Validation of an organ mapping antibody panel for cyclical immunofluorescence microscopy on normal human kidneys.

The lack of standardization in antibody validation remains a major contributor to irreproducibility of human research. To address this, we have applied a standardized approach to validate a panel of antibodies to identify 18 major cell types and 5 extracellular matrix compartments in the human kidney by immunofluorescence (IF) microscopy. We have used these to generate an organ mapping antibody panel for two-dimensional (2-D) and three-dimensional (3-D) cyclical IF (CyCIF) to provide a more detailed method for evaluating tissue segmentation and volumes using a larger panel of markers than would normally be possible using standard fluorescence microscopy. CyCIF also makes it possible to perform multiplexed IF microscopy of whole slide images, which is a distinct advantage over other multiplexed imaging technologies that are applicable to limited fields of view. This enables a broader view of cell distributions across larger anatomical regions, allowing a better chance to capture localized regions of dysfunction in diseased tissues. These methods are broadly accessible to any laboratory with a fluorescence microscope, enabling spatial cellular phenotyping in normal and disease states. We also provide a detailed solution for image alignment between CyCIF cycles that can be used by investigators to perform these studies without programming experience using open-sourced software. This ability to perform multiplexed imaging without specialized instrumentation or computational skills opens the door to integration with more highly dimensional molecular imaging modalities such as spatial transcriptomics and imaging mass spectrometry, enabling the discovery of molecular markers of specific cell types, and how these are altered in disease.NEW & NOTEWORTHY We describe here validation criteria used to define on organ mapping panel of antibodies that can be used to define 18 cell types and five extracellular matrix compartments using cyclical immunofluorescence (CyCIF) microscopy. As CyCIF does not require specialized instrumentation, and image registration required to assemble CyCIF images can be performed by any laboratory without specialized computational skills, this technology is accessible to any laboratory with access to a fluorescence microscope and digital scanner.

Hyperoxia Increases Kidney Injury During Renal Ischemia and Reperfusion in Mice.

BACKGROUND: Renal ischemia and reperfusion (IR) contribute to perioperative acute kidney injury, and oxygen is a key regulator of this process. We hypothesized that oxygen administration during surgery and renal IR would impact postoperative kidney function and injury in mice. METHODS: Mice were anesthetized, intubated, and mechanically ventilated with a fraction of inspired oxygen (F io2 ) 0.10 (hypoxia), 0.21 (normoxia), 0.60 (moderate hyperoxia), or 1.00 (severe hyperoxia) during 67 minutes of renal IR or sham IR surgery. Additional mice were treated before IR or sham IR surgery with 50 mg/kg tempol, a superoxide scavenger. At 24 hours, mice were sacrificed, and blood and kidney collected. We assessed and compared kidney function and injury across groups by measuring blood urea nitrogen (BUN, primary end point), renal histological injury, renal expression of neutrophil gelatinase-associated lipocalin (NGAL), and renal heme oxygenase 1 ( Ho-1 ), peroxisome proliferator-activated receptor gamma coactivator 1-α ( Pgc1-α ), and glutathione peroxidase 4 ( Gpx-4 ) transcripts, to explore potential mechanisms of any effect of oxygen. RESULTS: Hyperoxia and hypoxia during renal IR surgery decreased renal function and increased kidney injury compared to normoxia. Baseline median (interquartile range) BUN was 22.2 mg/dL (18.4-26.0), and 24 hours after IR surgery, BUN was 17.5 mg/dL (95% confidence interval [CI], 1.3-38.4; P = .034) higher in moderate hyperoxia-treated animals, 51.8 mg/dL (95% CI, 24.9-74.8; P < .001) higher in severe hyperoxia-treated animals, and 64.9 mg/dL (95% CI, 41.2-80.3; P < .001) higher in hypoxia-treated animals compared to animals treated with normoxia ( P < .001, overall effect of hyperoxia). Hyperoxia-induced injury, but not hypoxia-induced injury, was attenuated by pretreatment with tempol. Histological injury scores, renal NGAL staining, and renal transcription of Ho-1 and suppression of Pgc1- α followed the same pattern as BUN, in relation to the effects of oxygen treatment. CONCLUSIONS: In this controlled preclinical study of oxygen treatment during renal IR surgery, hyperoxia and hypoxia impaired renal function, increased renal injury, and impacted expression of genes that affect mitochondrial biogenesis and antioxidant response. These results might have implications for patients during surgery when high concentrations of oxygen are frequently administered, especially in cases involving renal IR.

