Tubulointerstitial Compartment Research Articles

#71 Dysregulation of ITGAM and ITGB2 in the pathogenesis of diabetic kidney disease—results from bioinformatics analysis and in vitro studies

Abstract Background and Aims Activated neutrophils can release web-like chromatin structures known as neutrophil extracellular traps (NETs), but their roles in the immunopathogenesis of diabetic kidney disease (DKD) remains elusive. Method The characteristic NETs associated targets of differentially expressed genes (DEGs) from human kidney biopsy datasets of patients with DKD (GSE30528 and GSE30529) were identified by machine learning method (LASSO and SVM-RFE). The genes expression was further validated using kidney biopsy dataset of GSE142025. The most abundant single cell of the targets in DKD was further selected by single-nucleus RNA sequencing datasets (GSE131882). Predictive pathway network of targeted genes was constructed using GENEMANIA database. The expressions and potential signaling pathway of these candidate genes were validated using flow cytometry and ELISA. Results Three predicting NETs related genes (ITGAM, ITGB2 and TLR7), identified by machine learning algorithms, which all showed significant upregulation in both glomerular and tubulointerstitial compartments in human DKD kidney samples. Single-cell analysis suggested that ITGAM, ITGB2 and TLR7 were most enriched in leucocytes. DKD patients (n = 18) showed significantly higher activated neutrophils as well as increased expression of ITGAM and ITGB2 compared to healthy controls (HCs, n = 9). DKD patients also exhibited higher serum levels of IL6 but lower IL-10 than HCs (all p < 0.01). The significant network of IL10, IL6, ITGAM and ITGB2 was generated. This network might be associated with leukocyte activation, macrophage activation, JAK-STAT pathway, acute inflammatory response and lymphocyte immune response (all p < 0.05). Conclusion Aberrant ITGAM and ITGB2 expressions in activated neutrophils contribute to DKD pathogenesis and may serve as novel therapeutic targets for DKD.

Metabolic Analysis and Renal Protective Effects of Linagliptin and Empagliflozin in Alport Syndrome.

We previously demonstrated that Empagliflozin (Empa), a sodium-glucose cotransporter-2 (SGLT2) inhibitor, reduces intrarenal lipid accumulation and slows kidney function decline in experimental Alport syndrome (AS). In this study, we aimed to evaluate the renal protective benefits of Linagliptin (Lina), a dipeptidyl peptidase-4 (DPP4) inhibitor in AS and compare it to Empa. Metabolite distribution in kidney cortices was assessed using mass spectrometry imaging. We examined albuminuria and histological changes in kidneys from AS mice treated with Lina and/or Empa or vehicle. Several metabolites, including adrenic acid (AdA) and glucose, were increased in renal cortices of AS mice when compared to wildtype (WT) mice, while eicosapentaenoic acid (EPA) levels were decreased. In addition, a redistribution of AdA from the glomerular compartment in WT mice to the tubulointerstitial compartment in AS mice was observed. Both Lina and Empa treatments were found to reduce albuminuria, to extend the survival of AS mice for about 10 days, and to decrease glomerulosclerosis and tubulointerstitial fibrosis compared to WT mice. There were no significant differences with regard to the renal phenotype observed between Empa and Lina treated AS mice, and the combination of Lina and Empa was not superior to individual treatments. In vitro experiments revealed that DPP4 is expressed in podocytes and tubular cells derived from both AS and WT mice. Differently from what we have reported for Empa, Lina treatment was found to reduce glucose-driven respiration in AS tubular cells, but not in AS podocytes. Renal expression patterns and spatial distribution of several metabolites differ in AS compared to WT mice. While Lina and Empa treatments similarly partially slow the progression of kidney disease in AS, the metabolic mechanisms conferring the protective effect may be different.

#2543 Complement receptor C5aR1 correlates with inflammatory responses driven by glomerular uPAR upregulation in renal vasculitides

Abstract Background and Aims Avacopan is a novel promising treatment for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) targeting the complement system by blocking C5a receptor C5aR1. We here pursued a transcriptome array-based approach to dissect molecular signatures associated with C5aR1 in renal vasculitis. Method C5aR1 mRNA expression levels (reporter ID: 210845_s_at, platform: Affymetrix Human Genome U133 Plus 2.0 Array, altCDF v10) were extracted from microdissected samples of renal vasculitis (glomerular: n = 23, tubulointerstitial: n = 21). Pathway analysis was performed for gene enrichment associated with PLAUR mRNA expression with a correlation threshold of ≥0.5 by using reactome (http://reactome.org), pathways with a predefined entities value of p≤0.001 were included. Results We here identified a predominant glomerular expression of C5aR1 in renal vasculitis as compared to the tubulointerstitial compartment (p < 0.0001). Furthermore, glomerular C5aR1 induction correlated with impaired kidney function (eGFR: 0.0193). Gene set enrichment identified various inflammatory signatures including neutrophil degranulation and innate immunity, and particularly enrichment of urokinase plasminogen activator receptor (uPAR). uPAR itself also correlated with impairment of kidney function in renal vasculitis and particularly active glomerular lesions (cellular crescents and necrosis, p < 0.0001). Conclusion The therapeutic implications of targeting uPAR in renal vasculitis are promising, and ongoing research endeavors are aimed at unraveling the complexities of uPAR as a potential diagnostic marker and therapeutic target for this challenging renal pathology. We here expand our current knowledge and link C5aR1 to inflammatory responses driven by glomerular uPAR upregulation in renal vasculitis.

