The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. Comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measure dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We establish a spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we note distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3, KLF6, and KLF10 regulates the transition between health and injury, while in thick ascending limb cells this transition is regulated by NR2F1. Further, combined perturbation of ELF3, KLF6, and KLF10 distinguishes two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Molecular Signatures of Glomerular Neovascularization in a Patient with Diabetic Kidney Disease.

The Kidney Precision Medicine Project (KPMP) aims to create a kidney tissue atlas, define disease subgroups, and identify critical cells, pathways, and targets for novel therapies through molecular investigation of human kidney biopsies obtained from participants with acute kidney injury (AKI) or chronic kidney disease (CKD). We present the case of a 66-year-old woman with diabetic kidney disease who underwent a protocol KPMP kidney biopsy. Her clinical history included diabetes mellitus complicated by neuropathy and eye disease, increased insulin resistance, hypertension, albuminuria, and relatively preserved glomerular filtration rate (early CKD stage 3a). The patient's histopathology was consistent with diabetic nephropathy and arterial and arteriolar sclerosis. Three-dimensional, immunofluorescence imaging of the kidney biopsy specimen revealed extensive peri-glomerular neovascularization that was underestimated by standard histopathologic approaches. Spatial transcriptomics was performed to obtain gene expression signatures at discrete areas of the kidney biopsy. Gene expression in the areas of glomerular neovascularization revealed increased expression of genes involved in angiogenic signaling, proliferation and survival of endothelial cells, as well as new vessel maturation and stability. This molecular correlation provides additional insights into the development of kidney disease in patients with diabetes and spotlights how novel molecular techniques employed by the KPMP can supplement and enrich the histopathologic diagnosis obtained from a kidney biopsy.

A spatially anchored transcriptomic atlas of the human kidney papilla identifies significant immune injury in patients with stone disease.

Kidney stone disease causes significant morbidity and increases health care utilization. In this work, we decipher the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease and healthy subjects. In addition to identifying cell types important in papillary physiology, we characterize collecting duct cell subtypes and an undifferentiated epithelial cell type that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncover a global injury signature characterized by immune activation, oxidative stress and extracellular matrix remodeling. We also identify the association of MMP7 and MMP9 expression with stone disease and mineral deposition, respectively. MMP7 and MMP9 are significantly increased in the urine of patients with CaOx stone disease, and their levels correlate with disease activity. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla and identify associated urinary biomarkers.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

The chromatin landscape of healthy and injured cell types in the human kidney.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. However, comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measured dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We established a comprehensive and spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we noted distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3 , KLF6 , and KLF10 regulated the transition between health and injury, while in thick ascending limb cells this transition was regulated by NR2F1 . Further, combined perturbation of ELF3 , KLF6 , and KLF10 distinguished two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Integrated Cytometry With Machine Learning Applied to High-Content Imaging of Human Kidney Tissue for In Situ Cell Classification and Neighborhood Analysis.

The human kidney is a complex organ with various cell types that are intricately organized to perform key physiological functions and maintain homeostasis. New imaging modalities, such as mesoscale and highly multiplexed fluorescence microscopy, are increasingly being applied to human kidney tissue to create single-cell resolution data sets that are both spatially large and multidimensional. These single-cell resolution high-content imaging data sets have great potential to uncover the complex spatial organization and cellular makeup of the human kidney. Tissue cytometry is a novel approach used for the quantitative analysis of imaging data; however, the scale and complexity of such data sets pose unique challenges for processing and analysis. We have developed the Volumetric Tissue Exploration and Analysis (VTEA) software, a unique tool that integrates image processing, segmentation, and interactive cytometry analysis into a single framework on desktop computers. Supported by an extensible and open-source framework, VTEA's integrated pipeline now includes enhanced analytical tools, such as machine learning, data visualization, and neighborhood analyses, for hyperdimensional large-scale imaging data sets. These novel capabilities enable the analysis of mesoscale 2- and 3-dimensional multiplexed human kidney imaging data sets (such as co-detection by indexing and 3-dimensional confocal multiplexed fluorescence imaging). We demonstrate the utility of this approach in identifying cell subtypes in the kidney on the basis of labels, spatial association, and their microenvironment or neighborhood membership. VTEA provides an integrated and intuitive approach to decipher the cellular and spatial complexity of the human kidney and complements other transcriptomics and epigenetic efforts to define the landscape of kidney cell types.

