The gold standard for diagnosing lupus nephritis is the renal biopsy. While it has several significant benefits, this procedure is not without risk (i.e. bleeding) and only interrogates a minute portion of the renal parenchyma. Recently, near-infrared fluorescent (NIRF) probes have been utilized to image protein activity noninvasively in animals and humans. We hypothesized that specific inflammatory processes observed in nephritis can be noninvasively detected and monitored using NIRF-based optical imaging approaches. Using a probe that becomes fluorescent in the presence of the protease cathepsin B (CatB), we tested the ability of using NIR optical imaging to assess renal inflammation as a noninvasive marker for early-stage glomerulonephritis (GN).
Methods
Experimental GN was induced in 129 mice by nephrotoxic serum (NTS) delivered intravenously. Proteinuria was quantified using albumin ELISA and chromogenic creatinine assay. NIR optical imaging of anesthetized mice was performed following intravenous administration of a cleavable sensor for CatB and fluorescence intensity of kidney regions quantified using fluorescence molecular tomography (FMT)−3000 instrument at days 1 to 10 post-NTS administration.
Results
In NTS-treated mice, a strong signal from the CatB-activatable probe was observed as early as day 1, which associated with the onset of proteinuria (figure 1). This signal could be detected for at least 10 days. In contrast, control mice were devoid of any CatB signal. To assess the specificity of CatB signal to GN, we examined CD2AP KO mice that develop nephrotic syndrome in the absence of inflammation. CD2AP KO had no CatB signal despite ongoing nephrosis.
Conclusions
Induction of GN by NTS was specifically detected noninvasively using a CatB-activatable probe and NIR optical imaging. These data establish the proof-of-principle that novel noninvasive tomographic approaches may represent a translatable approach to establishing early stages of GN. We believe that this approach can be expanded to other experimental imaging approaches, such as photoacoustics, as a novel method for detecting lupus nephritis in humans.
Funding Source(s):
Department of Defense Discovery Award #W81XWH-17–1–0128 (PI: Kim) Representative images of a control mouse and a mouse treated with nephrotoxic serum to induce glomerulonephritis (NTS). A strong fluorescence signal induced by cathepsin B is observed in NTS-treated mice only.
Abstract Introduction Kidney resident macrophages (KRMs) are important in renal homeostasis and the response to acute kidney injury. Preliminary data suggests that the KRM population consists of several undescribed subpopulations. Here, we combined single-cell RNA sequencing (scRNAseq) and spatial transcriptomics to identify and localize KRM subpopulations during homeostasis and injury. Methods KRMs were isolated from C57BL/6J mice without treatment and after bilateral ischemia-reperfusion injury (BIRI) as well as from a human donor kidney. ScRNAseq was performed using the 10X Genomics Chromium platform and spatial transcriptomics using the 10X Visium platform. Gene expression data were integrated and analyzed using the R package, Seurat 4.0. Results UMAP plots of integrated data revealed seven major clusters of mouse KRMs with unique transcriptional profiles associated with distinct functions. Spatial transcriptomics revealed that these clusters reside in distinct cellular compartments within the kidney and appear to associate with specific renal structures. Following BIRI, these subpopulations appear in cellular compartments distinct from those occupied in the controls. Several human KRM clusters were correlated with those of the mouse and localized to specific regions of the kidney. Conclusion Transcriptionally distinct subpopulations of mouse KRMs reside within specific kidney microenvironments and change location as a function of injury. Similar subpopulations of KRMs were identified in the human kidney as well. Therefore, further study of the temporal and spatial characteristics and signaling pathways of these subpopulations in the context of homeostasis and injury is warranted. Supported by grants from NIH (R01-DK-59600, T32-AI007051)
AKI is a common sequela of infection with SARS-CoV-2 and contributes to the severity and mortality from COVID-19. Here, we tested the hypothesis that kidney alterations induced by COVID-19-associated AKI could be detected in cells collected from urine.
Abstract Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression may be able to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.
The success of B cell depletion therapies and identification of leptomeningeal ectopic lymphoid tissue (ELT) in patients with multiple sclerosis (MS) has renewed interest in the antibody-independent pathogenic functions of B cells during neuroinflammation. The timing and location of B cell antigen presentation during MS and its animal model experimental autoimmune encephalomyelitis (EAE) remain undefined. Using a new EAE system that incorporates temporal regulation of MHCII expression by myelin-specific B cells, we observed the rapid formation of large B cell clusters in the spinal cord subarachnoid space. Neutrophils preceded the accumulation of meningeal B cell clusters, and inhibition of CXCR2-mediated granulocyte trafficking to the central nervous system reduced pathogenic B cell clusters and disease severity. Further, B cell-restricted very late antigen-4 (VLA-4) deficiency abrogated EAE dependent on B cell antigen presentation. Together, our findings demonstrate that neutrophils coordinate VLA-4–dependent B cell accumulation within the meninges during neuroinflammation, a key early step in the formation of ELT observed in MS.
