the pathogenic responses observed in IgA nephropathy, such as inflammation, tissue remodeling, immune system activation, and fibrosis (Fig. 3C&D).
In contrast, the downregulated genes were associated with functions in renal absorption, ATP metabolism, response to nutrient levels, metal ion transport, mRNA processing, blood circulation, morphogenesis of cellular anatomical entities, intra-Golgi transport, kidney epithelium development, cellular homeostasis, actin cytoskeleton organization, amino acid transport, alpha-amino acid metabolism, endocytosis regulation, and muscle cell differentiation (Fig. 3E&F). The suppression of these pathways could reflect the compromised renal functionality and structural integrity that are characteristic of IgA nephropathy.
This comprehensive analysis of gene expression changes at the cellular level provides insights into the biological significance of these alterations within the IgA nephropathy milieu. The data suggest a state of heightened immune activity and fibrotic processes in the kidneys, along with a decline in metabolic and structural maintenance activities, offering potential avenues for targeted therapeutic strategies.
Altered Cell-Cell Interactions During Nephritis Progression and Implications for Tissue Functionality
Through cell-cell interaction analysis, we examined the microenvironmental changes occurring during the progression of IgA nephropathy. A notable decrease in interactions among parenchymal cells suggests a decline in intrinsic kidney function, reflecting the damage inflicted by the ongoing disease process (Fig. 4A&B). This reduction in cellular cooperation within the kidney's functional units likely contributes to the impaired filtration and regulatory abilities observed in nephritis.
Conversely, we observed a marked increase in interactions between immune cells and other renal cell types, emphasizing the heightened inflammatory milieu (Fig. 4A&B). This upregulation points to the immune system's active engagement in the pathological state, which, while necessary for healing, may paradoxically exacerbate tissue damage if left unchecked19.
Fibroblast interactions with other cells also showed an uptick (Fig. 4A&B), suggesting an increased risk of tissue fibrosis. Fibroblasts play a crucial role in wound healing by depositing extracellular matrix components, but their persistent activation can lead to pathological fibrosis, compromising organ function20.
Particularly striking was the surge in interactions between podocytes, immune cells, and fibroblasts. Given the non-proliferative nature of podocytes, their increased engagement with inflammatory and fibrotic processes highlights their vulnerability and potential role in disease exacerbation. This is further supported by the upregulation of pathways related to 'Extracellular matrix organization', 'Inflammatory response pathway', and 'Collagen formation', all of which are involved in the structural alterations seen in nephritis (Fig. 4C&D).
The increase in interactions is also reflective of the activation of pathways such as 'Necroptosis' and 'EMT (epithelial-mesenchymal transition)', which are typically associated with cell death and transformation, respectively. This indicates a transition from a homeostatic state to one of tissue stress and potential scarring. 'Platelet activation and aggregation' and 'Notch signaling pathway' interactions suggest an increase in tissue repair mechanisms, which can have both protective and deleterious consequences.
The upregulation of 'Cytokine Signaling in Immune system' and 'Cell-matrix adhesion', alongside 'Blood vessel development' and 'Leukocyte differentiation', underscores a complex interplay between inflammation, angiogenesis, and immune cell recruitment. This complex web of interactions orchestrates the inflammatory response, tissue repair, and potential pathological remodeling within the nephritic kidney.
In summary, our data depict a dynamic landscape of cell-cell interactions in nephritis, with a shift towards inflammatory and fibrotic communication networks. These findings underscore the importance of balanced cell-cell crosstalk for maintaining kidney integrity and function, and they illuminate potential therapeutic avenues to mitigate tissue damage and preserve renal health in the context of nephritis.
Pseudotemporal Dynamics of Podocyte Gene Expression in IgA Nephropathy
In advancing our understanding of IgA nephropathy, we constructed a pseudotemporal trajectory of podocyte differentiation across the disease spectrum (Fig. 5A). Healthy tissue-derived podocytes appeared predominantly at the early to middle stages of this trajectory, while those from diseased tissue were situated towards the later stages (Fig. 5A&B), enabling a comparative analysis of podocyte state changes throughout disease progression.