Pretreatment with a novel Toll-like receptor 4 agonist attenuates renal ischemia-reperfusion injury.

Acute kidney injury (AKI) is common in surgical and critically ill patients. This study examined whether pretreatment with a novel Toll-like receptor 4 agonist attenuated ischemia-reperfusion injury (IRI)-induced AKI (IRI-AKI). We performed a blinded, randomized-controlled study in mice pretreated with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD), a synthetic Toll-like receptor 4 agonist. Two cohorts of male BALB/c mice received intravenous vehicle or PHAD (2, 20, or 200 µg) at 48 and 24 h before unilateral renal pedicle clamping and simultaneous contralateral nephrectomy. A separate cohort of mice received intravenous vehicle or 200 µg PHAD followed by bilateral IRI-AKI. Mice were monitored for evidence of kidney injury for 3 days postreperfusion. Kidney function was assessed by serum blood urea nitrogen and creatinine measurements. Kidney tubular injury was assessed by semiquantitative analysis of tubular morphology on periodic acid-Schiff (PAS)-stained kidney sections and by kidney mRNA quantification of injury [neutrophil gelatinase-associated lipocalin (Ngal), kidney injury molecule-1 (Kim-1), and heme oxygenase-1 (Ho-1)] and inflammation [interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (Tnf-α)] using quantitative RT-PCR. Immunohistochemistry was used to quantify proximal tubular cell injury and renal macrophages by quantifying the areas stained with Kim-1 and F4/80 antibodies, respectively, and TUNEL staining to detect the apoptotic nuclei. PHAD pretreatment yielded dose-dependent kidney function preservation after unilateral IRI-AKI. Histological injury, apoptosis, Kim-1 staining, and Ngal mRNA were lower in PHAD-treated mice and IL-1ß mRNA was higher in PHAD-treated mice. Similar pretreatment protection was noted with 200 mg PHAD after bilateral IRI-AKI, with significantly reduced Kim-1 immunostaining in the outer medulla of mice treated with PHAD after bilateral IRI-AKI. In conclusion, PHAD pretreatment leads to dose-dependent protection from renal injury after unilateral and bilateral IRI-AKI in mice.NEW & NOTEWORTHY Pretreatment with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide; a novel synthetic Toll-like receptor 4 agonist, preserves kidney function during ischemia-reperfusion injury-induced acute kidney injury.

Supervised Deep Generation of High-Resolution Arterial Phase Computed Tomography Kidney Substructure Atlas.

The Human BioMolecular Atlas Program (HuBMAP) provides an opportunity to contextualize findings across cellular to organ systems levels. Constructing an atlas target is the primary endpoint for generalizing anatomical information across scales and populations. An initial target of HuBMAP is the kidney organ and arterial phase contrast-enhanced computed tomography (CT) provides distinctive appearance and anatomical context on the internal substructure of kidney organs such as renal context, medulla, and pelvicalyceal system. With the confounding effects of demographics and morphological characteristics of the kidney across large-scale imaging surveys, substantial variation is demonstrated with the internal substructure morphometry and the intensity contrast due to the variance of imaging protocols. Such variability increases the level of difficulty to localize the anatomical features of the kidney substructure in a well-defined spatial reference for clinical analysis. In order to stabilize the localization of kidney substructures in the context of this variability, we propose a high-resolution CT kidney substructure atlas template. Briefly, we introduce a deep learning preprocessing technique to extract the volumetric interest of the abdominal regions and further perform a deep supervised registration pipeline to stably adapt the anatomical context of the kidney internal substructure. To generate and evaluate the atlas template, arterial phase CT scans of 500 control subjects are de-identified and registered to the atlas template with a complete end-to-end pipeline. With stable registration to the abdominal wall and kidney organs, the internal substructure of both left and right kidneys are substantially localized in the high-resolution atlas space. The atlas average template successfully demonstrated the contextual details of the internal structure and was applicable to generalize the morphological variation of internal substructure across patients.

Multiparametric magnetic resonance imaging in diagnosis of long-term renal atrophy and fibrosis after ischemia reperfusion induced acute kidney injury in mice.