The atypical chemokine receptor 2 reduces T cell expansion and tertiary lymphoid tissue but does not limit autoimmune organ injury in lupus-prone B6lpr mice.

The atypical chemokine receptor 2 (ACKR2) is a chemokine scavenger receptor, which limits inflammation and organ damage in several experimental disease models including kidney diseases. However, potential roles of ACKR2 in reducing inflammation and tissue injury in autoimmune disorders like systemic lupus erythematosus (SLE) and lupus nephritis are unknown, as well as its effects on systemic autoimmunity. To characterize functional roles of ACKR2 in SLE, genetic Ackr2 deficiency was introduced into lupus-prone C57BL/6lpr (Ackr2-/- B6lpr) mice. Upon inflammatory stimulation in vitro, secreted chemokine levels increased in Ackr2 deficient tubulointerstitial tissue but not glomeruli. Moreover, Ackr2 expression was induced in kidneys and lungs of female C57BL/6lpr mice developing SLE. However, female Ackr2-/- B6lpr mice at 28 weeks of age showed similar renal functional parameters as wildtype (WT)-B6lpr mice. Consistently, assessment of activity and chronicity indices for lupus nephritis revealed comparable renal injury. Interestingly, Ackr2-/- B6lpr mice showed significantly increased renal infiltrates of CD3+ T and B cells, but not neutrophils, macrophages or dendritic cells, with T cells predominantly accumulating in the tubulointerstitial compartment of Ackr2-/- B6lpr mice. In addition, histology demonstrated significantly increased peribronchial lung infiltrates of CD3+ T cells in Ackr2-/- B6lpr mice. Despite this, protein levels of pro-inflammatory chemokines and mRNA expression of inflammatory mediators were not different in kidneys and lungs of WT- and Ackr2-/- B6lpr mice. This data suggests compensatory mechanisms for sufficient chemokine clearance in Ackr2-deficient B6lpr mice in vivo. Analysis of systemic autoimmune responses revealed comparable levels of circulating lupus-associated autoantibodies and glomerular immunoglobulin deposition in the two genotypes. Interestingly, similar to kidney and lung CD4+ T cell numbers and activation were significantly increased in spleens of Ackr2-deficient B6lpr mice. In lymph nodes of Ackr2-/- B6lpr mice abundance of activated dendritic cells decreased, but CD4+ T cell numbers were comparable to WT. Moreover, increased plasma levels of CCL2 were present in Ackr2-/- B6lpr mice, which may facilitate T cell mobilization into spleens and peripheral organs. In summary, we show that ACKR2 prevents expansion of T cells and formation of tertiary lymphoid tissue, but is not essential to limit autoimmune tissue injury in lupus-prone B6lpr mice.

Computational pathology model to assess acute and chronic transformations of the tubulointerstitial compartment in renal allograft biopsies

Managing patients with kidney allografts largely depends on biopsy diagnosis which is based on semiquantitative assessments of rejection features and extent of acute and chronic changes within the renal parenchyma. Current methods lack reproducibility while digital image data-driven computational models enable comprehensive and quantitative assays. In this study we aimed to develop a computational method for automated assessment of histopathology transformations within the tubulointerstitial compartment of the renal cortex. Whole slide images of modified Picrosirius red-stained biopsy slides were used for the training (n = 852) and both internal (n = 172) and external (n = 94) tests datasets. The pipeline utilizes deep learning segmentations of renal tubules, interstitium, and peritubular capillaries from which morphometry features were extracted. Seven indicators were selected for exploring the intrinsic spatial interactions within the tubulointerstitial compartment. A principal component analysis revealed two independent factors which can be interpreted as representing chronic and acute tubulointerstitial injury. A K-means clustering classified biopsies according to potential phenotypes of combined acute and chronic transformations of various degrees. We conclude that multivariate analyses of tubulointerstitial morphometry transformations enable extraction of and quantification of acute and chronic components of injury. The method is developed for renal allograft biopsies; however, the principle can be applied more broadly for kidney pathology assessment.

