BackgroundMacrophage infiltration in lupus nephritis is associated with fibrosis and kidney damage. Prior histologic studies lacked the specificity of single-cell RNA sequencing (scRNA-seq) for ...macrophage classification, so it was impossible to determine how the spatial organization of each subset related to kidney remodeling. Our recent scRNA-seq of macrophages has defined 3 novel subsets enriched in lupus nephritis over healthy kidneys: ‘inflammatory’ macrophages likely enter lupus kidneys from blood and shift to ‘phagocytic’ and ‘reparative-like’ states. Here, we mapped their positions in kidney sections from 20 different lupus nephritis patients using subset-specific transcripts from our scRNA-seq data to reveal new macrophage spatial phenotypes.MethodsWe collected and sectioned archived FFPE class III or IV index lupus nephritis kidney biopsies from Brigham and Women’s Hospital. After standard FFPE antigen retrieval, we used commercial RNA probes against 2-3 highly specific genes based on scRNA-seq to stain each novel subset (inflammatory CSF1R+/CD300E+/CD36-, phagocytic CSF1R+/CD300E+/CD36+; reparative CSF1R+/RNASE1+), and probes against non-mammalian genes as a negative control. We identified our macrophage subsets based on the presence of probes above the background and within 3 microns of the DAPI-stained nucleus as a cell boundary estimate. For spatial mapping, we transferred annotated histologic features from an adjacent H&E section to sections stained for cells. For cell distance and density measurements we used HALO (Indica Labs).ResultsOur in situ staining approach confirmed the presence of the novel inflammatory, phagocytic, and reparative macrophages discovered by scRNA-seq in class III and IV lupus nephritis kidney sections. Most inflammatory and phagocytic macrophages were localized to positions inside glomeruli while a smaller proportion in the tubulointerstitium formed a gradient toward the glomerular borders. Reparative macrophages were the most abundant macrophage subset in situ and were mostly in the tubulointerstitium arranged as a gradient toward glomerular borders. The abundance of reparative macrophages inside glomeruli varied across patients.ConclusionsMacrophage subsets were spatially localized to and around the glomerulus in lupus nephritis kidney sections. Inflammatory and phagocytic macrophage subsets were mostly inside the glomerulus, suggesting that glomerular factors supported their recruitment from blood and in situ differentiation. The most abundant subset - reparative macrophages - were localized to the tubulointerstitial space and arranged in a gradient toward glomerular borders, indicating a chemical attraction to nephritic glomeruli and the presence of factors that promote reparative differentiation. Interestingly, the abundance of reparative macrophages inside glomeruli varied considerably across patients, raising the possibility that interpatient variability reflects differences in kidney function that we are now testing in an expanded cohort.AcknowledgmentsRheumatology Research Foundation, Lupus Research Alliance, Lupus Foundation of America
The current classification system for focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) does not fully capture the complex structural changes in kidney biopsies nor the ...clinical and molecular heterogeneity of these diseases.
Prospective observational cohort study.
221 MCD and FSGS patients enrolled in the Nephrotic Syndrome Study Network (NEPTUNE).
The NEPTUNE Digital Pathology Scoring System (NDPSS) was applied to generate scores for 37 glomerular descriptors.
Time from biopsy to complete proteinuria remission, time from biopsy to kidney disease progression (40% estimated glomerular filtration rate eGFR decline or kidney failure), and eGFR over time.
Cluster analysis was used to group patients with similar morphologic characteristics. Glomerular descriptors and patient clusters were assessed for associations with outcomes using adjusted Cox models and linear mixed models. Messenger RNA from glomerular tissue was used to assess differentially expressed genes between clusters and identify genes associated with individual descriptors driving cluster membership.
Three clusters were identified: X (n = 56), Y (n = 68), and Z (n = 97). Clusters Y and Z had higher probabilities of proteinuria remission (HRs of 1.95 95% CI, 0.99-3.85 and 3.29 95% CI, 1.52-7.13, respectively), lower hazards of disease progression (HRs of 0.22 95% CI, 0.08-0.57 and 0.11 95% CI, 0.03-0.45, respectively), and lower loss of eGFR over time compared with X. Cluster X had 1,920 genes that were differentially expressed compared with Y+Z; these reflected activation of pathways of immune response and inflammation. Six descriptors driving the clusters individually correlated with clinical outcomes and gene expression.
