Odorant Receptors Expressing and Antennal Lobes Architecture Are Linked to Caste Dimorphism in Asian Honeybee, Apis cerana (Hymenoptera: Apidae).

Insects heavily rely on the olfactory system for food, mating, and predator evasion. However, the caste-related olfactory differences in Apis cerana, a eusocial insect, remain unclear. To explore the peripheral and primary center of the olfactory system link to the caste dimorphism in A. cerana, transcriptome and immunohistochemistry studies on the odorant receptors (ORs) and architecture of antennal lobes (ALs) were performed on different castes. Through transcriptomesis, we found more olfactory receptor genes in queens and workers than in drones, which were further validated by RT-qPCR, indicating caste dimorphism. Meanwhile, ALs structure, including volume, surface area, and the number of glomeruli, demonstrated a close association with caste dimorphism. Particularly, drones had more macroglomeruli possibly for pheromone recognition. Interestingly, we found that the number of ORs and glomeruli ratio was nearly 1:1. Also, the ORs expression distribution pattern was very similar to the distribution of glomeruli volume. Our results suggest the existence of concurrent plasticity in both the peripheral olfactory system and ALs among different castes of A. cerana, highlighting the role of the olfactory system in labor division in insects.

Circuit formation and sensory perception in the mouse olfactory system.

In the mouse olfactory system, odor information is converted to a topographic map of activated glomeruli in the olfactory bulb (OB). Although the arrangement of glomeruli is genetically determined, the glomerular structure is plastic and can be modified by environmental stimuli. If the pups are exposed to a particular odorant, responding glomeruli become larger recruiting the dendrites of connecting projection neurons and interneurons. This imprinting not only increases the sensitivity to the exposed odor, but also imposes the positive quality on imprinted memory. External odor information represented as an odor map in the OB is transmitted to the olfactory cortex (OC) and amygdala for decision making to elicit emotional and behavioral outputs using two distinct neural pathways, innate and learned. Innate olfactory circuits start to work right after birth, whereas learned circuits become functional later on. In this paper, the recent progress will be summarized in the study of olfactory circuit formation and odor perception in mice. We will also propose new hypotheses on the timing and gating of olfactory circuit activity in relation to the respiration cycle.

Development of Drug Efficacy Testing Platform for Glomerulonephritis.

We developed a 3D glomeruli tissue chip for glomerulonephritis (GN) testing, featuring a gravity-driven glomerular filtration barrier (GFB) with human podocytes and endothelial cells with a bidirectional flow in the bottom channel. Using puromycin-induced GN, we observed decreased cell viability, increased albumin permeability, and reduced WT1 and nephrin compared to the normal GFB. Tacrolimus restored cell viability, reduced albumin permeability, and increased WT1 expression. Using serum from five membranous nephropathy (MN) patients, we created MN models using a GFB-mimicking chip. A notable decline in cell viability was observed in the serum-induced MN1 and MN2 models. However, tacrolimus restored it. Albumin permeability was reduced in the MN1, MN2, and MN5 models by tacrolimus treatment. MN1 displayed the best clinical response to tacrolimus, exhibiting increased expression of WT1 in chip-based evaluations after tacrolimus treatment. We successfully evaluated the efficacy of tacrolimus using puromycin-induced and serum-induced GN models on a chip that mimicked the structure and function of the GFB. The GFB-mimicking chip holds promise as a personalized platform for assessing drug efficacy using patient serum samples.

Evaluation of the Risk Prediction Models in Predicting Kidney Outcomes in Antiglomerular Basement Membrane Disease.

