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Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.

Patients with systemic sclerosis and systemic lupus erythematosus serologies present a unique challenge to the clinician when hypertension is detected in the outpatient setting. Treatment choices for non-renal crisis hypertension are different for systemic sclerosis versus systemic lupus erythematosus. Urgent laboratory studies and, in the presence of certain symptoms, imaging assessment are indicated in systemic sclerosis and systemic lupus erythematosus overlap patients with systemic hypertension. Long-term assessment of systemic hypertension may be enhanced by advances in non-contrast imaging that serve as valuable biomarkers for progressive vasculopathy. In this review, the diagnostic approach to systemic sclerosis and systemic lupus erythematosus overlap patients presenting with hypertension is discussed.

Supplementary Table 1: Clinical characteristics of patients whose tumors were utilized for phosphotyrosine peptide survey. Supplementary Table 2: Phosphorylated Proteins in RCC Cell Line and Tumor Samples. Supplementary Table 3: Phosphopeptides identified using the Andromeda search engine of 10 renal cell carcinoma cell lines. Supplementary Table 4: Supplementary Table 4. Label-free quantification of phosphopeptides identified using the Andromeda search engine of 15 clear cell and 15 papillary renal cell carcinoma tumors. Supplementary Figure 1: Tyrosine kinase inhibitors (0.5 μM) provide functional correlation. Supplementary Figure 2: Tyrosine kinase inhibitors (2.5 μM) provide functional correlation. Please note that this data, due to its large size, is represented best in Microsoft Excel.

Supplementary Figures 3-5 Legends

Immunohistochemistry demonstrates robust DDR1 phosphorylation in papillary RCC tumors.

Supplementary Materials and Methods

This file contains the primer sequence of CYP2C9* variants (Supplementary Table S1); the SNPs genotyped in the BioVU NSCLC study (Supplementary Table S2); quality control measures of SNPs in the BioVU NSCLC study (Supplementary Table S3); clinical and demographic description of NSCLC cases (Supplementary Table S4); the association of clinical and demographic variables with survival (Supplementary Table S5); the genotypes in the NSCLC cases (Supplementary Table S6); the spectra of CYP2C9* variants and the effect of arachidonic acid on CYP2C9* variant (Supplementary Figure S1); the association between CYP2C9* variants and platinum chemotherapy (Supplementary Figure S2).

Internal (Supplementary Figure 3) and external (Supplementary Figure 4) tumor LC-MS/MS controls.

Increased expression of cytochrome P450 CYP2C9, together with elevated levels of its products epoxyeicosatrienoic acids (EET), is associated with aggressiveness in cancer. Cytochrome P450 variants CYP2C9*2 and CYP2C9*3 encode proteins with reduced enzymatic activity, and individuals carrying these variants metabolize drugs more slowly than individuals with wild-type CYP2C9*1, potentially affecting their response to drugs and altering their risk of disease. Although genetic differences in CYP2C9-dependent oxidation of arachidonic acid (AA) have been reported, the roles of CYP2C9*2 and CYP2C9*3 in EET biosynthesis and their relevance to disease are unknown. Here, we report that CYP2C9*2 and CYP2C9*3 metabolize AA less efficiently than CYP2C9*1 and that they play a role in the progression of non–small cell lung cancer (NSCLC) via impaired EET biosynthesis. When injected into mice, NSCLC cells expressing CYP2C9*2 and CYP2C9*3 produced lower levels of EETs and developed fewer, smaller, and less vascularized tumors than cells expressing CYP2C9*1. Moreover, endothelial cells expressing these two variants proliferated and migrated less than cells expressing CYP2C*1. Purified CYP2C9*2 and CYP2C9*3 exhibited attenuated catalytic efficiency in producing EETs, primarily due to impaired reduction of these two variants by NADPH-P450 reductase. Loss-of-function SNPs within CYP2C9*2 and CYP2C9*3 were associated with improved survival in female cases of NSCLC. Thus, decreased EET biosynthesis represents a novel mechanism whereby CYPC29*2 and CYP2C9*3 exert a direct protective role in NSCLC development.Significance: These findings report single nucleotide polymorphisms in the human CYP2C9 genes, CYP2C9*2 and CYP2C9*3, exert a direct protective role in tumorigenesis by impairing EET biosynthesis. Cancer Res; 78(17); 4865–77. ©2018 AACR.