Low prevalence of some descriptors and biopsy at a single time point.
The NDPSS allows for categorization of FSGS/MCD patients into clinically and biologically relevant subgroups, and uncovers histologic parameters associated with clinical outcomes and molecular signatures not included in current classification systems.
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Maintenance of systemic homeostasis by kidney requires the coordinated response of diverse cell types. The use of single‐cell RNA sequencing (scRNAseq) for patient tissue samples remains fraught with ...difficulties with cell isolation, purity, and experimental bias. The ability to characterize immune and parenchymal cells during transplant rejection will be invaluable in defining transplant pathology where tissue availability is restricted to needle biopsy fragments. Herein, we present feasibility data for multiplexing approach for droplet scRNAseq (Mux‐Seq). Mux‐Seq has the potential to minimize experimental batch bias and variation even with very small sample input. In this first proof‐of‐concept study for this approach, explant tissues from six normal and two transplant recipients after multiple early post‐transplant rejection episodes leading to nephrectomy due to aggressive antibody mediated rejection, were pooled for Mux‐Seq. A computational tool, Demuxlet was applied for demultiplexing the individual cells from the pooled experiment. Each sample was also applied individually in a single microfluidic run (singleplex) to correlate results with the pooled data from the same sample. Our applied protocol demonstrated that data from Mux‐Seq correlated highly with singleplex (Pearson coefficient 0.982) sequencing results, with the ability to identify many known and novel kidney cell types including different infiltrating immune cells. Trajectory analysis of proximal tubule and endothelial cells demonstrated separation between healthy and injured kidney from transplant explant suggesting evolving stages of cell‐ specific differentiation in alloimmune injury. This study provides the technical groundwork for understanding the pathogenesis of alloimmune injury and host tissue response in transplant rejection and normal human kidney and provides a protocol for optimized processing precious and low input human kidney biopsy tissue for larger scale studies.
Multi‐ and singleplex single cell RNA sequencing of normal compared to allograft rejection human kidney tissue uncovers multiple novel kidney cell types and states, with perturbations of cell fate in transplant rejection.
Apolipoprotein L1 (APOL1)-associated focal segmental glomerulosclerosis (FSGS) is the dominant form of FSGS in Black individuals. There are no targeted therapies for this condition, in part because ...the molecular mechanisms underlying APOL1’s pathogenic contribution to FSGS are incompletely understood. Studying the transcriptomic landscape of APOL1 FSGS in patient kidneys is an important way to discover genes and molecular behaviors that are unique or most relevant to the human disease. With the hypothesis that the pathology driven by the high-risk APOL1 genotype is reflected in alteration of gene expression across the glomerular transcriptome, we compared expression and co-expression profiles of 15,703 genes in 16 Black patients with FSGS at high-risk vs 14 Black patients with a low-risk APOL1 genotype. Expression data from APOL1-inducible HEK293 cells and normal human glomeruli were used to pursue genes and molecular pathways uncovered in these studies. We discovered increased expression of APOL1 and nine other significant differentially expressed genes in high-risk patients. This included stanniocalcin, which has a role in mitochondrial and calcium-related processes along with differential correlations between high- and low-risk APOL1 and metabolism pathway genes. There were similar correlations with extracellular matrix- and immune-related genes, but significant loss of co-expression of mitochondrial genes in high-risk FSGS, and an NF-κB-down regulating gene, NKIRAS1, as the most significant hub gene with strong differential correlations with NDUF family (mitochondrial respiratory genes) and immune-related (JAK-STAT) genes. Thus, differences in mitochondrial gene regulation appear to underlie many differences observed between high- and low-risk Black patients with FSGS.
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Background
In the current study, longitudinal BP and lipid measurements were examined in a NEPTUNE cohort of children with newly diagnosed nephrotic syndrome (cNEPTUNE). We hypothesized that ...hypertensive BP and dyslipidemia would persist in children with nephrotic syndrome, regardless of steroid treatment response.
