Identification of Conserved Gene Expression Changes Across Common Glomerular Diseases by Spatial Transcriptomics

Background: STAT3 signaling is activated in podocytes and parietal epithelial cells (PECs) in murine models of proliferative glomerulopathy and in human RPGN and subtypes of FSGS. We previously showed that podocyte-specific loss of STAT3 preserves podocyte loss and attenuates PEC activation. However, the mechanism by which STAT3 activation triggers PEC activation, proliferation, and crescent formation and whether its inhibition as a therapeutic target in proliferative glomerulopathies remains poorly understood. Methods: PECs were treated with increasing doses of IL-6 (5, 10, 20, 40ng/ml) to explore the role of STAT3 activation in cell viability. We generated mouse PECs with deletion of STAT3 using Crispr/Cas9. MTT assay was performed on Cas9 (wildtype) and STAT3 knockout (STAT3-/-) PECs. Cell migration of Cas9 and C7 cells were measured using a scratch assay. Mitochondrial respiration, glycolytic rate, and ATP production were performed using seahorse analyzer. Glutathione, superoxide, and reactive oxygen species (ROS) levels were measured. RNA sequencing was conducted in the Cas9 and STAT3-/- PECs. PEC-specific STAT3-/- mice were generated as well as STAT3 inhibitor treatment in mice post-nephrotoxic serum (NTS) administration. Results: STAT3-/- PECs exhibited reduced cell proliferation, activation, oxygen consumption, and ATP production. RNAseq demonstrated a downregulation of differential expressed genes (DEGs) involved in glutathione metabolism with an upregulation in focal adhesion DEGs. STAT3-/- PECs had reduced cellular glutathione pool leading to increased levels of superoxide levels leading to increased oxidative stress and DHE expression. In silico analysis showed that pSTAT3 occupies the promoter region of key glutathione synthesis genes, suggesting potential direct regulation. PEC-specific STAT3-/- mice or treatment with STAT3 inhibitor reduced proteinuria, PEC activation (CD44, Akap12), crescent formation with increased oxidative stress (8-oxo-G, OGG1) as compared to their respective controls post-NTS treatment. Conclusions: To date, this is the first study to demonstrate the mechanism by which STAT3 activation in PECs enhances glutathione metabolism to maintain a balance in ROS and exacerbate PEC activation and crescent formation in proliferative glomerulopathies.

Background: C3 glomerulopathy (C3G) is caused by a dysregulation of the complement system leading to C3 deposition and formation of glomerular deposits.Several C3G patients harbor mutations or copy number variations in the human Factor H (FH) and/or Factor H-Related (FHRs) genes.Therefore, FH and FHRs are emerging immune targets for inhibition of the complement cascade, as well as markers to monitor patients on complement regulatory drugs to test their efficiency.
Methods: Here, we focused our study on FHR2, known to inhibit in vitro formation of the terminal complement complex.We identified new variants for the FHR2 gene in a cohort of C3G patients and performed detailed functional studies on the novel variant FHR2 L46 , which has the Pro at position 46 replaced by Leu.Patients with FHR2 L46 variant presented increased FHR2 plasma level, as compared to controls and displayed FHR2 deposits in glomeruli.We generated a recombinant FHR2 L46 mutant protein to gain insight into the effect of this novel FHR2 variant on complement regulation.
Results: As the amino acid exchange occurred in the first short consensus repeat (SCR1), we first tested if the Leu at position 46 altered FHR2 homodimerization and heterodimerization of FHR2 with FHR1 and FHR5.We observed that FHR2 L46 binds significantly less to FHR2 and FHR1 but more to FHR5.Furthermore, FHR2 46L acquired the capacity to bind to cell surfaces by interacting with glycosaminoglycans heparin and malondialdehyde (MDA)-modified amino group (MAA) epitopes.FHR2 L46 also bound substantially more to necrotic cells compared to wild-type FHR2 (FHR2 WT ).In contrast, no difference was observed between FHR2 L46 and FHR2 WT binding to C3 and C5.

Conclusions:
Taken together, the present study identified a novel FHR2 L46 variant in a C3G patient and suggests that the FHR2 L46 mutant forms stable oligomers with FHR5 and enhances complement activation.

TH-PO541 Poster Thursday
Glomerular Diseases: From Inflammation to Fibrosis -I Background: The use of kidney organoids as a model for studying kidney disease shows great promise, but their potential is limited by our limited understanding of the proteins they express and their functional profiles.In this study, we aimed to address this limitation by examining the proteome and transcriptome of organoids throughout their culture period and in response to TNFα, a cytokine stressor.
Methods: In our study, we employed proteomic analysis to compare kidney organoids with other established model systems and native tissues, including native glomeruli and cultured podocytes.We examined the developmental trajectory of organoids and explored their innate immune responses, thereby expanding the applicability of organoids as a valuable model system in the field of nephrology.Additionally, we performed a comprehensive comparison of our proteomic data with both bulk and single-cell transcriptomics data, providing a more comprehensive understanding of the molecular landscape of kidney organoids.
Results: We found that older organoids displayed increased accumulation of extracellular matrix while showing decreased expression of glomerular proteins.By integrating single-cell transcriptome data, we discovered that most changes in the proteome were localized to podocytes, tubular cells, and stromal cells.Treatment of the organoids with TNFα resulted in the differential expression of 322 proteins, including cytokines and complement components.Importantly, the transcript expression of these 322 proteins was significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease.Notably, key proteins associated with TNFα (C3 and VCAM1) were found to be increased in both human tubular and organoid kidney cell populations, indicating the potential of organoids to advance the development of biomarkers.VCAM1 was localized to the descending thin limb (DTL) of proteinuric patients with kidney disease.
Conclusions: By integrating various "omic" layers of kidney organoids, incorporating a relevant cytokine stressor, and comparing with human data, we highlight the significance of kidney organoid modeling in understanding and studying complex human kidney disease.
Funding: NIDDK Support, Other NIH Support -The Nephrotic Syndrome Study Network (NEPTUNE) is part of the Rare Diseases Clinical Research Network (RDCRN), which is funded by the National Institutes of Health (NIH) and led by the National Center for Advancing Translational Sciences (NCATS) through its Division of Rare Diseases Research Innovation (DRDRI)., Government Support -Non-U.S.

TH-PO542 Poster Thursday
Glomerular Diseases: From Inflammation to Fibrosis -I
Background: Glomerular diseases encompass a group of kidney diseases that may share common gene expression pathways.We aimed to analyze glomerular-specific gene expression profiles across various glomerular diseases.
Methods: We performed spatial transcriptomic profiling using formalin-fixed paraffin-embedded kidney biopsy specimens of controls and patients with five types of glomerular diseases using the GeoMx Digital Spatial Profiler.We identified common differentially expressed genes (DEGs) across glomerular diseases and performed Gene Ontology (GO) annotation using the ToppGene suite.
Results: A total of 35 DEGs were consistently downregulated in glomeruli across the disease compared to the control, while none of the DEGs were consistently upregulated.Twelve of 35 downregulated DEGs, including the two hub genes FOS and JUN, were annotated with molecular function GO terms related to DNA-binding transcription factor activity.The annotated biological process GO terms included response to lipid-related (17/35 DEGs), response to steroid hormone (12/35 DEGs), or cell cycle regulation (10/35 DEGs).
Conclusions: Identifying common DEGs by spatial transcriptomic analysis provides insights into underlying molecular mechanisms of glomerular diseases and may lead to novel assessment or therapeutic strategies.