Supplementary Figure 1 from Transforming Growth Factor β Induces Clustering of HER2 and Integrins by Activating Src-Focal Adhesion Kinase and Receptor Association to the Cytoskeleton

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PDF file - 24K, Analysis of p60.5 and KrasLA2 tumor cells.

PDF file - 99K, Analysis of human GFP-A549 NSCLC tumor cells.

PDF file - 40K, Treatment of athymic mice orthotopically injected with human GFP-A549 cells with Bezafibrate.

Supplementary Figure 1 from Loss of Integrin α1β1 Ameliorates Kras-Induced Lung Cancer

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its

During alveolar repair, alveolar type 2 (AT2) epithelial cell progenitors rapidly proliferate and differentiate into flat type 1 alveolar epithelial cells. Failure of normal alveolar repair mechanisms can lead to loss of alveolar structure (emphysema) or development of fibrosis, depending on the type and severity of injury. To test if β1-containing integrins are required during repair following acute injury, we administeredE. colilipopolysaccharide (LPS) by intratracheal injection to mice with a post-developmental deletion of β1 integrin in AT2 cells. While control mice recovered from LPS injury without structural abnormalities, β1-deficient mice had more severe inflammation and developed emphysema. In addition, recovering alveoli were repopulated with an abundance of rounded epithelial cells co-expressing type 2, type 1, and mixed intermediate cell state markers, with few mature type 1 cells. β1-deficient AT2 cells showed persistently increased proliferation after injury, which was blocked by inhibiting NF-κB activation in these cells. Lineage tracing experiments revealed that β1-deficient AT2 cells failed to differentiate into mature type 1 alveolar epithelial cells. Together, these findings demonstrate that functional alveolar repair after injury with terminal alveolar epithelial differentiation requires β1-containing integrins.

A unique and complex microstructure underlies the diverse functions of the liver. Breakdown of this organization, as occurs in fibrosis and cirrhosis, impairs liver function and leads to disease. The role of integrin β1 was examined both in establishing liver microstructure and recreating it after injury. Embryonic deletion of integrin β1 in the liver disrupts the normal development of hepatocyte polarity, specification of cell–cell junctions, and canalicular formation. This in turn leads to the expression of transforming growth factor β (TGF-β) and widespread fibrosis. Targeted deletion of integrin β1 in adult hepatocytes prevents recreation of normal hepatocyte architecture after liver injury, with resultant fibrosis. In vitro, integrin β1 is essential for canalicular formation and is needed to prevent stellate cell activation by modulating TGF-β. Taken together, these findings identify integrin β1 as a key determinant of liver architecture with a critical role as a regulator of TGF-β secretion. These results suggest that disrupting the hepatocyte–extracellular matrix interaction is sufficient to drive fibrosis.

Pressure overload of the heart is characterized by concentric hypertrophy and interstitial fibrosis. Cardiac fibroblasts (CFs) in the ventricular wall become activated during injury and synthesize and compact the extracellular matrix, which causes interstitial fibrosis and stiffening of the ventricular heart walls. Talin1 (Tln1) and Talin2 (Tln2) are mechanosensitive proteins that participate in focal adhesion transmission of signals from the extracellular environment to the actin cytoskeleton of CFs. The aim of the present study was to determine whether the removal of Tln1 and Tln2 from CFs would reduce interstitial fibrosis and cardiac hypertrophy. Twelve-week-old male and female Tln2-null (Tln2(−/−)) and Tln2-null, CF-specific Tln1 knockout (Tln2(−/−);Tln1(CF−/−)) mice were given angiotensin-II (ANG II) (1.5 mg/kg/day) or saline through osmotic pumps for 8 wk. Cardiomyocyte area and measures of heart thickness were increased in the male ANG II-infused Tln2(−/−);Tln1(CF−/−) mice, whereas there was no increase in interstitial fibrosis. Systolic blood pressure was increased in the female Tln2(−/−);Tln1(CF−/−) mice after ANG II infusion compared with the Tln2(−/−) mice. However, there was no increase in cardiac hypertrophy in the Tln2(−/−);Tln1(CF−/−) mice, which was seen in the Tln2(−/−) mice. Collectively, these data indicate that in male mice, the absence of Tln1 and Tln2 in CFs leads to cardiomyocyte hypertrophy in response to ANG II, whereas it results in a hypertrophy-resistant phenotype in female mice. These findings have important implications for the role of mechanosensitive proteins in CFs and their impact on cardiomyocyte function in the pathogenesis of hypertension and cardiac hypertrophy. NEW & NOTEWORTHY The role of talins has been previously studied in cardiomyocytes; however, these mechanotransductive proteins that are members of the focal adhesion complex have not been examined in cardiac fibroblasts previously. We hypothesized that loss of talins in cardiac fibroblasts would reduce interstitial fibrosis in the heart with a pressure overload model. However, we found that although loss of talins did not alter fibrosis, it did result in cardiomyocyte and ventricular hypertrophy.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires ...