Kidney resident macrophages have distinct subsets and multifunctional roles

BackgroundThe kidney contains distinct glomerular and tubulointerstitial compartments with diverse cell types and extracellular matrix components. The role of immune cells in glomerular environment is crucial for dampening inflammation and maintaining homeostasis. Macrophages are innate immune cells that are influenced by their tissue microenvironment. However, the multifunctional role of kidney macrophages remains unclear. MethodsFlow and imaging cytometry were used to determine the relative expression of CD81 and CX3CR1 (C-X3-C motif chemokine receptor 1) in kidney macrophages. Monocyte replenishment was assessed in Cx3cr1CreER X R26-yfp-reporter and shielded chimeric mice. Bulk RNA-sequencing and mass spectrometry-based proteomics were performed on isolated kidney macrophages from wild type and Col4a5−/− (Alport) mice. RNAscope was used to visualize transcripts and macrophage purity in bulk RNA assessed by CIBERSORTx analyses. ResultsIn wild type mice we identified three distinct kidney macrophage subsets using CD81 and CX3CR1 and these subsets showed dependence on monocyte replenishment. In addition to their immune function, bulk RNA-sequencing of macrophages showed enrichment of biological processes associated with extracellular matrix. Proteomics identified collagen IV and laminins in kidney macrophages from wild type mice whilst other extracellular matrix proteins including cathepsins, ANXA2 and LAMP2 were enriched in Col4a5−/− (Alport) mice. A subset of kidney macrophages co-expressed matrix and macrophage transcripts. ConclusionsWe identified CD81 and CX3CR1 positive kidney macrophage subsets with distinct dependence for monocyte replenishment. Multiomic analysis demonstrated that these cells have diverse functions that underscore the importance of macrophages in kidney health and disease.

A Transcriptome Array-Based Approach to Link SGLT-2 and Intrarenal Complement C5 Synthesis in Diabetic Nephropathy.

Diabetic nephropathy is a common microvascular complication of diabetes mellitus. It is characterized by progressive chronic kidney disease (CKD) with decline of kidney function by hyperfiltration. On a mechanistic level, activation of the complement system has been implicated in the pathogenesis of diabetic nephropathy. Therefore, here we pursued a transcriptome array-based approach to link intrarenal SGLT-2 and the synthesis of distinct complement components in diabetic nephropathy. Publicly available datasets for SLC5A2 (encoding SGLT-2) and complement system components were extracted specifically from microdissected tubulointerstitial (healthy controls: n = 31, diabetic nephropathy: n = 17) and glomerular compartments (healthy controls: n = 21, diabetic nephropathy: n = 12). First, we compared tubulointerstitial and glomerular log2SLC5A2 mRNA expression levels and confirmed a predominant synthesis within the tubulointerstitial compartment. Among various complement components and receptors, the only significant finding was a positive association between SLC5A2 and the tubulointerstitial synthesis of the complement component C5 in diabetic nephropathy (p = 0.0109). Finally, intrarenal expression of SLC5A2 was associated predominantly with pathways involved in metabolic processes. Interestingly, intrarenal complement C5 synthesis was also associated with enrichment of metabolic signaling pathways, overlapping with SLC5A2 for "metabolism" and "biological oxidations". These observations could be of relevance in the pathogenesis of diabetic nephropathy and implicate a mechanistic link between SGLT-2 and intrarenal complement synthesis.

The Renal Tubulointerstitial Compartment: Site of Nephrotoxin-Induced Injury

The Renal Tubulointerstitial Compartment: Site of Nephrotoxin-Induced Injury

Histologic and Molecular Correlates in Patients with AL Amyloidosis in Hematologic Remission but with Persistent Renal Disease

ABSTRACT: Despite the high prevalence of kidney involvement in patients with light chain (AL) amyloidosis, a significant knowledge gap exists in understanding how immunoglobulin light chains drive kidney injury. The chief reason is the lack of molecular data from high-quality kidney tissue samples from this patient population. The RAIN trial (NCT03168906) was a phase 2b randomized placebo-controlled, clinical trial that evaluated the efficacy of NEOD001, a monoclonal antibody targeting amyloid fibrils, versus placebo in patients with AL nephropathy. Before drug administration, renal biopsies were taken and analyzed. OBJECTIVE: Identifying amyloid-specific molecular signatures in kidney biopsy tissue. MATERIALS & METHODS: Renal biopsies were collected on all RAIN participants before randomization to NEOD001 or placebo. Clinical data was collected and correlated to histologic and molecular findings. Histologic assessment included a composite scarring injury score (CSIC) as determined by the degree of glomerular and tubulointerstitial scarring, and a novel amyloid score (AS), as quantified by the extent and distribution of amyloid deposition. Glomerular (G) and tubulointerstitial (TI) compartments were micro-dissected and sequenced separately. To identify enriched pathways in AL nephropathy, the expression data was compared to the glomerular and tubular expression profiles of healthy living donor (LD) from the Nephrotic Syndrome Study Network (NEPTUNE) study. Differentially expressed genes (DEGs) were determined . RESULTS: Ten patients were enrolled, randomized and treated in the RAIN trial (5 NEOD001 and five placeboes) before trial closure in April 2018. Kidney tissue was available in nine of the ten patients. Cluster analysis revealed two distinct patient clusters (G1 and G2) within the glomerular and TI compartments ( See Figure 1). Key clinical data pertaining to kidney function and biopsy morphology by compartment and cluster type were documented. Despite similar eGFR and degree of proteinuria, there was a significantly higher AS score in the TI compartment (6.5 vs. 4.5; p=0.0290) of G2 which was driven most entirely by an increase in mesangial and capillary wall amyloid deposition. Pathway Enrichment in G2 versus G1: Glomeruli showed activation of fibrotic pathways accompanied by a reduction in metabolic processes including gluconeogenesis, lipid transport and xenobiotics. There was an increase in canonical signaling of LPS/IL-1 that drives increased IL1 and TNF release from macrophages. Transcriptomic evidence of TNF activation was also noted in the TI compartment. Other enriched ingenuity canonical pathways to note in the TI included ‘coagulation system’, ‘GADD45 signaling’, ‘Wnt/Ca+ pathway’, ‘intrinsic prothrombin activation pathway’ among others. Of note, genes like BRCA and CDKN1A were enriched between G1 and G2, both of which have been associated with molecular mechanisms that underly certain neoplasms ( See Figure 2). Pathway enrichment in Amyloid kidney disease versus LD: IPA analysis identified unique activation signatures in the glomeruli and tubules. In the glomeruli, there was increased signaling of TNF, NFKB, IL33, IL-8 and IL15, consistent with an important inflammatory response. There was also an enrichment in pathways related to migration, adhesion and integrin signaling. PI3K/Akt signaling was also noted. Tubules showed increased activation of transcriptional regulators TP53, TP73 and PI3K/AKT signaling. Some gene regulators of cellular proliferation such as ABL1, BCL2 and AKT1 were found to be enriched in the amyloid group. CONCLUSIONS: While the RAIN trial closed prematurely and NEOD001 cannot be evaluated, the molecular data revealed the presence of two distinct molecular subtypes of AL nephropathy. Whether these distinct molecular subtypes reflect different stages of the disease along a continuous spectrum or are of unique prognostic entities in of themselves remains unclear. Detailed studies, of a larger cohort of patients including investigating diseased tissue at a single cell resolution can help identify the response of individual kidney cell types. Understanding the cellular responses of key cell types to injury from amyloid deposits could provide novel therapeutic targets to mitigate renal injury and improve patient outcomes. Based on this early work, targeting the PI3K/Akt pathway may be a worthy pursuit in this disease.