Introduction: A previous study showed that the renal risk score (RRS) was transferrable to antiglomerular basement membrane (anti-GBM) disease and proposed a risk stratification according to the need of renal replacement therapy (RRT) and the percentage of normal glomeruli (N). Herein, we analyzed the risk factors associated with kidney outcomes in patients with biopsy-proven anti-GBM disease and evaluated these 2 prognosis systems. Methods: A total of 120 patients with biopsy-proven anti-GBM disease with complete clinicopathologic and outcome data were analyzed. Results: The median time to kidney biopsy was 41 days (interquartile range [IQR]: 22-63 days). RRT and N were the only independent predictors of end-stage kidney disease (ESKD). Patients with N ≥10% were more likely to achieve ESKD-free outcomes, even in the subcohort of patients who underwent posttreatment biopsies (P < 0.001). N and serum creatinine at presentation (cut-off values 750 µmol/l and 1300 µmol/l) were 2 independent factors for predicting kidney recovery. The RRS and the risk stratification tool exhibited predictive value for ESKD and could be transferred to patients with kidney biopsy following treatment (Harrell's C statistic [C] = 0.738 and C = 0.817, respectively). However, a cross-over of outcomes among groups was observed in the risk stratification tool in long-term follow-up, when patients with RRT and N ≥10% achieved better kidney outcomes than those without RRT but N <10%. Conclusion: Normal glomeruli percentage, even posttreatment, was a strong indicator for kidney outcomes, especially on long-term prognosis. Serum creatinine is a predictor for kidney recovery, independent of biopsy findings. The risk stratification tool for kidney survival was transferrable to patients with anti-GBM disease with biopsy following treatment in our cohort; however, this needs further validations for long-term outcomes.

What are olfaction and gustation, and do all animals have them?

Different animals have distinctive anatomical and physiological properties to their chemical senses that enhance detection and discrimination of relevant chemical cues. Humans and other vertebrates are recognized as having 2 main chemical senses, olfaction and gustation, distinguished from each other by their evolutionarily conserved neuroanatomical organization. This distinction between olfaction and gustation in vertebrates is not based on the medium in which they live because the most ancestral and numerous vertebrates, the fishes, live in an aquatic habitat and thus both olfaction and gustation occur in water and both can be of high sensitivity. The terms olfaction and gustation have also often been applied to the invertebrates, though not based on homology. Consequently, any similarities between olfaction and gustation in the vertebrates and invertebrates have resulted from convergent adaptations or shared constraints during evolution. The untidiness of assigning olfaction and gustation to invertebrates has led some to recommend abandoning the use of these terms and instead unifying them and others into a single category-chemical sense. In our essay, we compare the nature of the chemical senses of diverse animal types and consider their designation as olfaction, oral gustation, extra-oral gustation, or simply chemoreception. Properties that we have found useful in categorizing chemical senses of vertebrates and invertebrates include the nature of peripheral sensory cells, organization of the neuropil in the processing centers, molecular receptor specificity, and function.

Spatial top-down proteomics for the functional characterization of human kidney.

Background: The Human Proteome Project has credibly detected nearly 93% of the roughly 20,000 proteins which are predicted by the human genome. However, the proteome is enigmatic, where alterations in amino acid sequences from polymorphisms and alternative splicing, errors in translation, and post-translational modifications result in a proteome depth estimated at several million unique proteoforms. Recently mass spectrometry has been demonstrated in several landmark efforts mapping the human proteoform landscape in bulk analyses. Herein, we developed an integrated workflow for characterizing proteoforms from human tissue in a spatially resolved manner by coupling laser capture microdissection, nanoliter-scale sample preparation, and mass spectrometry imaging. Results: Using healthy human kidney sections as the case study, we focused our analyses on the major functional tissue units including glomeruli, tubules, and medullary rays. After laser capture microdissection, these isolated functional tissue units were processed with microPOTS (microdroplet processing in one-pot for trace samples) for sensitive top-down proteomics measurement. This provided a quantitative database of 616 proteoforms that was further leveraged as a library for mass spectrometry imaging with near-cellular spatial resolution over the entire section. Notably, several mitochondrial proteoforms were found to be differentially abundant between glomeruli and convoluted tubules, and further spatial contextualization was provided by mass spectrometry imaging confirming unique differences identified by microPOTS, and further expanding the field-of-view for unique distributions such as enhanced abundance of a truncated form (1-74) of ubiquitin within cortical regions. Conclusions: We developed an integrated workflow to directly identify proteoforms and reveal their spatial distributions. Where of the 20 differentially abundant proteoforms identified as discriminate between tubules and glomeruli by microPOTS, the vast majority of tubular proteoforms were of mitochondrial origin (8 of 10) where discriminate proteoforms in glomeruli were primarily hemoglobin subunits (9 of 10). These trends were also identified within ion images demonstrating spatially resolved characterization of proteoforms that has the potential to reshape discovery-based proteomics because the proteoforms are the ultimate effector of cellular functions. Applications of this technology have the potential to unravel etiology and pathophysiology of disease states, informing on biologically active proteoforms, which remodel the proteomic landscape in chronic and acute disorders.