Nearly 6 million Americans suffer from heart failure. Increased fibrosis contributes to functional decline of the heart that leads to heart failure. Previously, we identified a mechanosensitive protein, small proline‐rich repeat 3 (SPRR3), in vascular smooth muscle cells of atheromas. In this study, we demonstrate SPRR3 expression in cardiac fibroblasts which is induced in activated fibroblasts following pressure‐induced heart failure. Sprr3 deletion in mice showed preserved cardiac function and reduced interstitial fibrosis in vivo and reduced fibroblast proliferation and collagen expression in vitro. SPRR3 loss resulted in reduced activation of Akt, FAK, ERK, and p38 signaling pathways, which are coordinately regulated by integrins and growth factors. SPRR3 deletion did not impede integrin‐associated functions including cell adhesion, migration, or contraction. SPRR3 loss resulted in reduced activation of PDGFRβ in fibroblasts. This was not due to the reduced PDGFRβ expression levels or decreased binding of the PDGF ligand to PDGFRβ. SPRR3 facilitated the association of integrin β1 with PDGFRβ and subsequently fibroblast proliferation, suggesting a role in PDGFRβ‐Integrin synergy. We postulate that SPRR3 may function as a conduit for the coordinated activation of PDGFRβ by integrin β1, leading to augmentation of fibroblast proliferation and matrix synthesis downstream of biomechanical and growth factor signals.

Basement membranes (BMs) are complex macromolecular networks underlying all continuous layers of cells. Essential components include collagen IV and laminins, which are affected by human genetic variants leading to a range of debilitating conditions including kidney, muscle, and cerebrovascular phenotypes. We investigated the dynamics of BM assembly in human pluripotent stem cell-derived kidney organoids. We resolved their global BM composition and discovered a conserved temporal sequence in BM assembly that paralleled mammalian fetal kidneys. We identified the emergence of key BM isoforms, which were altered by a pathogenic variant in COL4A5. Integrating organoid, fetal, and adult kidney proteomes, we found dynamic regulation of BM composition through development to adulthood, and with single-cell transcriptomic analysis we mapped the cellular origins of BM components. Overall, we define the complex and dynamic nature of kidney organoid BM assembly and provide a platform for understanding its wider relevance in human development and disease.

The main laminin-binding integrins α3β1, α6β1 and α6β4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-κB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-κB-dependent signaling.

We previously showed that global deletion of the cytochrome P450 epoxygenase Cyp2c44, a major epoxyeicosatrienoic acid (EET) producing enzyme in mice, leads to impaired hepatic insulin signaling resulting in insulin resistance. This finding led us to investigate whether administration of a water soluble EET analog restores insulin signaling in vivo in Cyp2c44(-/-) mice and investigated the underlying mechanisms by which this effect is exerted. Cyp2c44(-/-) mice treated with the analog EET-A for 4 weeks improved fasting glucose and glucose tolerance compared to Cyp2c44(-/-) mice treated with vehicle alone. This beneficial effect was accompanied by enhanced hepatic insulin signaling, decreased expression of gluconeogenic genes and increased expression of glycogenic genes. Mechanistically, we show that insulin-stimulated phosphorylation of insulin receptor β (IRβ) is impaired in primary Cyp2c44(-/-) hepatocytes and this can be restored by cotreatment with EET-A and insulin. Plasma membrane fractionations of livers indicated that EET-A enhances the retention of IRβ in membrane rich fractions, thus potentiating its activation. Altogether, EET analogs ameliorate insulin signaling in a genetic model of hepatic insulin resistance by stabilizing membrane-associated IRβ and potentiating insulin signaling.