Our gene expression pattern analysis over pseudotime identified two clusters with divergent trends (Fig. 5C). The first cluster, with genes that increased in expression along the pseudotime continuum, included functions primarily associated with response to injury and cellular stress, such as the Toll-like receptor signaling pathway and Extracellular Matrix Organization. These upregulated genes suggest an environment favoring inflammatory responses and fibrotic remodeling in diseased podocytes (Fig. 5D).
In contrast, the second cluster, which showed a decrease in gene expression over pseudotime, was significantly enriched for functions crucial to podocyte health and kidney function (Fig. 5D). This included Podocyte differentiation, the backbone of podocyte identity and function, and Actin cytoskeleton organization, vital for maintaining the structural framework necessary for the podocyte's filtration capabilities21. Additionally, Oxidative phosphorylation, a pathway indicative of cellular energy status, showed decreased expression, potentially signaling a metabolic crisis within diseased podocytes that could compromise their viability and function22.
Further downregulated functions like Podocyte cell migration and Cell morphogenesis are particularly telling, as they reflect the podocytes' reduced capacity to respond to and repair glomerular injury, a critical aspect of disease pathology. The decline in gene expression related to Cell morphogenesis also suggests an impairment in the adaptive structural changes required for podocyte survival under stress.
The downregulation of genes within these pathways paints a picture of the progressive loss of podocyte functionality, structural integrity, and reparative capacity in IgA nephropathy. This decline is juxtaposed against a backdrop of increased cellular stress responses, highlighting a dual process of cellular dysfunction and compensatory mechanisms that may ultimately lead to podocyte depletion and glomerular failure23. Understanding these changes offers potential pathways for therapeutic intervention, aiming to restore podocyte function and slow disease progression.
Pseudotemporal Trajectory and Gene Expression Patterns of Fibroblasts in IgA Nephropathy
In our analysis, pseudotemporal trajectory mapping of fibroblasts revealed distinct states of cell differentiation between healthy and diseased kidney tissues (Fig. 6A). Fibroblasts from healthy tissue were mainly positioned at the early to mid-stages of the trajectory, while those from diseased tissue were more frequently found at the mid to later stages, allowing for a detailed examination of the cellular state transitions of fibroblasts during the course of IgA nephropathy (Fig. 6A&B).
Gene expression pattern analysis along the pseudotemporal trajectory showed that certain genes possibly indicative of fibroblast activation increased in expression (Fig. 6C). These genes were associated with biological functions such as Collagen fibril organization, Cell activation, Positive Regulation of Cell Migration, Fibroblast activation, and TGFβ receptor signaling pathway (Fig. 6D). The elevated expression of these genes over pseudotime suggests an ongoing process of fibrotic remodeling, which is characteristic of IgA nephropathy progression. The increase in Collagen fibril organization and Fibroblast activation points to the fibroblasts' central role in extracellular matrix deposition and scar formation. Furthermore, the upregulation of the TGFβ receptor signaling pathway is a well-known driver of fibrosis, promoting fibroblast differentiation into myofibroblasts, a state associated with enhanced fibrotic activity24.
Conversely, the second cluster of genes, which decreased in expression over pseudotime, was linked to functions including the MAPK Signaling Pathway, PDGFR-beta pathway, Rhythmic process, Apoptosis, and Negative regulation of locomotion (Fig. 6E). The downregulation of the MAPK and PDGFR-beta pathways may reflect alterations in cell signaling that typically mediate cell growth and survival, potentially leading to a dysregulated fibroblast response under pathological conditions. The decrease in genes governing Apoptosis and Negative regulation of locomotion might be indicative of a reduced capacity for apoptosis, contributing to the accumulation of fibroblasts and impaired cellular motility, respectively.
The insights gained from this pseudotemporal analysis are crucial for understanding the dynamic changes in fibroblast behavior and function during the development and progression of IgA nephropathy. The observed gene expression trends provide valuable clues to the underlying molecular mechanisms that may be targeted to mitigate fibrosis and preserve kidney function in patients with IgA nephropathy.