Complement Cascade Proteins Correlate with Fibrosis and Inflammation in Early-Stage Type 1 Diabetic Kidney Disease in the Ins2Akita Mouse Model.

Diabetic kidney disease (DKD) is characterized by histological changes including fibrosis and inflammation. Evidence supports that DKD is mediated by the innate immune system and more specifically by the complement system. Using Ins2Akita T1D diabetic mice, we studied the connection between the complement cascade, inflammation, and fibrosis in early DKD. Data were extracted from a previously published quantitative-mass-spectrometry-based proteomics analysis of kidney glomeruli of 2 (early DKD) and 4 months (moderately advanced DKD)-old Ins2Akita mice and their controls A Spearman rho correlation analysis of complement- versus inflammation- and fibrosis-related protein expression was performed. A cross-omics validation of the correlation analyses' results was performed using public-domain transcriptomics datasets (Nephroseq). Tissue sections from 43 patients with DKD were analyzed using immunofluorescence. Among the differentially expressed proteins, the complement cascade proteins C3, C4B, and IGHM were significantly increased in both early and later stages of DKD. Inflammation-related proteins were mainly upregulated in early DKD, and fibrotic proteins were induced in moderately advanced stages of DKD. The abundance of complement proteins with fibrosis- and inflammation-related proteins was mostly positively correlated in early stages of DKD. This was confirmed in seven additional human and mouse transcriptomics DKD datasets. Moreover, C3 and IGHM mRNA levels were found to be negatively correlated with the estimated glomerular filtration rate (range for C3 rs = -0.58 to -0.842 and range for IGHM rs = -0.6 to -0.74) in these datasets. Immunohistology of human kidney biopsies revealed that C3, C1q, and IGM proteins were induced in patients with DKD and were correlated with fibrosis and inflammation. Our study shows for the first time the potential activation of the complement cascade associated with inflammation-mediated kidney fibrosis in the Ins2Akita T1D mouse model. Our findings could provide new perspectives for the treatment of early DKD as well as support the use of Ins2Akita T1D in pre-clinical studies.

Adverse effects of acute tubular injury on the glomerulus: contributing factors and mechanisms.

The intricate relationship between tubular injury and glomerular dysfunction in kidney diseases has been a subject of extensive research. While the impact of glomerular injury on downstream tubules has been well-studied, the reverse influence of tubular injury on the glomerulus remains less explored. This paper provides a comprehensive review of recent advances in the field, focusing on key pathways and players implicated in the pathogenesis of tubular injury on glomerular dysfunction. Anatomical and physiological evidence supports the possibility of crosstalk from the tubule to the glomerulus, whereby various mechanisms contribute to glomerular injury following tubular injury. These mechanisms include tubular backleak, dysfunctional tubuloglomerular feedback, capillary rarefaction, atubular glomeruli, and the secretion of factors from damaged tubular epithelial cells. Clinical evidence further supports the association between even mild or recovered acute kidney injury and an increased risk of chronic kidney disease, including glomerular diseases. We also discuss potential therapeutic interventions aimed at mitigating acute tubular injury, thereby reducing the detrimental effects on glomerular function. By unraveling the complex interplay from tubular injury to glomerular dysfunction, we aim to provide insights that can enhance clinical management strategies and improve outcomes for patients with kidney disease.

Combined colour deconvolution and artificial intelligence approach for region-selective immunohistochemical labelling quantification: The example of alpha smooth muscle actin in mouse kidney.