Tissue Cytometry With Machine Learning in Kidney: From Small Specimens to Big Data.

Advances in cellular and molecular interrogation of kidney tissue have ushered a new era of understanding the pathogenesis of kidney disease and potentially identifying molecular targets for therapeutic intervention. Classifying cells in situ and identifying subtypes and states induced by injury is a foundational task in this context. High resolution Imaging-based approaches such as large-scale fluorescence 3D imaging offer significant advantages because they allow preservation of tissue architecture and provide a definition of the spatial context of each cell. We recently described the Volumetric Tissue Exploration and Analysis cytometry tool which enables an interactive analysis, quantitation and semiautomated classification of labeled cells in 3D image volumes. We also established and demonstrated an imaging-based classification using deep learning of cells in intact tissue using 3D nuclear staining with 4',6-diamidino-2-phenylindole (DAPI). In this mini-review, we will discuss recent advancements in analyzing 3D imaging of kidney tissue, and how combining machine learning with cytometry is a powerful approach to leverage the depth of content provided by high resolution imaging into a highly informative analytical output. Therefore, imaging a small tissue specimen will yield big scale data that will enable cell classification in a spatial context and provide novel insights on pathological changes induced by kidney disease.

Integration of spatial and single-cell transcriptomics localizes epithelial cell-immune cross-talk in kidney injury.

Single-cell sequencing studies have characterized the transcriptomic signature of cell types within the kidney. However, the spatial distribution of acute kidney injury (AKI) is regional and affects cells heterogeneously. We first optimized coordination of spatial transcriptomics and single-nuclear sequencing data sets, mapping 30 dominant cell types to a human nephrectomy. The predicted cell-type spots corresponded with the underlying histopathology. To study the implications of AKI on transcript expression, we then characterized the spatial transcriptomic signature of 2 murine AKI models: ischemia/reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were associated with injury pathways. Using single-cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. Neutrophils infiltrated the renal medulla in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubules. In the CLP model, infiltrating macrophages dominated the outer cortical signature, and Mdk was identified as a corresponding chemotactic factor. The regional distribution of these immune cells was validated with multiplexed CO-Detection by indEXing (CODEX) immunofluorescence. Spatial transcriptomic sequencing complemented single-cell sequencing by uncovering mechanisms driving immune cell infiltration and detection of relevant cell subpopulations.

Molecular characterization of the human kidney interstitium in health and disease.

The gene expression signature of the human kidney interstitium is incompletely understood. The cortical interstitium (excluding tubules, glomeruli, and vessels) in reference nephrectomies (N = 9) and diabetic kidney biopsy specimens (N = 6) was laser microdissected (LMD) and sequenced. Samples underwent RNA sequencing. Gene signatures were deconvolved using single nuclear RNA sequencing (snRNAseq) data derived from overlapping specimens. Interstitial LMD transcriptomics uncovered previously unidentified markers including KISS1, validated with in situ hybridization. LMD transcriptomics and snRNAseq revealed strong correlation of gene expression within corresponding kidney regions. Relevant enriched interstitial pathways included G-protein coupled receptor. binding and collagen biosynthesis. The diabetic interstitium was enriched for extracellular matrix organization and small-molecule catabolism. Cell type markers with unchanged expression (NOTCH3, EGFR, and HEG1) and those down-regulated in diabetic nephropathy (MYH11, LUM, and CCDC3) were identified. LMD transcriptomics complements snRNAseq; together, they facilitate mapping of interstitial marker genes to aid interpretation of pathophysiology in precision medicine studies.