Glomerulonephritis (GN) represents a major cause of morbidity and mortality in many conditions such as systemic lupus erythematosus. The standard for diagnosing GN is through renal biopsy, but this is not performed uniformly across many centers. Furthermore, it is well established that early treatment of GN improves complication and survival outcomes (1,2). While proteinuria and/or hematuria is typically the first clinical sign of GN, immunologic changes in the kidney such as macrophage infiltration is observed well before. Thus, there is an unmet need to identify a noninvasive approach for recognizing GN, especially the preclinical stages. Recent advances in deep tissue imaging using probes detected by near-infrared (NIR) wavelengths have enabled the noninvasive probing of biologic activity. Activated macrophages that infiltrate the kidney early in GN express the papain-like cysteine protease cathepsin B (3). Thus, renal macrophage activation can be assessed using an NIR probe that becomes fluorescent upon cleavage by cathepsin B.
Objectives
We tested whether NIR optical imaging can assess renal macrophage activation as a noninvasive marker for early-stage GN.
Methods
GN was induced in 129 mice by nephrotoxic serum (NTS) delivered intravenously. Proteinuria was quantitated using albumin ELISA and chromogenic creatinine assay. Presence of renal macrophages was confirmed using FACS. NIR optical imaging of anesthetized mice was performed following intravenous administration of a cleavable sensor for cathepsin B and fluorescence intensity of kidney regions quantified using the Perkin Elmer FMT4000 fluorescence molecular tomography device.
Results
In mice with uninflamed kidneys, we confirmed the paucity of renal macrophages. Accordingly, there was minimal renal fluorescence signal as determined by fluorescent molecular imaging of cathepsin B activity. 3 days post-NTS administration, we observed a massive influx of macrophages into the kidney by flow cytometry, which correlated with a significant increase in renal fluorescence intensity signal in NTS mice compared to control mice. This was followed by the onset of proteinuria on days 5–7 in NTS mice.
Conclusions
Induction of GN by NTS caused significant macrophage infiltration, which could be detected noninvasively by a cathepsin B-activatable probe and NIR optical imaging. Importantly, detection of activated macrophages preceded the onset of proteinuria by several days. These data establish the proof-of-principle that NIR optical imaging may represent a translatable approach to detecting the preclinical stages of GN.
References
Fiehn C, et al., Ann Rheum Dis, 62:435, 2003. Faurschou M, et al., J Rheumatol, 33:8, 2006. Bethunaickan R, et al., J Immunol, 186:4994, 2011.
Acknowledgement
A.H.J.K. would like to acknowledge the Rheumatology Research Foudation for funding through an Investigator Award.
Disclosure of Interest
S. Braehler: None declared, M. Cheung: None declared, D. Huang: None declared, W. Akers: None declared, A. Kim Grant/research support from: Rheumatology Research Foundation, Kypha Inc.
IntroductionAcute kidney injury (AKI) is common in COVID-19 and associated with increased morbidity and mortality. We investigated alterations in the urine metabolome to test the hypothesis that impaired nicotinamide adenine dinucleotide (NAD+) biosynthesis and other deficiencies in energy metabolism in the kidney, previously characterized in ischemic, toxic, and inflammatory etiologies of AKI, will be present in COVID-19–associated AKI.MethodsThis is a case-control study among the following 2 independent populations of adults hospitalized with COVID-19: a critically ill population in Boston, Massachusetts, and a general population in Birmingham, Alabama. The cases had AKI stages 2 or 3 by Kidney Disease Improving Global Outcomes (KDIGO) criteria; the controls had no AKI. Metabolites were measured by liquid chromatography–mass spectrometry.ResultsA total of 14 cases and 14 controls were included from Boston and 8 cases and 10 controls from Birmingham. Increased urinary quinolinate-to-tryptophan ratio (Q/T), found with impaired NAD+ biosynthesis, was present in the cases at each location and pooled across locations (median [interquartile range]: 1.34 [0.59–2.96] in cases, 0.31 [0.13–1.63] in controls, P = 0.0013). Altered energy metabolism and purine metabolism contributed to a distinct urinary metabolomic signature that differentiated patients with and without AKI (supervised random forest class error: 2 of 28 in Boston, 0 of 18 in Birmingham).ConclusionUrinary metabolites spanning multiple biochemical pathways differentiate AKI versus non-AKI in patients hospitalized with COVID-19 and suggest a conserved impairment in NAD+ biosynthesis, which may present a novel therapeutic target to mitigate COVID-19–associated AKI.