Immunohistochemical (IHC) localisation of protein expression is a widely used tool in pathology. This is semi-quantitative and exhibits substantial intra- and inter-observer variability. Digital approaches based on stain quantification applied to IHC are precise but still operator-dependent and time-consuming when regions of interest (ROIs) must be defined to quantify protein expression in a specific tissue area. This study aimed at developing an IHC quantification workflow that benefits from colour deconvolution for stain quantification and artificial intelligence for automatic ROI definition. The method was tested on 10 whole slide images (WSI) of alpha-smooth muscle actin (aSMA) stained mouse kidney sections. The task was to identify aSMA-positive areas within the glomeruli automatically. Total aSMA detection was performed using two channels (DAB, haematoxylin) colour deconvolution. Glomeruli segmentation within the same IHC WSI was performed by training a convolutional neural network with annotated examples of glomeruli. For both aSMA and glomeruli, binary masks were created. Co-localisation was performed by overlaying the masks and assigning red/green colours, with yellow indicative of a co-localised signal. The workflow described and exemplified using the case of aSMA expression in glomeruli can be applied to quantify the expression of IHC markers within different structures of immunohistochemically stained slides. The technique is objective, has a fully automated threshold approach (colour deconvolution phase) and uses AI to eliminate operator-dependent steps.

The olfactory system of Pieris brassicae caterpillars: from receptors to glomeruli.

The olfactory system of adult lepidopterans is among the best described neuronal circuits. However, comparatively little is known about the organization of the olfactory system in the larval stage of these insects. Here, we explore the expression of olfactory receptors and the organization of olfactory sensory neurons in caterpillars of Pieris brassicae, a significant pest species in Europe and a well-studied species for its chemical ecology. To describe the larval olfactory system in this species, we first analyzed the head transcriptome of third-instar larvae (L3) and identified 16 odorant receptors (ORs) including the OR coreceptor (Orco), 13 ionotropic receptors (IRs), and 8 gustatory receptors (GRs). We then quantified the expression of these 16 ORs in different life stages, using qPCR, and found that the majority of ORs had significantly higher expression in the L4 stage than in the L3 and L5 stages, indicating that the larval olfactory system is not static throughout caterpillar development. Using an Orco-specific antibody, we identified all olfactory receptor neurons (ORNs) expressing the Orco protein in L3, L4, and L5 caterpillars and found a total of 34 Orco-positive ORNs, distributed among three sensilla on the antenna. The number of Orco-positive ORNs did not differ among the three larval instars. Finally, we used retrograde axon tracing of the antennal nerve and identified a mean of 15 glomeruli in the larval antennal center (LAC), suggesting that the caterpillar olfactory system follows a similar design as the adult olfactory system, although with a lower numerical redundancy. Taken together, our results provide a detailed analysis of the larval olfactory neurons in P. brassicae, highlighting both the differences as well as the commonalities with the adult olfactory system. These findings contribute to a better understanding of the development of the olfactory system in insects and its life-stage-specific adaptations.

Neutrophil Extracellular Traps Aggravate Contrast-Induced Acute Kidney Injury by Damaging Glomeruli and Peritubular Capillaries.

Background: Contrast-induced acute kidney injury (CI-AKI) is considered to be the third leading cause of hospital-acquired kidney injury. Current studies mostly suggest that contrast agents mainly harm renal tubular epithelial cells, but we hypothesized that the development of CI-AKI should be the result of the interaction of renal vascular and tubular injury. Methods: First we constructed a CI-AKI mouse model and verified the success of the model by pathological injury and serum creatinine level. Immunohistochemistry, protein quantification and qRT-PCR were used to detect the location and level of expression of neutrophil extracellular traps (NETs) in the kidney. Then, we blocked the in vivo accumulation of NETs using GSK484 and DNase I and detected the expression of NETs and the damage of glomerular and peritubular capillaries. Results: We first identified the presence of NETs in CI-AKI mice, and NETs were mainly accumulated in glomeruli and peritubular capillaries. The expression of NETs was positively correlated with the severity of CI-AKI kidney. After inhibition of NETs release or promotion of NETs degradation by drugs, renal vascular endothelial cell injury was reduced and renal pathological changes and creatinine levels were reversed in CI-AKI mice. In addition, inhibition of NETs reduced apoptosis and pyroptosis of renal cells and attenuated inflammation in vivo. Conclusion: These findings suggest that NETs are involved in the development of CI-AKI by damaging glomerular and peritubular capillary endothelial cells. This study will provide a new strategy for clinical prevention and treatment of CI-AKI.