Methods
A multi-center longitudinal observational analysis of data obtained from children < 19 years of age with new onset nephrotic syndrome enrolled in the Nephrotic Syndrome Study Network (cNEPTUNE) was conducted. BP and lipid data were examined over time stratified by disease activity and steroid exposure. Generalized estimating equation regressions were used to find determinants of hypertensive BP and dyslipidemia.
Results
Among 122 children, the prevalence of hypertensive BP at any visit ranged from 17.4% to 57.4%, while dyslipidemia prevalence ranged from 40.0% to 96.2% over a median of 30 months of follow-up. Hypertensive BP was found in 46.2% (116/251) of study visits during active disease compared with 31.0% (84/271) of visits while in remission. Dyslipidemia was present in 88.2% (120/136) of study visits during active disease and in 66.0% (101/153) while in remission. Neither dyslipidemia nor hypertensive BP were significantly different with/without medication exposure (steroids and/or CNI). In regression analysis, male sex and urine protein:creatinine ratio (UPC) were significant determinants of hypertensive BP over time, while eGFR was found to be a determinant of dyslipidemia over time.
Conclusions
Results demonstrate persistent hypertensive BPs and unfavorable lipid profiles in the cNEPTUNE cohort regardless of remission status or concurrent steroid or calcineurin inhibitor treatment.
Graphical abstract
A higher resolution version of the Graphical abstract is available as
Supplementary information
Lupus nephritis (LN) is a pathologically heterogenous autoimmune disease linked to end-stage kidney disease and mortality. Better therapeutic strategies are needed as only 30%-40% of patients ...completely respond to treatment. Noninvasive biomarkers of intrarenal inflammation may guide more precise approaches. Because urine collects the byproducts of kidney inflammation, we studied the urine proteomic profiles of 225 patients with LN (573 samples) in the longitudinal Accelerating Medicines Partnership in RA/SLE cohort. Urinary biomarkers of monocyte/neutrophil degranulation (i.e., PR3, S100A8, azurocidin, catalase, cathepsins, MMP8), macrophage activation (i.e., CD163, CD206, galectin-1), wound healing/matrix degradation (i.e., nidogen-1, decorin), and IL-16 characterized the aggressive proliferative LN classes and significantly correlated with histological activity. A decline of these biomarkers after 3 months of treatment predicted the 1-year response more robustly than proteinuria, the standard of care (AUC: CD206 0.91, EGFR 0.9, CD163 0.89, proteinuria 0.8). Candidate biomarkers were validated and provide potentially treatable targets. We propose these biomarkers of intrarenal immunological activity as noninvasive tools to diagnose LN and guide treatment and as surrogate endpoints for clinical trials. These findings provide insights into the processes involved in LN activity. This data set is a public resource to generate and test hypotheses and validate biomarkers.
We conducted this study to test the hypothesis that plasma zonulin levels are elevated in pediatric patients with nephrotic syndrome compared to healthy controls.
Plasma zonulin levels were measured ...by ELISA in 114 children enrolled in the NEPTUNE study. Clinical and laboratory data were retrieved from the NEPTUNE database.
The median age of the patients was 10 (IQR = 5 to 14) years, 59 were male, 64 had minimal change disease, 47 focal segmental glomerulosclerosis, median eGFR was 96 (IQR = 80 to 114) ml/min/1.73 m
, and median urine protein:creatinine ratio was 0.5 (IQR = 0.1 to 3.4) (g:g). The plasma zonulin level was 14.2 ± 5.0 vs. 10.2 ± 2.5 ng/ml in healthy adults in a report using the same assay kit,
= 0.0025. These findings were confirmed in an independent cohort of children with nephrotic syndrome compared to healthy age-matched controls,
= 0.01. Zonulin concentrations did not differ in children with minimal change disease vs. focal segmental glomerulosclerosis, frequently relapsing vs. steroid-dependent vs. steroid-resistant clinical course, and were not influenced by the immunosuppressive treatment regimen. There was no relationship between plasma zonulin levels and the absolute or percentage change in proteinuria from enrollment until the time of the zonulin assay.
Plasma zonulin levels are elevated in childhood nephrotic syndrome regardless of level of proteinuria or specific treatment. The cause of the high plasma zonulin levels and whether zonulin contributes to glomerular injury requires further study.
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