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2–Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.

Background Electronic data capture (EDC) in academic health care organizations provides an opportunity for the management, aggregation, and secondary use of research and clinical data. It is especially important in resource-constrained environments such as the South African public health care sector, where paper records are still the main form of clinical record keeping. Objective The aim of this study was to describe the strategies followed by the University of the Witwatersrand Faculty of Health Sciences (Wits FHS) during the period from 2013 to 2021 to overcome resistance to, and encourage the adoption of, the REDCap (Research Electronic Data Capture; Vanderbilt University) system by academic and clinical staff. REDCap has found wide use in varying domains, including clinical studies and research projects as well as administrative, financial, and human resource applications. Given REDCap’s global footprint in >5000 institutions worldwide and potential for future growth, the strategies followed by the Wits FHS to support users and encourage adoption may be of importance to others using the system, particularly in resource-constrained settings. Methods The strategies to support users and encourage adoption included top-down organizational support; secure and reliable application, hosting infrastructure, and systems administration; an enabling and accessible REDCap support team; regular hands-on training workshops covering REDCap project setup and data collection instrument design techniques; annual local symposia to promote networking and awareness of all the latest software features and best practices for using them; participation in REDCap Consortium activities; and regular and ongoing mentorship from members of the Vanderbilt University Medical Center. Results During the period from 2013 to 2021, the use of the REDCap EDC system by individuals at the Wits FHS increased, respectively, from 129 active user accounts to 3447 active user accounts. The number of REDCap projects increased from 149 in 2013 to 12,865 in 2021. REDCap at Wits also supported various publications and research outputs, including journal articles and postgraduate monographs. As of 2020, a total of 233 journal articles and 87 postgraduate monographs acknowledged the use of the Wits REDCap system. Conclusions By providing reliable infrastructure and accessible support resources, we were able to successfully implement and grow the REDCap EDC system at the Wits FHS and its associated academic medical centers. We believe that the increase in the use of REDCap was driven by offering a dependable, secure service with a strong end-user training and support model. This model may be applied by other academic and health care organizations in resource-constrained environments planning to implement EDC technology.

BACKGROUND Electronic data capture (EDC) in academic healthcare organizations provides an opportunity for the management, aggregation and secondary use of research and clinical data. It is especially important in resource constrained environments such as the South African public healthcare sector, where paper records are still the main form of clinical record-keeping. OBJECTIVE We describe the strategies followed by the Faculty of Health Sciences, University of the Witwatersrand (Wits FHS), during the period of 2013-2021 to overcome resistance and encourage adoption of the Research Electronic Data Capture (REDCap) system by academic and clinical staff. REDCap has found wide use in varying domains including clinical studies, research projects and administrative, financial and human resource applications. Given REDCap’s global footprint in more than 5 000 institutions worldwide and potential for future growth, the strategies followed by the Wits FHS to support users and encourage adoption may be of interest to others utilizing the system, particularly in resource-constrained setting. METHODS The strategies to support users and encourage adoption included: 1) Top-down organizational support; 2) Secure and reliable hosting infrastructure and systems administration 3) An enabling and accessible REDCap support team 4) Regular hands-on training workshops covering data collection instrument design and use of REDCap; 5) Annual local symposia to promote networking and awareness of all the latest software features and best practices for using them 6) Participation in REDCap Consortium activities; and 7) Regular and ongoing mentorship from members of Vanderbilt University Medical Center. RESULTS During the period 2013-2021, utilization of the REDCap EDC system by individuals in the Wits FHS increased from 129 active user accounts in 2013 to 3 447 active user accounts respectively in 2021. The number of REDCap projects numbered 149 in 2013 and increased to 12 865 in 2021. REDCap at Wits also supported various publications and research outputs including journal articles and postgraduate monographs. As of 2020, a total of 233 journal articles and 87 postgraduate monographs acknowledged the use of the Wits REDCap system. CONCLUSIONS By providing reliable infrastructure and accessible support resources, we were able to successfully implement and grow the REDCap electronic data capture system at the Wits FHS and its associated academic medical centers. We believe that the increase in use of REDCap was driven by offering a dependable, secure service with a strong end-user training and support model. This model may be applied by other academic and healthcare organizations in resource-constrained environments planning to implement EDC technology.