BlobCUT: A Contrastive Learning Method to Support Small Blob Detection in Medical Imaging.

Medical imaging-based biomarkers derived from small objects (e.g., cell nuclei) play a crucial role in medical applications. However, detecting and segmenting small objects (a.k.a. blobs) remains a challenging task. In this research, we propose a novel 3D small blob detector called BlobCUT. BlobCUT is an unpaired image-to-image (I2I) translation model that falls under the Contrastive Unpaired Translation paradigm. It employs a blob synthesis module to generate synthetic 3D blobs with corresponding masks. This is incorporated into the iterative model training as the ground truth. The I2I translation process is designed with two constraints: (1) a convexity consistency constraint that relies on Hessian analysis to preserve the geometric properties and (2) an intensity distribution consistency constraint based on Kullback-Leibler divergence to preserve the intensity distribution of blobs. BlobCUT learns the inherent noise distribution from the target noisy blob images and performs image translation from the noisy domain to the clean domain, effectively functioning as a denoising process to support blob identification. To validate the performance of BlobCUT, we evaluate it on a 3D simulated dataset of blobs and a 3D MRI dataset of mouse kidneys. We conduct a comparative analysis involving six state-of-the-art methods. Our findings reveal that BlobCUT exhibits superior performance and training efficiency, utilizing only 56.6% of the training time required by the state-of-the-art BlobDetGAN. This underscores the effectiveness of BlobCUT in accurately segmenting small blobs while achieving notable gains in training efficiency.

General Morphology of the Mammalian Carotid Body.

The carotid body (CB) is the main peripheral arterial chemoreceptor that registers the levels of pO2, pCO2 and pH in the blood and responds to their changes by regulating breathing. It is strategically located in the bifurcation of each common carotid artery. The organ consists of "glomera" composed of two cell types, glomus and sustentacular cells, interspersed by blood vessels and nerve bundles and separated by connective tissue. The neuron-like glomus or type I cells are considered as the chemosensory cells of the CB. They contain numerous cytoplasmic organelles and dense-cored vesicles that store and release neurotransmitters. They also form both conventional chemical and electrical synapses between each other and are contacted by peripheral nerve endings of petrosal ganglion neurons. The glomus cells are dually innervated by both sensory nerve fibers through the carotid sinus nerve and autonomic fibers of sympathetic origin via the ganglioglomerular nerve. The parasympathetic efferent innervation is relayed by vasomotor fibers of ganglion cells located around or inside the CB. The glial-like sustentacular or type II cells are regarded to be supporting cells although they sustain physiologic neurogenesis in the adult CB and are thus supposed to be progenitor cells as well. The CB is a highly vascularized organ and its intraorgan hemodynamics possibly plays a role in the process of chemoreception.

Spatially Aware Transformer Networks for Contextual Prediction of Diabetic Nephropathy Progression from Whole Slide Images.

Diabetic nephropathy (DN) in the context of type 2 diabetes is the leading cause of end-stage renal disease (ESRD) in the United States. DN is graded based on glomerular morphology and has a spatially heterogeneous presentation in kidney biopsies that complicates pathologists' predictions of disease progression. Artificial intelligence and deep learning methods for pathology have shown promise for quantitative pathological evaluation and clinical trajectory estimation; but, they often fail to capture large-scale spatial anatomy and relationships found in whole slide images (WSIs). In this study, we present a transformer-based, multi-stage ESRD prediction framework built upon nonlinear dimensionality reduction, relative Euclidean pixel distance embeddings between every pair of observable glomeruli, and a corresponding spatial self-attention mechanism for a robust contextual representation. We developed a deep transformer network for encoding WSI and predicting future ESRD using a dataset of 56 kidney biopsy WSIs from DN patients at Seoul National University Hospital. Using a leave-one-out cross-validation scheme, our modified transformer framework outperformed RNNs, XGBoost, and logistic regression baseline models, and resulted in an area under the receiver operating characteristic curve (AUC) of 0.97 (95% CI: 0.90-1.00) for predicting two-year ESRD, compared with an AUC of 0.86 (95% CI: 0.66-0.99) without our relative distance embedding, and an AUC of 0.76 (95% CI: 0.59-0.92) without a denoising autoencoder module. While the variability and generalizability induced by smaller sample sizes are challenging, our distance-based embedding approach and overfitting mitigation techniques yielded results that suggest opportunities for future spatially aware WSI research using limited pathology datasets.