Clinical, histopathologic and molecular features of idiopathic and diabetic nodular mesangial sclerosis in humans.

BACKGROUND: Idiopathic nodular mesangial sclerosis, also called idiopathic nodular glomerulosclerosis (ING), is a rare clinical entity with an unclear pathogenesis. The hallmark of this disease is the presence of nodular mesangial sclerosis on histology without clinical evidence of diabetes mellitus or other predisposing diagnoses. To achieve insights into its pathogenesis, we queried the clinical, histopathologic and transcriptomic features of ING and nodular diabetic nephropathy (DN). METHODS: All renal biopsy reports accessioned at Indiana University Health from 2001 to 2016 were reviewed to identify 48 ING cases. Clinical and histopathologic features were compared between individuals with ING and DN (n = 751). Glomeruli of ING (n = 5), DN (n = 18) and reference (REF) nephrectomy (n = 9) samples were isolated by laser microdissection and RNA was sequenced. Immunohistochemistry of proline-rich 36 (PRR36) protein was performed. RESULTS: ING subjects were frequently hypertensive (95.8%) with a smoking history (66.7%). ING subjects were older, had lower proteinuria and had less hyaline arteriolosclerosis than DN subjects. Butanoate metabolism was an enriched pathway in ING samples compared with either REF or DN samples. The top differentially expressed gene, PRR36, had increased expression in glomeruli 248-fold [false discovery rate (FDR) P = 5.93 × 10-6] compared with the REF and increased 109-fold (FDR P = 1.85 × 10-6) compared with DN samples. Immunohistochemistry revealed a reduced proportion of cells with perinuclear reaction in ING samples as compared to DN. CONCLUSIONS: Despite similar clinical and histopathologic characteristics in ING and DN, the uncovered transcriptomic signature suggests that ING has distinct molecular features from nodular DN. Further study is warranted to understand these relationships.

Large-scale, three-dimensional tissue cytometry of the human kidney: a complete and accessible pipeline.

The advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell's physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ.

In Situ Classification of Cell Types in Human Kidney Tissue Using 3D Nuclear Staining.

To understand the physiology and pathology of disease, capturing the heterogeneity of cell types within their tissue environment is fundamental. In such an endeavor, the human kidney presents a formidable challenge because its complex organizational structure is tightly linked to key physiological functions. Advances in imaging-based cell classification may be limited by the need to incorporate specific markers that can link classification to function. Multiplex imaging can mitigate these limitations, but requires cumulative incorporation of markers, which may lead to tissue exhaustion. Furthermore, the application of such strategies in large scale 3-dimensional (3D) imaging is challenging. Here, we propose that 3D nuclear signatures from a DNA stain, DAPI, which could be incorporated in most experimental imaging, can be used for classifying cells in intact human kidney tissue. We developed an unsupervised approach that uses 3D tissue cytometry to generate a large training dataset of nuclei images (NephNuc), where each nucleus is associated with a cell type label. We then devised various supervised machine learning approaches for kidney cell classification and demonstrated that a deep learning approach outperforms classical machine learning or shape-based classifiers. Specifically, a custom 3D convolutional neural network (NephNet3D) trained on nuclei image volumes achieved a balanced accuracy of 80.26%. Importantly, integrating NephNet3D classification with tissue cytometry allowed in situ visualization of cell type classifications in kidney tissue. In conclusion, we present a tissue cytometry and deep learning approach for in situ classification of cell types in human kidney tissue using only a DNA stain. This methodology is generalizable to other tissues and has potential advantages on tissue economy and non-exhaustive classification of different cell types.

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue.

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

Quantitative Large-Scale Three-Dimensional Imaging of Human Kidney Biopsies: A Bridge to Precision Medicine in Kidney Disease.