Abstract 13695: Finerenone Added to RAS/SGLT2 Blockade for Non-Diabetic Chronic Kidney Disease. Results of a Preclinical Double-Blinded Randomized Controlled Trial

Introduction: Dual inhibition of the renin-angiotensin system (RAS) plus sodium-glucose transporter (SGLT)-2 or plus the mineralocorticoid receptor (MR) demonstrated additive renoprotective effects in large clinical trials. Hypothesis: We hypothesized that triple therapy with a combination of RAS/SGLT2/MR inhibitors would be superior to dual RAS/SGLT2 blockade in attenuating CKD progression Methods: We performed a preclinical randomized controlled trial (PCTE0000266) in Col4a3-deficient mice with established Alport nephropathy. Treatment was initiated late (6 weeks of age) in mice with elevated serum creatinine and albuminuria and in presence of glomerulosclerosis, interstitial fibrosis and tubular atrophy. We block-randomized 40 male and 40 female mice to either nil (vehicle) or late onset food admixes of ramipril monotherapy (10 mg/kg), ramipril plus empagliflozin (30 mg/kg), or ramipril plus empagliflozin plus finerenone (10 mg/kg). Primary endpoint was mean survival. Results: Mean survival was 63.7 ± 10.0 days (vehicle), 77.3 ± 5.3 days (ramipril), 80.3 ± 11.0 days (dual), and 103.1 ± 20.3 days (triple), respectively. Sex did not affect outcome. Histopathology, pathomics, and RNA sequencing revealed that finerenone mainly suppressed the residual interstitial inflammation and fibrosis despite dual RAS/SGLT2 inhibition. Conclusions: This preclinical randomized controlled trial suggests that triple RAS/SGLT2/MR blockade may substantially improve renal outcomes in Alport syndrome and possibly other progressive chronic kidney disorders for synergistic effects on the glomerular and tubulointerstitial compartment, respectively.

Study of Sequence of Histopathological Changes in Hyperglycaemia-Induced Experimental Diabetic Nephropathy in Wistar Rat Model

The sequence of histopathological changes in hyperglycaemia-induced nephrotoxicity as well as alteration in renal parameters is still not well established in diabetic models. Hyperglycaemic ambience has been shown to generate oxidative stress which becomes a trigger for further degenerative changes that occur in the microenvironment of the kidney. This study was therefore intended to investigate histopathological alterations in vascular, glomerular and tubulointerstitial compartments of the renal tissues, and the corresponding changes in values of oxidative stress markers, creatinine clearance, proteinuria and serum creatinine concentration in a duration of three, seven and twelve weeks of sustained hyperglycaemia in diabetic. The experiment included four groups of adult Wistar rat, Group A (Normal Control, treated with normal saline), Groups B, C and D were induced with diabetes (treated with 65mg/kg body weight of streptozotocin) and allowed for 3 weeks 7 weeks and 12 weeks respectively. At termination, Oxidative stress markers were analyzed using Oxidative stress marker kits. A 24 hours urine collection was obtained from metabolic cages few hours before sacrifice and used for renal analysis and histopathological examination was done using a light microscope. Results reveals that oxidative stress was climaxed at 7th week and was maintained at a constant level while histopathological changes in glomerulus first presented on the 3rd week accompanied by vascular changes. Tubulointerstitial changes were noticed on the 7th week. On the 12th week renal parameters were significantly altered when compared to the animals sacrificed on 3th and 7th week. In conclusion, the sequence in diabetic–induced renal dysfunction begins with changes in vascular and glomerular compartment followed by distortion in tubulointerstitial compartment. An alteration in renal parameters presents lastly and correlates with the histopathological changes. These findings can be adopted in clinical management and treatment of diabetes-induced kidney dysfunction. J. Bio-Sci. 31(1): 29-37, 2023