Neuronal scaling in the olfactory system of bats.

Comparative studies are a common way to address large-scale questions in sensory biology. For studies that investigate olfactory abilities, the most commonly used metric is olfactory bulb size. However, recent work has called into question the broad-scale use of olfactory bulb size. In this paper, we use three neuroanatomical measures with a more mechanistic link to olfactory function (number of olfactory sensory neurons (OSNs), number of mitral cells (MCs), and number of glomeruli) to ask how species with different diets may differ with respect to olfactory ability. We use phyllostomid bats as our study system because behavioral and physiological work has shown that fruit- and nectar-feeding phyllostomids rely on odors for detecting, localizing, and assessing potential foods, while insect-eating species do not. Therefore, we predicted that fruit- and nectar-feeding bats would have larger numbers of these three neuroanatomical measures than insect-eating species. In general, our results supported the predictions. We found that fruit-eaters had greater numbers of OSNs and glomeruli than insect-eaters, but we found no difference between groups in number of MCs. We also examined the allometric relationship between the three neuroanatomical variables and olfactory bulb volume, and we found isometry in all cases. These findings lend support to the notion that neuroanatomical measures can offer valuable insights into comparative olfactory abilities, and suggest that the size of the olfactory bulb may be an informative parameter to use at the whole-organism level.

The method for assessment of local permutations in the glomerular patterns of the rat olfactory bulb by aligning interindividual odor maps.

The comparison of odor functional maps in rodents demonstrates a high degree of inter-individual variability in glomerular activity patterns. There are substantial methodological difficulties in the interindividual assessment of local permutations in the glomerular patterns, since the position of anatomical extracranial landmarks, as well as the size, shape and angular orientation of olfactory bulbs can vary significantly. A new method for defining anatomical coordinates of active glomeruli in the rat olfactory bulb has been developed. The method compares the interindividual odor functional maps and calculates probabilistic maps of glomerular activity with adjustment. This adjustment involves rotation, scaling and shift of the functional map relative to its expected position in probabilistic map, computed according to the anatomical coordinates. The calculation of the probabilistic map of the odorant-specific response compensates for potential anatoamical errors due to individual variability in olfactory bulb dimensions and angular orientation. We show its efficiency on real data from a large animal sample recorded by two-photon calcium imaging in dorsal surface of the rat olfactory bulb. The proposed method with probabilistic map calculation enables the spatial consistency of the effects of individual odorants in different rats to be assessed and allow stereotypical positions of odor-specific clusters in the glomerular layer of the olfactory bulb to be identified.

Glomeruli Count and Arterial Injury in Real-Time Ultrasound-Guided Needle Kidney Biopsy Specimens: Adequacy of Three Approaches for Pig Kidneys.

INTRODUCTION: Real-time ultrasound-guided percutaneous kidney biopsy is essential for diagnosis and treatment planning; nonetheless, the optimal puncture approach has yet to be established. In vivo, performing different approaches on the same patient at once is not possible. This study aimed to determine the impact of different approaches on the number of obtained glomeruli and their potential to cause arterial injury using pig kidneys, which are similar to humans. METHODS: A total of 120 pig kidneys (60 right-sided kidneys and 60 left-sided kidneys) for research were obtained from a slaughterhouse. The specimens were collected from the lower pole on the sagittal plane of the kidney using three different approaches on the same kidney: caudocranial approach, caudal to cranial; craniocaudal approach, cranial to caudal; and vertical approach, through the surface cortex. Five blinded pediatric nephrologists assessed the number of glomeruli and arterial injuries. RESULTS: Overall, 360 specimens were collected from the kidneys through biopsy using a 16-gauge needle (mean vertical kidney length, 11.2 ± 0.7 cm; mean depth, 3.47 ± 0.23 cm). No significant difference in the incidence of arterial injury was observed between the three approaches (caudocranial vs. craniocaudal vs. vertical approaches: 78% vs. 87% vs. 87%, p = 0.14). In contrast, the vertical approach retrieved significantly more glomeruli than the caudocranial and craniocaudal approaches (caudocranial approach: 7.5 ± 2.8, craniocaudal approach: 7.8 ± 2.7, and vertical approach: 8.9 ± 3.3, p < 0.001). CONCLUSIONS: Considering its efficacy and safety profile, the vertical approach may be preferred, as more glomeruli can be obtained without increasing the incidence of arterial injury. Although the results cannot be directly extrapolated to humans due to the differences between species, they still offer important insights into the characteristics of each approach.