Kidney biopsy remains the gold standard for uncovering the pathogenesis of acute and chronic kidney diseases. However, the ability to perform high resolution, quantitative, molecular and cellular interrogation of this precious tissue is still at a developing stage compared to other fields such as oncology. Here, we discuss recent advances in performing large-scale, three-dimensional (3D), multi-fluorescence imaging of kidney biopsies and quantitative analysis referred to as 3D tissue cytometry. This approach allows the accurate measurement of specific cell types and their spatial distribution in a thick section spanning the entire length of the biopsy. By uncovering specific disease signatures, including rare occurrences, and linking them to the biology in situ, this approach will enhance our understanding of disease pathogenesis. Furthermore, by providing accurate quantitation of cellular events, 3D cytometry may improve the accuracy of prognosticating the clinical course and response to therapy. Therefore, large-scale 3D imaging and cytometry of kidney biopsy is poised to become a bridge towards personalized medicine for patients with kidney disease.

Loss of endothelial nitric oxide synthase exacerbates intestinal and lung injury in experimental necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) continues to be a devastating condition among preterm infants. Nitric oxide, which is synthesized in the intestine by endothelial nitric oxide synthase (eNOS), acts as a potent vasodilator and antioxidant within the mesentery and may play a role in prevention of NEC. We hypothesized that loss of endothelial nitric oxide would worsen both intestinal and associated lung injury and increase local and systemic inflammation during experimental NEC. METHODS: NEC was induced in five-day-old wild type (WT) and eNOS-knockout (eNOSKO) mouse pups. Experimental groups (n=10) were formula fed and subjected to intermittent hypoxic and hypothermic stress, while control groups (n=10) remained with their mother to breastfeed. Pups were monitored by daily clinical assessment. After sacrifice on day nine, intestine and lung were assessed for injury, and cytokines were measured in tissue homogenates by ELISA. Data were compared with Mann-Whitney, and p<0.05 was significant. RESULTS: Each NEC group was compared to its respective strain's breastfed control to facilitate comparisons between the groups. Both NEC groups were significantly sicker than their breastfed controls. eNOSKO NEC animals had a median clinical assessment score of 3 (IQR=1-5), and the WT NEC animal's median score was 3 (IQR=2-5). Despite similar clinical scores, intestinal injury was significantly worse in the eNOSKO NEC groups compared to WT NEC groups (median injury scores of 3.25 (IQR=2.25-3.625) and 2 (IQR=1-3), respectively (p=0.0474). Associated lung injury was significantly worse in the eNOSKO NEC group as compared to the WT NEC group (median scores of 8.5 (IQR=6.75-11.25) and 6.5 (IQR=5-7.5), respectively, p=0.0391). Interestingly, cytokines in both tissues were very different between the two groups, with varying effects noted for each cytokine (IL-6, IL-1ß, VEGF, and IL-12) in both tissues. CONCLUSION: Nitric oxide from eNOS plays a key role in preventing the development of NEC. Without eNOS function, both intestinal and lung injuries are more severe, and the inflammatory cascade is significantly altered. Further studies are needed to determine how eNOS-derived nitric oxide facilitates these beneficial effects.

The route and timing of hydrogen sulfide therapy critically impacts intestinal recovery following ischemia and reperfusion injury.

