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.

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.

Serving on a Graduate Medical Education Diversity, Equity, Inclusion, and Justice Committee: Lessons Learned From a Journey of Growth and Healing.

Efforts toward achieving diversity, equity, inclusion, and justice (DEIJ) within graduate medical education (GME) often begin with the formation of a DEIJ committee that steers the work. Little is known about the experiences and the challenges faced by those serving on such committees. We sought to describe the experiences of members of our institutional GME DEIJ committee to gain knowledge that would propel this work forward. An open-ended survey was electronically administered to members of our institutional GME DEIJ committee. Responses were analyzed using a rapid qualitative analytical approach. Eighteen members (58%) responded. Of these, (67%) were women and five (28%) were Black. Six domains emerged: "motivation," "challenges," "emotional response," "highs," "facilitators," and "advice." Black respondents more often cited the need to increase diversity as a motivator to join this work. Women and Black respondents more often identified time constraints as a challenge to participation. Some members found the work emotionally draining; others described it as uplifting. Two themes emerged as high points of participation-pride and achievement around the work completed and the personal benefits of building a community with a shared purpose. Three themes emerged as facilitators: effective leadership, support, and establishing psychological safety during the meetings. Many arrived at the realization that change would take time and advocated for patience and perseverance. Protected time and DEIJ expertise were identified as integral to successful committee work. Our findings provide novel insights into the experience of serving on a GME DEIJ committee and highlights infrastructural and institutional prerequisites for success.

Alterations in Protein Translation and Carboxylic Acid Catabolic Processes in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease despite decades of study. Alterations in the glomerulus and kidney tubules both contribute to the pathogenesis of DKD although the majority of investigative efforts have focused on the glomerulus. We sought to examine the differential expression signature of human DKD in the glomerulus and proximal tubule and corroborate our findings in the db/db mouse model of diabetes. A transcriptogram network analysis of RNAseq data from laser microdissected (LMD) human glomerulus and proximal tubule of DKD and reference nephrectomy samples revealed enriched pathways including rhodopsin-like receptors, olfactory signaling, and ribosome (protein translation) in the proximal tubule of human DKD biopsy samples. The translation pathway was also enriched in the glomerulus. Increased translation in diabetic kidneys was validated using polyribosomal profiling in the db/db mouse model of diabetes. Using single nuclear RNA sequencing (snRNAseq) of kidneys from db/db mice, we prioritized additional pathways identified in human DKD. The top overlapping pathway identified in the murine snRNAseq proximal tubule clusters and the human LMD proximal tubule compartment was carboxylic acid catabolism. Using ultra-performance liquid chromatography-mass spectrometry, the fatty acid catabolism pathway was also found to be dysregulated in the db/db mouse model. The Acetyl-CoA metabolite was down-regulated in db/db mice, aligning with the human differential expression of the genes ACOX1 and ACACB. In summary, our findings demonstrate that proximal tubular alterations in protein translation and carboxylic acid catabolism are key features in both human and murine DKD.

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.

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.

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.

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.

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.

Live-Animal Imaging of Renal Function by Multiphoton Microscopy.

Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high-speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis. © 2018 by John Wiley & Sons, Inc.

Endothelial STAT3 Modulates Protective Mechanisms in a Mouse Ischemia-Reperfusion Model of Acute Kidney Injury.

STAT3 is a transcriptional regulator that plays an important role in coordinating inflammation and immunity. In addition, there is a growing appreciation of the role STAT3 signaling plays in response to organ injury following diverse insults. Acute kidney injury (AKI) from ischemia-reperfusion injury is a common clinical entity with devastating consequences, and the recognition that endothelial alterations contribute to kidney dysfunction in this setting is of growing interest. Consequently, we used a mouse with a genetic deletion of Stat3 restricted to the endothelium to examine the role of STAT3 signaling in the pathophysiology of ischemic AKI. In a mouse model of ischemic AKI, the loss of endothelial STAT3 signaling significantly exacerbated kidney dysfunction, morphologic injury, and proximal tubular oxidative stress. The increased severity of ischemic AKI was associated with more robust endothelial-leukocyte adhesion and increased tissue accumulation of F4/80+ macrophages. Moreover, important proximal tubular adaptive mechanisms to injury were diminished in association with decreased tissue mRNA levels of the epithelial cell survival cytokine IL-22. In aggregate, these findings suggest that the endothelial STAT3 signaling plays an important role in limiting kidney dysfunction in ischemic AKI and that selective pharmacologic activation of endothelial STAT3 signaling could serve as a potential therapeutic target.