Peptide location fingerprinting identifies structural alterations within basement membrane components in ageing kidney

During ageing, the glomerular and tubular basement membranes (BM) of the kidney undergo a progressive decline in function that is underpinned by histological changes, including glomerulosclerosis and tubular interstitial fibrosis and atrophy. This BM-specific ageing is thought to result from damage accumulation to long-lived extracellular matrix (ECM) protein structures. Determining which BM proteins are susceptible to these structure-associated changes, and the possible mechanisms and downstream consequences, is critical to understand age-related kidney degeneration and to identify markers for therapeutic intervention. Peptide location fingerprinting (PLF) is an emerging proteomic mass spectrometry analysis technique capable of identifying ECM proteins with structure-associated differences that may occur by damage modifications in ageing. Here, we apply PLF as a bioinformatic screening tool to identify BM proteins with structure-associated differences between young and aged human glomerular and tubulointerstitial compartments. Several functional regions within key BM components displayed alterations in tryptic peptide yield, reflecting potential age-dependent shifts in molecular (e.g. laminin-binding regions in agrin) and cellular (e.g. integrin-binding regions in laminins 521 and 511) interactions, oxidation (e.g. collagen IV) and the fragmentation and release of matrikines (e.g. canstatin and endostatin from collagens IV and XVIII). Furthermore, we found that periostin and the collagen IV α2 chain exhibited structure-associated differences in ageing that were conserved between human kidney and previously analysed mouse lung, revealing BM components that harbour shared susceptibilities across species and organs.

Vascular, valvular and kidney calcification manifested in mouse models of adenine-induced chronic kidney disease

Background Ectopic calcification (EC) involves multiple organ systems in chronic kidney disease (CKD). Previous CKD-animal models primarily focused on a certain histological abnormality but did not show the correlation with calcified development among various tissues. This study compared calcified deposition in various tissues during CKD progression in mice. Methods Male 8-week-old C57BL/6J mice were randomly allocated to the seven groups: a basic, adenine, high-phosphorus, or adenine and high-phosphorus diet for 12–16 weeks (Ctl16, A12, P16, or AP16, respectively); an adenine diet for 4–6 weeks; and a high-phosphorus or adenine and high-phosphorus diet for 10–12 weeks (A6 + P10, A4 + P12, or A4 + AP12, respectively). Results Compared to the Ctl16 mice, the P16 mice only displayed a slight abnormality in serum calcium and phosphorus; the A12 mice had the most serious kidney impairment; the A4 + P12 and A6 + P10 mice had similar conditions of CKD, mineral abnormalities, and mild calcification in the kidney and aortic valves; the A4 + AP12 and AP16 groups had severe kidney impairment, mineral abnormalities and calcification in the kidneys, aortic valves and aortas. Furthermore, calcium-phosphate particles were deposited not only in the tubulointerstitial compartment but in the glomerular and tubular basement membrane. The elemental composition of EC in various tissues matched the calcification of human cardiovascular tissue as determined by energy dispersive spectroscopy. Conclusions The severity of CKD was unparalleled with the progression of mineral metabolism disorder and EC. Calcification was closely related in different tissues and observed in the glomerular and tubular basement membranes.

Basic nephropathology for pathologists-part2 : Non-inflammatory lesions

The evaluation of kidney biopsies for specific renal diseases or kidney transplant biopsies is mainly restricted to specialized centers. Lesions in nonneoplastic renal tissue in partial nephrectomies or nephrectomies due to renal tumors, especially noninflammatory, ischemic, vascular changes or diabetic nephropathy can be of greater prognostic significance than the tumor itself in patients with alocalized tumor and good tumor-associated survival. In this part of basic nephropathology for pathologists, the most common noninflammatory lesions of the vascular, glomerular and tubulo-interstitial compartment are discussed.

