In this study, we identified DEGs within the glomeruli of patients with common glomerular diseases compared to those of healthy controls by spatial transcriptomic analysis to characterize the consistently dysregulated molecular pathways in glomerular diseases. Using GeoMx DSP, we identified a set of 35 DEGs that were universally downregulated across five glomerular diseases. These conserved DEGs, including FOS, JUN, EGR1, NR4A1, NR4A3, KLF4, ATF3, SOCS3, DUSP1, and ZFP36, were assessed via functional enrichment analysis and GO term annotation to interpret the complex interactions and functions between genes and gene products. Seventeen of 35 DEGs, including the two hub genes JUN and FOS, which were annotated to the AP-1 transcription factor complex,10 were annotated to lipid metabolism. Additionally, we observed significant upregulation of ubiquitin-specific peptidase genes in MN, MCD, FSGS, and DN samples compared to control samples, while their expression was downregulated in IgAN samples.
Spatial transcriptomic profiling using a microdissection-based method, including GeoMX DSP, has particular strength in the study of glomerular diseases, as the main focus of interest is usually on pathological changes in the glomerulus where most of the pathophysiological processes occur.6,9,11 Although there are other types of RNA-seq-based spatial transcriptomic methods (e.g., Visium), their current resolution is not sufficient to capture the glomerulus-specific transcriptome.12 On the other hand, single-cell RNA-seq is another useful transcriptomic analysis method for investigating cell-specific mRNA expression,13 yet most single-cell human data include a relatively low portion of glomerulus cells with low feature variance.14,15 Previously, we performed manual microdissection to investigate the glomerulus-specific transcriptome;16 however, laborious hand microdissection and sample availability limited the expansion of the data. In the current study, we used GeoMx DSP, which has been trialed in transplant rejection8 and collapsing glomerulopathy,9 to study the common transcriptomic alteration of the glomerulus in patients with various glomerular diseases. By implementing this advanced technique, we identified universal mRNA expression changes in diverse types of glomerular diseases, which may expand our knowledge of the common pathophysiological features of glomerular diseases. Another strength of the current study is the collection of kidney donor biopsy tissue during transplantation surgery, which closely approximates the physiological state of normal kidney tissue and facilitates the identification of DEGs in our study.
Nephrin, a podocyte-adhesive signaling molecule located in the slit diaphragm, is crucial for maintaining the structure and function of the glomerular filtration barrier. The slit diaphragm is a highly specialized lipid-raft structured cell junction that bridges adjacent podocyte foot processes and is commonly disrupted in nephrotic diseases.17,18 The AP-1 transcription factor is a dimeric molecule that plays a crucial role in cell proliferation, differentiation, and apoptosis by modulating the cell cycle.10,19 Nephrin is essential for activating the AP-1 signal transduction pathways20,21 along with Src family members22–24. In nephrin-expressing NHP15 cells, higher c-Jun activity has been observed than in Finn-minor cells that lack human nephrin expression, indicating that nephrin is involved in activating the AP-1 signaling pathway through c-Jun activation.20 It has also been suggested that nephrin-triggered cellular signaling cascades may preserve the structural integrity of the podocyte slit diaphragm.21 In this study, we identified consistent downregulation of DEGs, including JUN, FOS, JUNB, and ATF3, which are components of the AP-1 transcription factor, in common glomerular diseases. Therefore, we suggest that the nephrin-induced AP-1 signaling pathway is universally downregulated in glomerular diseases accompanied by disruption of the slit diaphragm and dislocation of nephrin.
Furthermore, c-Fos is known to activate lipid synthesis, including phospholipids25 and glycolipids26, and multiple DEGs, including JUN, FOS, KLF9, ATF3, and CDKN1A, were annotated to the ‘response to lipid’ biological GO term in this study. The slit diaphragm has been proposed as a lipid raft functioning as a signaling hub with the interaction of proteins including podocin and nephrin.17,27,28 Although the structures and metabolic mechanisms of the slit diaphragm are largely unknown, we infer that the downregulation of the above genes may interfere with lipid-protein interactions that are important in maintaining the function of the slit diaphragm.
Consistent with our findings, downregulation of FOS or JUN was detected in patients with IgAN, lupus nephritis, MCD, FSGS, MN, and DN in a study that compared transcriptomic profiles between patients with glomerular diseases and healthy controls.29 Additionally, there was a report that c-FOS expression was scant or c-FOS was not expressed in glomeruli but that c-FOS expression was detected in tubules, small vessels, or interstitial cells in crescentic IgAN patient samples.30 Despite several reports indicating AP-1 activation with an inflammatory response in kidney injury models or polycystic kidney diseases, the upregulation of AP-1 was mainly exhibited in tubules and interstitium, not in glomeruli.31–33 In most chronic glomerular diseases, the downregulation of JUN/FOS and AP-1 was predominant within the glomeruli consisting of a disrupted slit diaphragm and altered protein signaling.
We also identified that the glomerular mRNA expression of USP17L family genes in IgAN samples showed reverse directional changes when compared to the other types of glomerular diseases. Previous studies have reported the upregulation of the ubiquitin‒proteasome pathway in IgAN, which demonstrated the overexpression of the proteasome and its switch to the immunoproteasome in peripheral blood mononuclear cells of IgAN patients.34 As the ubiquitin‒proteasome system regulates the antigen processing and presentation of major histocompatibility complex (MHC) class I molecules,35 the enhanced immune response to certain MHC-1 antigens in IgAN has also been suggested.34 In addition, the association between genetic polymorphisms within the MHC regions and susceptibility to IgAN was identified in a previous genome-wide association study of IgAN.36 Our findings are in accordance with previous studies linking ubiquitin‒proteasome system activation and the T-cell response in the pathogenesis of IgAN. Further investigation is needed to determine the significance of the difference in the direction of the ubiquitin‒proteasome pathway in IgAN compared to other glomerular diseases.
There are some limitations in our study. First, the downregulated DEGs were not assessed by further experimental investigations. An additional mechanistic experiment is warranted to confirm the clinical implication of our findings, along with transcriptomic analysis focusing on disease-specific glomerular changes. Second, although challenging, identifying the cell specificity of the identified DEGs would enrich the meaning of our findings. With the advancement in platforms for spatial transcriptomic strategies, a cellular- or subcellular-level investigation should be widely available in the future, and our study data may provide background information for future studies. Third, the issue of generalizability should be considered, as the study participants were Asian.
In conclusion, we revealed universally downregulated DEGs annotated to AP-1 or lipid metabolism in the glomeruli of various glomerular diseases through spatial transcriptomic profiling. Our study elucidates the potential for common molecular mechanisms underlying the pathogenesis of various glomerular diseases.