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.

Novel roles for mucin 1 in the kidney.

PURPOSE OF REVIEW: Recent studies in the kidney have revealed that the well characterized tumor antigen mucin 1 (MUC1/Muc1) also has numerous functions in the normal and injured kidney. RECENT FINDINGS: Mucin 1 is a transmembrane mucin with a robust glycan-dependent apical targeting signal and efficient recycling from endosomes. It was recently reported that the TRPV5 calcium channel is stabilized on the cell surface by galectin-dependent cross-linking to mucin 1, providing a novel mechanism for regulation of ion channels and normal electrolyte balance.Our recent studies in mice show that Muc 1 is induced after ischemia, stabilizing hypoxia-inducible factor 1 (HIF-1)α and ß-catenin levels, and transactivating the HIF-1 and ß-catenin protective pathways. However, prolonged induction of either pathway in the injured kidney can proceed from apparent full recovery to chronic kidney disease. A very recent report indicates that aberrant activation of mucin 1 signaling after ischemic injury in mice and humans is associated with development of chronic kidney disease and fibrosis. A frameshift mutation in MUC1 was recently identified as the genetic lesion causing medullary cystic kidney disease type 1, now appropriately renamed MUC1 Kidney Disease. SUMMARY: Studies of mucin 1 in the kidney now reveal significant functions for the extracellular mucin-like domain and signaling through the cytoplasmic tail.

Quantitative Three-Dimensional Tissue Cytometry to Study Kidney Tissue and Resident Immune Cells.

Analysis of the immune system in the kidney relies predominantly on flow cytometry. Although powerful, the process of tissue homogenization necessary for flow cytometry analysis introduces bias and results in the loss of morphologic landmarks needed to determine the spatial distribution of immune cells. An ideal approach would support three-dimensional (3D) tissue cytometry: an automated quantitation of immune cells and associated spatial parameters in 3D image volumes collected from intact kidney tissue. However, widespread application of this approach is limited by the lack of accessible software tools for digital analysis of large 3D microscopy data. Here, we describe Volumetric Tissue Exploration and Analysis (VTEA) image analysis software designed for efficient exploration and quantitative analysis of large, complex 3D microscopy datasets. In analyses of images collected from fixed kidney tissue, VTEA replicated the results of flow cytometry while providing detailed analysis of the spatial distribution of immune cells in different regions of the kidney and in relation to specific renal structures. Unbiased exploration with VTEA enabled us to discover a population of tubular epithelial cells that expresses CD11C, a marker typically expressed on dendritic cells. Finally, we show the use of VTEA for large-scale quantitation of immune cells in entire human kidney biopsies. In summary, we show that VTEA is a simple and effective tool that supports unique digital interrogation and analysis of kidney tissue from animal models or biobanked human kidney biopsies. We have made VTEA freely available to interested investigators via electronic download.

Inhibition of Toll-Like Receptor 4 Signaling Mitigates Microvascular Loss but Not Fibrosis in a Model of Ischemic Acute Kidney Injury.