A Transcriptome Array-Based Approach Links Proteinuria and Distinct Molecular Signatures to Intrarenal Expression of Type I Interferon IFNA5 in Lupus Nephritis

In systemic lupus erythematosus (SLE), the relevance of non-hematopoietic sources of type I interferon in human autoimmunity has recently been recognized. Particularly, type I interferon production precedes autoimmunity in early skin lesions related to SLE. However, the relevance of intrarenal type I interferon expression has not been shown in lupus nephritis. From transcriptome array datasets, median-centered log2 mRNA expression levels of IFNα (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21), IFNω (IFNW1), and IFNβ (IFNB1) in lupus nephritis were extracted specifically from microdissected tubulointerstitial (n = 32) and glomerular compartments (n = 32). We found an association between proteinuria and tubulointerstitial expression of type I interferon IFNA5 (p = 0.0142), while all others were not significantly associated. By contrast, no such correlation was observed between proteinuria and any type I interferon expression in the glomerular compartment in lupus nephritis. Interestingly, there was no difference between female and male patients (p = 0.8237) and no association between type I interferon IFNA5 expression and kidney function or lupus nephritis progression. Finally, we identified distinct molecular signatures involved in transcriptional regulation (GLI protein-regulated transcription, IRF7 activation, and HSF1-dependent transactivation) and receptor signaling (BMP signaling and GPCR ligand binding) in association with tubulointerstitial expression of type I interferon IFNA5 in the kidney. In summary, this transcriptome array-based approach links proteinuria to the tubulointerstitial expression of type I interferon IFNA5 in lupus nephritis. Because type I interferon receptor subunit I antagonism has recently been investigated in active SLE, the current study further emphasizes the role of type I interferons in lupus nephritis and might also be of relevance for mechanistic studies.

#3802 ROLE OF NEUTROPHIL EXTRACELLULAR TRAPS IN DIABETIC KIDNEY DISEASE: DATA FROM INTEGRATION OF BULK RNA AND SINGLE-NUCLEUS RNA SEQUENCING ANALYSES

Abstract Background and Aims Neutrophil extracellular traps (NETs) is an important immune response against infections that is implicated in various immune-mediated conditions. Growing evidence shows that inflammation has key pathogenic contributions in diabetic kidney disease (DKD) but the role of NETs remains unclear. Method We evaluated the differentially expressed genes (DEGs) of NETs in human DKD using the bulk RNA sequencing datasets of kidney biopsy from DKD patients (GSE30528 and GSE30529). The candidate genes were further selected based on the machine learning algorithms (LASSO and SVM-RFE). The single-nucleus RNA sequencing (GSE195460 and GSE131882) and DKD bulk RNA sequencing (GSE30122) were used to validate the results. Receiver Operating Characteristics (ROC) curve were constructed to assess the diagnostic performance of the identified genes for DKD. Candidate drugs were further screened from the DSigDB database. Results Three characteristic genes (ITGAM, ITGB2 and TLR7) were selected using machine learning approach, which were all upregulated in human DKD glomerular and tubulointerstitial portion compared to healthy controls (all P <0.05). Single-cell analysis further demonstrated that ITGAM, ITGB2 and TLR7 were mainly overexpressed in leucocytes. ITGAM, ITGB2 and TLR7 expression in tubulointerstitial compartment also showed good diagnostic performance for DKD (ROC AUC 0.983, 0.975 and 0.958 respectively). Candidate drugs that target ITGAM, ITGB2 or TLR7 genes in DKD include ropivacaine, lidocaine and sulfasalazine. Conclusion ITGAM, ITGB2 and TLR7 may serve as biomarkers for DKD and drugs that target these genes may have therapeutic potential for DKD.

#3053 CORRELATION OF SYSTEMIC AND IN SITU BIOMARKERS OF COMPLEMENT ACTIVATION WITH HISTOPATHOLOGICAL FEATURES AND PROGNOSIS IN C3 GLOMERULOPATHY

Abstract Background and Aims C3 glomerulopathy is a rare complement mediated disease, resulting from complement alternative pathway (AP) activation. In addition to AP activation, there are emerging evidence for terminal pathway (TP) implication in kidney damage. A C3G histopathologic index has recently been proposed to evaluate both activity and chronicity parameters. Only a chronicity index > 4 has been independently validated as prognosis factor for end stage kidney disease. We aimed to determine the impact of local activation of the TP on disease phenotype. Method C3G histopathological index grading were evaluated via central review of 53 kidney biopsies from 43 patients carrying C3G. C5b-9 staining was performed on 53 FFPE kidney sections and quantified (scoring from grade 0 to 3 for C5b-9 deposits in glomeruli and from 0 to 2 in the tubulointerstitial compartment). Clinical data, C3 and sC5b-9 levels at the time of KB were retrospectively collected. Results KB were performed at diagnosis for n = 31. In 22 cases, KB was performed during follow up, under specific treatment (n = 10), without treatment (n = 7), or at disease relapse (n = 5). Twenty-five (47%) were children. Median[Q1-Q3] C3 level at KB was 623 mg/l [236-931]mg/l. Median[Q1-Q3] sC5b-9 level at KB, available in 16, was 374 ng/ml [203-1295]. Median [Q1-Q3] activity and chronicity index were respectively 9[6-12] and 1[0-6]. We confirmed the prognostic impact of chronicity index (p = 0.03) (Figure 1A) without impact of the activity index. Glomerular C5b-9 staining was positive in 47/53 (87%) (grade 1: n = 16 (30%); 2: n = 17 (32%); 3: n = 14 (26%)) and n = 40 (75%) had interstitial C5b-9 staining (grade 1: n = 28 (53%); 2: n = 12 (23%)). Soluble C5b-9 measurement correlated with intensity of C5b-9 glomerular deposit (coef corr = 0.69). As compared to patients with normal C3 level, patients with AP activation had higher proteinuria but kidney function at KB was similar. We found distinct histological features according to C3 plasmatic level (Table 1): patients with low C3 level had higher histological activity index (p = 0.03) and higher glomerular and tubulointerstitial C5b-9 staining (p = 0.001 and p = 0.002 respectively). C5b-9 glomeruli staining allows the identification of 3 groups of KB (1: no or low, 2: medium and 3: high C5b-9 staining) with distinct histological and clinical features. Activity index were higher in groups 2 & 3 (p = 0.05) and chronicity index in groups 1 & 3 (p = 0.003) (Table 2). Renal prognosis was poorer in patients with higher glomerular C5b-9 deposits (Figure 1B). Patients with chronicity index >4 and high C5b-9 staining had the poorest renal survival (Figure 1C). Conclusion Our results suggest that TP activation in glomeruli may have an impact on histological features, contributing therefore to renal prognosis. Deeper exploration of correlation between systemic and in situ complement activation and histological features could allow to better identify patients with worst prognosis or who could benefit from emerging complement inhibition therapeutics.