Integrins - the principal extracellular matrix (ECM) receptors of the cell - promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin β1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin β1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin β1-mediated adhesion to ECM but are dependent on integrin β1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin β1 in lung tumorigenesis that is mediated through constitutive, ECM binding-independent signaling involving the cytoplasmic tail.

SUMMARYBasement membranes (BMs) are complex macromolecular networks underlying all continuous layers of cells. Essential components include type IV collagen and laminins, which are affected by human genetic defects leading to a range of debilitating conditions including kidney, muscle, and cerebrovascular phenotypes. We investigated the dynamics of BM assembly in human pluripotent stem cell-derived kidney organoids. We resolved their global BM composition and discovered a conserved temporal sequence in BM assembly that paralleled mammalian fetal kidneys. We identified the emergence of key BM isoforms, which were altered by a pathogenic variant in COL4A5. Integrating organoid, fetal and adult kidney proteomes we found dynamic regulation of BM composition through development to adulthood, and with single-cell transcriptomic analysis we mapped the cellular origins of BM components. Overall, we define the complex and dynamic nature of vertebrate BM assembly and provide a platform for understanding its wider relevance in human development and disease.

International audience; Integrins, the major receptors for cell-extracellular matrix (ECM) interactions, regulate multiple cell biological processes including adhesion, migration, proliferation and growth factor-dependent signaling. The principal laminin (LM) binding integrins α3β1, α6β1 and α6β4 are usually coexpressed in cells and bind to multiple laminins with different affinities making it difficult to define their specific function. In this study, we generated kidney epithelial collecting duct (CD) cells that lack both the α3 and α6 integrin subunits. This deletion impaired cell adhesion and migration to LM-332 and LM-511 more than deleting α3 or α6 alone. Cell adhesion mediated by both α3β1 and α6 integrins was PI3K independent, but required K63-linked polyubiquitination of Akt by the ubiquitin-modifying enzyme TRAF6. Moreover, we provide evidence that glial-derived neurotrophic factor (GDNF) and fibroblast growth factor 10 (FGF10)-mediated cell signaling, spreading and proliferation were severely compromised in double integrin α3/α6but not single α3or α6-null CD cells. Interestingly, these growth factor-dependent cell functions required both PI3K-and TRAF6-dependent Akt activation. These data suggest that expression of the integrin α3 or α6 subunit is sufficient to mediate GDNF-and FGF10-dependent spreading, proliferation and signaling on LM-511. Thus, our study shows that α3 and α6 containing integrins promote distinct functions and signaling by CD cells on laminin substrata.

Proteinuria, the most common symptom of renal injury, is an independent factor for renal tubular injury. However, the underlying mechanism remains to be fully elucidated. Mitochondrion is an important target for proteinuria-induced renal tubular cell injury. Insufficient mitophagy exacerbates cell injury by initiating mitochondrial dysfunction-related cell apoptosis. In the experiment, the role of NIP3-like protein X (NIX)-mediated mitophagy was investigated in proteinuria-induced renal injury. In this study, we demonstrated that NIX expression was reduced in renal tubules and correlated with the decline of estimated glomerular filtration rate and increase of the proteinuria in patients. In proteinuric mice, NIX-mediated mitophagy was significantly suppressed. Meanwhile, the proteinuric mice exhibited renal dysfunction, increased mitochondrial fragmentation, and tubular cell apoptosis. Overexpression of NIX attenuated those disruptions in proteinuric mice. In cultured renal tubular epithelial cells, albumin induced a decrease in NIX-mediated mitophagy and an increase in cell apoptosis. Overexpression of NIX attenuated albumin-induced cell apoptosis, whereas NIX siRNA aggravated these perturbations. These results indicate that proteinuria suppresses NIX-mediated mitophagy in the renal tubular epithelial cell, which triggers the cell undergoing mitochondria-dependent cell apoptosis. Collectively, our finding suggests that restoration of NIX-mediated mitophagy might be a novel therapeutic target for alleviating proteinuria-induced kidney injury.