The development of chronic kidney disease (CKD) following an episode of acute kidney injury (AKI) is an increasingly recognized clinical problem. Inhibition of toll-like receptor 4 (TLR4) protects renal function in animal models of AKI and has become a viable therapeutic strategy in AKI. However, the impact of TLR4 inhibition on the chronic sequelae of AKI is unknown. Consequently, we examined the chronic effects of TLR4 inhibition in a model of ischemic AKI. Mice with a TLR4-deletion on a C57BL/6 background and wild-type (WT) background control mice (C57BL/6) were subjected to bilateral renal artery clamping for 19 min and reperfusion for up to 6 weeks. Despite the acute protective effect of TLR4 inhibition on renal function (serum creatinine 1.6 ± 0.4 mg/dL TLR4-deletion vs. 2.8 ± 0.3 mg/dL·WT) and rates of tubular apoptosis following ischemic AKI, we found no difference in neutrophil or macrophage infiltration. Furthermore, we observed significant protection from microvascular rarefaction at six weeks following injury with TLR4-deletion, but this did not alter development of fibrosis. In conclusion, we validate the acute protective effect of TLR4 signal inhibition in AKI but demonstrate that this protective effect does not mitigate the sequential fibrogenic response in this model of ischemic AKI.

Novel application of complementary imaging techniques to examine in vivo glucose metabolism in the kidney.

The metabolic status of the kidney is a determinant of injury susceptibility and a measure of progression for many disease processes; however, noninvasive modalities to assess kidney metabolism are lacking. In this study, we employed positron emission tomography (PET) and intravital multiphoton microscopy (MPM) to assess cortical and proximal tubule glucose tracer uptake, respectively, following experimental perturbations of kidney metabolism. Applying dynamic image acquisition PET with 2-18fluoro-2-deoxyglucose (18F-FDG) and tracer kinetic modeling, we found that an intracellular compartment in the cortex of the kidney could be distinguished from the blood and urine compartments in animals. Given emerging literature that the tumor suppressor protein p53 is an important regulator of cellular metabolism, we demonstrated that PET imaging was able to discern a threefold increase in cortical 18F-FDG uptake following the pharmacological inhibition of p53 in animals. Intravital MPM with the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) provided increased resolution and corroborated these findings at the level of the proximal tubule. Extending our observation of p53 inhibition on proximal tubule glucose tracer uptake, we demonstrated by intravital MPM that pharmacological inhibition of p53 diminishes mitochondrial potential difference. We provide additional evidence that inhibition of p53 alters key metabolic enzymes regulating glycolysis and increases intermediates of glycolysis. In summary, we provide evidence that PET is a valuable tool for examining kidney metabolism in preclinical and clinical studies, intravital MPM is a powerful adjunct to PET in preclinical studies of metabolism, and p53 inhibition alters basal kidney metabolism.

Muc1 enhances the ß-catenin protective pathway during ischemia-reperfusion injury.

The hypoxia-inducible factor (HIF)-1 and ß-catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine mucin (Muc)1 protects kidney function and morphology in a mouse model of ischemia-reperfusion injury (IRI) by stabilizing HIF-1α, enhancing HIF-1 downstream signaling, and thereby preventing metabolic stress (Pastor-Soler et al. Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308: F1452-F1462, 2015). We asked if Muc1 regulates the ß-catenin protective pathway during IRI as 1) ß-catenin nuclear targeting is MUC1 dependent in cultured human cells, 2) ß-catenin is found in coimmunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, and 3) MUC1 prevents ß-catenin phosphorylation by glycogen synthase kinase (GSK)3ß and thereby ß-catenin degradation. Using the same mouse model of IRI, we found that levels of active GSK3ß were significantly lower in kidneys of control mice compared with Muc1 knockout (KO) mice. Consequently, ß-catenin was significantly upregulated at 24 and 72 h of recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both ß-catenin induction and nuclear targeting were absent in Muc1 KO mice. We also found downstream induction of ß-catenin prosurvival factors (activated Akt, survivin, transcription factor T cell factor 4 (TCF4), and its downstream target cyclin D1) and repression of proapoptotic factors (p53, active Bax, and cleaved caspase-3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during acute kidney injury proceeds by enhancing both the HIF-1 and ß-catenin protective pathways.