#5502 PRO-INFLAMMATORY EFFECTS OF BISPHENOL A AND POLYETHYLENE MICROPLASTICS ON HUMAN RENAL TUBULAR CELLS

Abstract Background Growing evidence shows that microplastics (MP) and their chemical derivates contaminate the environment and accumulate in the gut, liver, lungs, and kidneys. Furthermore, MP can adsorb a wide range of toxic substances (heavy metals, polycyclic aromatic hydrocarbons, Bisphenol A [B]), causing the accumulation of multiple pollutants and so, enhancing their toxicity (the so-called “Trojan Horse” effect). An epidemic of chronic kidney disease (CKD) of uncertain etiology is emerging worldwide and understanding the involvement of environmental pollution may be critical for health policies and public health responses. Inflammation is intimately linked to renal disease, and proinflammatory cytokines and chemokines are important mediators of fibrosis in the tubulointerstitial compartments. Aims To evaluate the effects of B and Polyethylene (Pe)-MPs and their combination B+Pe-MP on human renal tubular cells (HK-2). To this end, the activation of the Aryl hydrocarbon Receptor (AHR), a transcription factor binding xenobiotics, and the expression of inflammatory molecules were explored. Method HK-2 were exposed to 0 (No Treated cells: NT), 100 nM B, 0.2 mg/ml Pe-MP (diameter 1-4 µm), and B+Pe-MP for 5-24 hrs. Cell uptake of (FL)uorescent Pe-MP was visualized by microscope; viability was assessed by MTT; AHR, HSP90, CCL2, CCR2, CCL5, and CCR5 expression was studied by rt-PCR, Western blot, and immunofluorescence Results After 5 hrs exposure, a FLPe-MP cytoplasmatic and perinuclear deposition was observed (Figure 1). After 24 hrs, Pe-MP and B+Pe-MP exposition reduced cell viability (−20% vs NT p < 0.001). The treatments increased AHR expression (1.5-3-fold vs NT, p < 0.05-0.001), which had a nuclear localization both at 5 and 24 hrs. Conversely, HSP90 expression, a chaperone that keeps AHR inactive, decreased (−20-40% vs NT, p < 0.05) (Figure 2). After 5 hrs, CCL2 levels rose (1.8-3.5 fold, p<0.05-0.0001 vs NT) as well as its receptor CCR2 (2.3-6.7 fold, p<0.05-0.01). Similarly, CCL5 and CCR5 were, respectively, 1.6-2.7 and 3.8-8.4 fold overexpressed with respect to NT (p < 0.05-0.01). Interestingly, the co-exposure significantly enhanced the expression of these inflammatory molecules Conclusion Our data suggest that B and Pe-MPs induce a pro-inflammatory response in renal tubular cells and that the combined effect of B and Pe-MPs induces a worse effect on HK2 than B or MPs alone, supporting the theory of the “trojan horse” effect mediated by MPs. Therefore, MPs could be a trigger of kidney damage, thus confirming the potential impact of the environment on the pathogenesis of the renal disease. Moreover, these data could address the importance of investigating any health implications associated with MPs.