Isolation of Rat Glomeruli and Propagation of Mesangial Cells to Study the Kidney in Health and Disease.

Whole organ molecular analysis of the kidney potentially misses important factors involved in the pathogenesis of the glomerular disease. Organ-wide analysis therefore needs to be augmented by techniques that isolate enriched populations of glomeruli. Herein, we describe how differential sieving can be used to isolate a suspension of rat glomeruli from fresh tissue. Secondly, we also show how these can be used for the propagation of primary mesangial cell cultures. These protocols provide a practical approach for protein and RNA isolation for downstream analysis. These techniques are readily applicable to studies in isolated glomeruli in both experimental animal models and human kidney tissue.

Microscopy-Directed Imaging Mass Spectrometry for Rapid High Spatial Resolution Molecular Imaging of Glomeruli.

The glomerulus is a multicellular functional tissue unit (FTU) of the nephron that is responsible for blood filtration. Each glomerulus contains multiple substructures and cell types that are crucial for their function. To understand normal aging and disease in kidneys, methods for high spatial resolution molecular imaging within these FTUs across whole slide images is required. Here we demonstrate a workflow using microscopy-driven selected sampling to enable 5 µm pixel size matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) of all glomeruli within whole slide human kidney tissues. Such high spatial resolution imaging entails large numbers of pixels, increasing the data acquisition times. Automating FTU-specific tissue sampling enables high-resolution analysis of critical tissue structures, while concurrently maintaining throughput. Glomeruli were automatically segmented using coregistered autofluorescence microscopy data, and these segmentations were translated into MALDI IMS measurement regions. This allowed high-throughput acquisition of 268 glomeruli from a single whole slide human kidney tissue section. Unsupervised machine learning methods were used to discover molecular profiles of glomerular subregions and differentiate between healthy and diseased glomeruli. Average spectra for each glomerulus were analyzed using Uniform Manifold Approximation and Projection (UMAP) and k-means clustering, yielding 7 distinct groups of differentiated healthy and diseased glomeruli. Pixel-wise k-means clustering was applied to all glomeruli, showing unique molecular profiles localized to subregions within each glomerulus. Automated microscopy-driven, FTU-targeted acquisition for high spatial resolution molecular imaging maintains high-throughput and enables rapid assessment of whole slide images at cellular resolution and identification of tissue features associated with normal aging and disease.

The Glomerulus Multiomics Analysis Provides Deeper Insights into Diabetic Nephropathy.

Although diabetic nephropathy (DN) is the leading cause of the end-stage renal disease, the exact regulation mechanisms remain unknown. In this study, we integrated the transcriptomics and proteomics profiles of glomeruli isolated from 50 biopsy-proven DN patients and 25 controls to investigate the latest findings about DN pathogenesis. First, 1152 genes exhibited differential expression at the mRNA or protein level, and 364 showed significant association. These strong correlated genes were divided into four different functional modules. Moreover, a regulatory network of the transcription factors (TFs)-target genes (TGs) was constructed, with 30 TFs upregulated at the protein levels and 265 downstream TGs differentially expressed at the mRNA levels. These TFs are the integration centers of several signal transduction pathways and have tremendous therapeutic potential for regulating the aberrant production of TGs and the pathological process of DN. Furthermore, 29 new DN-specific splice-junction peptides were discovered with high confidence; these peptides may play novel functions in the pathological course of DN. So, our in-depth integrative transcriptomics-proteomics analysis provided deeper insights into the pathogenesis of DN and opened the potential avenue for finding new therapeutic interventions. MS raw files were deposited into the proteomeXchange with the dataset identifier PXD040617.