PURPOSE: Hydrogen sulfide (H2S) has many beneficial properties and may serve as a novel treatment in patients suffering from intestinal ischemia-reperfusion injury (I/R). The purpose of this study was to examine the method of delivery and timing of administration of H2S for intestinal therapy during ischemic injury. We hypothesized that 1) route of administration of hydrogen sulfide would impact intestinal recovery following acute mesenteric ischemia and 2) preischemic H2S conditioning using the optimal mode of administration as determined above would provide superior protection compared to postischemic application. METHODS: Male C57BL/6J mice underwent intestinal ischemia by temporary occlusion of the superior mesenteric artery. Following ischemia, animals were treated according to one of the following (N=6 per group): intraperitoneal or intravenous injection of GYY4137 (H2S-releasing donor, 50mg/kg in PBS), vehicle, inhalation of oxygen only, inhalation of 80ppm hydrogen sulfide gas. Following 24-h recovery, perfusion was assessed via laser Doppler imaging, and animals were euthanized. Perfusion and histology data were assessed, and terminal ileum samples were analyzed for cytokine production following ischemia. Once the optimal route of administration was determined, preischemic conditioning with H2S was undertaken using that route of administration. All data were analyzed using Mann-Whitney. P-values <0.05 were significant. RESULTS: Mesenteric perfusion following intestinal I/R was superior in mice treated with intraperitoneal (IP) GYY4137 (IP vehicle: 25.6±6.0 vs. IP GYY4137: 79.7±15.1; p=0.02) or intravenous (IV) GYY4137 (IV vehicle: 36.3±5.9 vs. IV GYY4137: 100.7±34.0; p=0.03). This benefit was not observed with inhaled H2S gas (O2 vehicle: 66.6±11.4 vs. H2S gas: 81.8±6.0; p=0.31). However, histological architecture was only preserved with intraperitoneal administration of GYY4127 (IP vehicle: 3.4±0.4 vs. IP GYY4137: 2±0.3; p=0.02). Additionally, IP GYY4137 allowed for significant attenuation of inflammatory chemokine production of IL-6, IP-10 and MIP-2. We then analyzed whether there was a difference between pre- and postischemic administration of IP GYY4137. We found that preconditioning of animals with intraperitoneal GYY4137 only added minor improvements in outcomes compared to postischemic application. CONCLUSION: Therapeutic benefits of H2S are superior with intraperitoneal application of an H2S donor compared to other administration routes. Additionally, while intraperitoneal treatment in both the pre- and postischemic period is beneficial, preischemic application of an H2S donor was found to be slightly better. Further studies are needed to examine long term outcomes and further mechanisms of action prior to widespread clinical application. TYPE OF STUDY: Basic science. LEVEL OF EVIDENCE: N/A.

Umbilical mesenchymal stromal cells provide intestinal protection through nitric oxide dependent pathways.

BACKGROUND: Umbilical-derived mesenchymal stromal cells (USCs) have shown promise in the protection of ischemic organs. We hypothesized that USCs would improve mesenteric perfusion, preserve intestinal histological architecture, and limit inflammation by nitric oxide-dependent mechanisms following intestinal ischemia/reperfusion (IR) injury. METHODS: Adult wild-type C57BL/6J (WT) and endothelial nitric oxide synthase knock out (eNOS KO) mice were used: (1) WT IR + vehicle, (2) WT IR + USC, (3) eNOS KO IR + vehicle, and (4) eNOS KO IR + USC. Mice were anesthetized, and a midline laparotomy was performed. The superior mesenteric artery was clamped with a nonoccluding clamp for 60-min. Following IR, mice were treated with an injection of 250 µL phosphate buffered saline or 2 × 106 USCs suspended in 250-µL phosphate buffered saline solution. Mesenteric perfusion images were acquired using laser Doppler imaging. Perfusion was analyzed as a percentage of baseline. At 24 h, mice were euthanized, and intestines were harvested. Intestines were evaluated for injury, and data were analyzed using the Mann-Whitney or Kruskal-Wallis tests. RESULTS: Intestinal mesenteric perfusion was significantly improved in WT mice treated with USC therapy compared with eNOS KOs. Intestinal histological architecture was preserved with USC therapy in WT mice. However, in eNOS KO mice, this benefit was abolished. Finally, the presence of several cytokines and growth factors were significantly improved in WT mice compared with eNOS KO mice treated with USCs. CONCLUSIONS: The benefits of USC-mediated therapy following intestinal IR injury likely occur via nitric oxide-dependent pathways. Further studies are required to define the molecular mechanisms by which USCs activate endothelial nitric oxide synthase to bring about their protective effects.