#5295 SECONDARY MEMBRANOUS NEPHROPATHY WITH IGA DEPOSITS IN A PATIENT WITH PRIMARY CILIARY DYSKINESIA

Abstract Background and Aims Membranous nephropathy and IgA nephropathy are two types of common glomerulonephritis worldwide. There are reports of the coexistence of these glomerulopathies, mostly with mesangial expansion and IgA deposits. Furthermore, primary ciliary dyskinesia is rarely associated to glomerulopathy. Thus, we report a case of a patient with primary ciliary dyskinesia with renal biopsy demonstrating membranous glomerulopathy associated with IgA and IgG deposits exclusively in glomerular basement membrane, without complement deposition. Method Case report. Results A 18 years-old man was referred from the nephropediatric outpatient clinic with subnephrotic hematuria and proteinuria. His medical history included a clinical diagnosis of primary ciliary dyskinesia due to a history of several respiratory infections since birth, asthma, rhinitis and bronchiectasis and a 15 years-old brother with a similar history. He was diagnosed with idiopathic thrombocytopenic purpura at age of one year-old and received prednisone. At 3 years-old he presented with nephrotic syndrome, hematuria and hypertension, and was treated with intravenous methylprednisolone and oral cyclophosphamide for three months. Renal biopsy demonstrated mesangial expansion and thickening of the glomerular basement membrane (GBM), without adequate material for immunofluorescence. Because proteinuria and hematuria persisted, the prednisone dose was increased to 2 mg/kg/day and cyclosporine was started. After 5 years on these medications, persisting with proteinuria, a second biopsy was performed and showed 16 glomeruli, three globally sclerosed and the others with diffuse thickening of the GBM, without other alterations, focal interstitial fibrosis and tubular atrophy and diffuse granular deposits of IgA 1+ and IgG 2+ along the GBM. Cyclosporine and prednisone were discontinued. At age of 18 years-old, he was admitted to adult glomerulonephritis clinic with significant developmental delay (weight 28.6kg and height 1.29m2), no edema and normal arterial blood pressure with RAAS inhibition. Laboratory tests: Hb 8.9 g/dL, serum iron 12 mcg/dL, Creatinine 0.7 mg/dL, albumine 2.3 g/dL. Serologies were negative for syphilis, hepatitis B and C, HIV, ANA and anti-DNA, with normal complement C3, C4 and C1q. Urinalysis revealed proteinuria 1+, hematuria 16.000/mL and proteinuria 1.400 mg/d. CT scan showed symmetrical bronchiectasis in both lungs, with mucoid endobronchial impingement and diffuse bronchial parietal thickening; and chronic post-thrombotic changes with calcified thrombi in the left renal vein, extending into the inferior vena cava. A third renal biopsy was performed showing 38 of 45 (84%) globally sclerosed glomeruli without mesangial matrix changes or capsular adhesions. GBM was thickened with spicules and holes and reactive podocytes. The tubulointerstitial compartment showed moderate atrophy and fibrosis (40-50%). Immunofluorescence microscopy evidenced strong immunoreactivity of IgG 3+/3+, IgA 3+/3+, Kappa 3+/3+ and Lambda 2+/3+, in granular pattern exclusively in GBM, without C3. Electron microscopy showed four sclerosed glomeruli, without deposits. Immunohistochemistry staining for IgG1 2+/3+, and it was negative for PLA2R, NELL-1, THSD7A, Exostosin-1 and 2, IgG 2, IgG3 and IgG4. The patient was maintained with supportive treatment, with RAAS inhibition and SGLT2 inhibitors, without immunosuppression. Conclusion This case highlights the codominance of IgG1 and IgA deposits along the MBG, without proliferation or mesangial deposition and without complement activation, in a patient with membranous nephropathy and primary ciliary dyskinesia, a condition rarely associated with glomerulopathies. Although rare, such renal biopsy findings reinforce the need for a better understanding of the different mechanisms of membranous nephritis, in addition to better understanding and management of treatment in this situation.

A case of acute tubulointerstitial nephritis following administration of the Oxford-AstraZeneca COVID-19 vaccine: a case report

BackgroundMore than 4 billion doses of the Coronavirus disease (COVID-19) vaccine have been administered worldwide but the relationship between the different vaccines and the development of renal disease is unknown. We present a case of tubulointerstitial nephritis following administration of the Oxford-AstraZeneca COVID-19 vaccine.Case presentationA previously fit and well 51-year-old female presented on 27th May 2021 with a one-month history of weight loss, fatigue, nausea, and a metallic taste. She had an acute kidney injury with a creatinine of 484 umol/L. She was on no regular medications and denied taking any over-the-counter or alternative medicines. She had received her first dose of the Oxford-AstraZeneca vaccine on 23rd March 2021 and her second dose on 20th May 2021. A renal biopsy was performed the following day. The 19 glomeruli appeared normal to light microscopy but the tubulointerstitial compartment contained a dense inflammatory infiltrate including many eosinophils. There was widespread acute tubular injury with tubulitis, but no established or longstanding atrophy. A diagnosis was made of an acute tubulointerstitial nephritis. She was commenced on oral prednisolone and her renal function improved. She did not require renal replacement therapy at any time.ConclusionsTo our knowledge, this was the first described case of acute tubulointerstitial nephritis following administration of the Oxford-AstraZeneca COVID-19 vaccine, although a number of cases have emerged more recently. In our case the patient was very fit and well, had no previous past medical history and had not taken any recent prescribed, over-the-counter or alternative medications. The absence of these provoking factors in our case makes the vaccine the most likely explanation for the development of tubulointerstitial nephritis although the pathophysiology behind this remains unknown. Given the unprecedented number of vaccinations being delivered around the world, nephrologists should be aware of this possible link although more research into the topic is required.