Hydrogen sulfide provides intestinal protection during a murine model of experimental necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) continues to be a morbid surgical condition among preterm infants. Novel therapies for this condition are desperately needed. Hydrogen sulfide (H2S) is an endogenous gasotransmitter that has been found to have beneficial properties. We therefore hypothesized that intraperitoneal injection of various H2S donors would improve clinical outcomes, increase intestinal perfusion, and reduce intestinal injury in an experimental mouse model of necrotizing enterocolitis. METHODS: NEC was induced in five-day-old mouse C57BL/6 mouse pups through maternal separation, formula feeding, and intermittent hypoxic and hypothermic stress. The control group (n=10) remained with their mother and breastfed ad lib. Experimental groups (n=10/group) received intraperitoneal injections of phosphate buffered saline (PBS) vehicle or one of the following H2S donors: (1) GYY4137, 50mg/kg daily; (2) Sodium sulfide (Na2S), 20mg/kg three times daily; (3) AP39, 0.16mg/kg daily. Pups were monitored for weight gain, clinical status, and intestinal perfusion via transcutaneous Laser Doppler Imaging (LDI). After sacrifice on day nine, intestinal appearance and histology were scored and cytokines were measured in tissue homogenates of intestine, liver, and lung. Data were compared with Mann-Whitney and p<0.05 was considered significant. RESULTS: Clinical score and weight gain were significantly improved in all three H2S-treated groups as compared to vehicle (p<0.05 for all groups). Intestinal perfusion of the vehicle group was 22% of baseline while the GYY4137 group was 38.7% (p=0.0103), Na2S was 47.0% (p=0.0040), and AP39 was 43.0% (p=0.0018). The vehicle group had a median histology score of 2.5, while the GYY4137 group's was 1 (p=0.0013), Na2S was 0.5 (p=0.0004), and AP39 was 0.5 (p=0.0001). Cytokine analysis of the intestine of the H2S-treated groups revealed levels closer to breastfed pups as compared to vehicle (p<0.05 for all groups). CONCLUSION: Intraperitoneal administration of H2S protects against development of NEC by improving mesenteric perfusion, and by limiting mucosal injury and altering the tissue inflammatory response. Further experimentation is necessary to elucidate downstream mechanisms prior to clinical implementation.

Large-scale 3-dimensional quantitative imaging of tissues: state-of-the-art and translational implications.

Recent developments in automated optical sectioning microscope systems have enabled researchers to conduct high resolution, three-dimensional (3D) microscopy at the scale of millimeters in various types of tissues. This powerful technology allows the exploration of tissues at an unprecedented level of detail, while preserving the spatial context. By doing so, such technology will also enable researchers to explore cellular and molecular signatures within tissue and correlate with disease course. This will allow an improved understanding of pathophysiology and facilitate a precision medicine approach to assess the response to treatment. The ability to perform large-scale imaging in 3D cannot be realized without the widespread availability of accessible quantitative analysis. In this review, we will outline recent advances in large-scale 3D imaging and discuss the available methodologies to perform meaningful analysis and potential applications in translational research.

Hydrogen Sulfide: A Potential Novel Therapy for the Treatment of Ischemia.

Hydrogen sulfide (H2S) is a novel signaling molecule most recently found to be of fundamental importance in cellular function as a regulator of apoptosis, inflammation, and perfusion. Mechanisms of endogenous H2S signaling are poorly understood; however, signal transmission is thought to occur via persulfidation at reactive cysteine residues on proteins. Although much has been discovered about how H2S is synthesized in the body, less is known about how it is metabolized. Recent studies have discovered a multitude of different targets for H2S therapy, including those related to protein modification, intracellular signaling, and ion channel depolarization. The most difficult part of studying hydrogen sulfide has been finding a way to accurately and reproducibly measure it. The purpose of this review is to: elaborate on the biosynthesis and catabolism of H2S in the human body, review current knowledge of the mechanisms of action of this gas in relation to ischemic injury, define strategies for physiological measurement of H2S in biological systems, and review potential novel therapies that use H2S for treatment.