Tubular atrophy and fibrosis are pathological changes that determine the prognosis of kidney disease induced by acute kidney injury (AKI). We aimed to evaluate multiple magnetic resonance imaging (MRI) parameters, including pool size ratio (PSR) from quantitative magnetization transfer, relaxation rates, and measures from spin-lock imaging ( R 1 ρ and S ρ ), for assessing the pathological changes associated with AKI-induced kidney disease. Eight-week-old male C57BL/6 J mice first underwent unilateral ischemia reperfusion injury (IRI) induced by reperfusion after 45 min of ischemia. They were imaged using a 7T MRI system 56 days after the injury. Paraffin tissue sections were stained using Masson trichrome and picrosirius red to identify histopathological changes such as tubular atrophy and fibrosis. Histology detected extensive tubular atrophy and moderate fibrosis in the cortex and outer stripe of the outer medulla (CR + OSOM) and more prominent fibrosis in the inner stripe of the outer medulla (ISOM) of IRI kidneys. In the CR + OSOM region, evident decreases in PSR, R 1 , R 2 , R 1 ρ , and S ρ showed in IRI compared with contralateral kidneys, with PSR and S ρ exhibiting the most significant changes. In addition, the exchange parameter S ρ dropped by the largest degree among all the MRI parameters, while R 2 * increased significantly. In the ISOM of IRI kidneys, PSR increased while S ρ kept decreasing. R 2 , R 1 ρ , and R 2 * all increased due to more severe fibrosis in this region. Among MRI measures, PSR and R 1 ρ showed the highest detectability of renal changes no matter whether tubular atrophy or fibrosis dominated. R 2 * and S ρ could be more specific to a single pathological event than other MRI measures because only R 2 * increased and S ρ decreased consistently when either fibrosis or tubular atrophy dominated, and their correlations with fibrosis scores were higher than other MRI measures. Multiparametric MRI may enable a more comprehensive analysis of histopathological changes following AKI.

Multi-contrast computed tomography healthy kidney atlas.

The construction of three-dimensional multi-modal tissue maps provides an opportunity to spur interdisciplinary innovations across temporal and spatial scales through information integration. While the preponderance of effort is allocated to the cellular level and explore the changes in cell interactions and organizations, contextualizing findings within organs and systems is essential to visualize and interpret higher resolution linkage across scales. There is a substantial normal variation of kidney morphometry and appearance across body size, sex, and imaging protocols in abdominal computed tomography (CT). A volumetric atlas framework is needed to integrate and visualize the variability across scales. However, there is no abdominal and retroperitoneal organs atlas framework for multi-contrast CT. Hence, we proposed a high-resolution CT retroperitoneal atlas specifically optimized for the kidney organ across non-contrast CT and early arterial, late arterial, venous and delayed contrast-enhanced CT. We introduce a deep learning-based volume interest extraction method by localizing the 2D slices with a representative score and crop within the range of the abdominal interest. An automated two-stage hierarchal registration pipeline is then performed to register abdominal volumes to a high-resolution CT atlas template with DEEDS affine and non-rigid registration. To generate and evaluate the atlas framework, multi-contrast modality CT scans of 500 subjects (without reported history of renal disease, age: 15-50 years, 250 males & 250 females) were processed. PDD-Net with affine registration achieved the best overall mean DICE for portal venous phase multi-organs label transfer with the registration pipeline (0.540 ± 0.275, p < 0.0001 Wilcoxon signed-rank test) comparing to the other registration tools. It also demonstrated the best performance with the median DICE over 0.8 in transferring the kidney information to the atlas space. DEEDS perform constantly with stable transferring performance in all phases average mapping including significant clear boundary of kidneys with contrastive characteristics, while PDD-Net only demonstrates a stable kidney registration in the average mapping of early and late arterial, and portal venous phase. The variance mappings demonstrate the low intensity variance in the kidney regions with DEEDS across all contrast phases and with PDD-Net across late arterial and portal venous phase. We demonstrate a stable generalizability of the atlas template for integrating the normal kidney variation from small to large, across contrast modalities and populations with great variability of demographics. The linkage of atlas and demographics provided a better understanding of the variation of kidney anatomy across populations.

Kidney repair and regeneration: perspectives of the NIDDK (Re)Building a Kidney consortium.

Acute kidney injury impacts âˆ¼13.3 million individuals and causes âˆ¼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK's open access data hub for accessing, downloading, and further analyzing data sets.

Experimental models of acute kidney injury for translational research.

Preclinical models of human disease provide powerful tools for therapeutic discovery but have limitations. This problem is especially apparent in the field of acute kidney injury (AKI), in which clinical trial failures have been attributed to inaccurate modelling performed largely in rodents. Multidisciplinary efforts such as the Kidney Precision Medicine Project are now starting to identify molecular subtypes of human AKI. In addition, over the past decade, there have been developments in human pluripotent stem cell-derived kidney organoids as well as zebrafish, rodent and large animal models of AKI. These organoid and AKI models are being deployed at different stages of preclinical therapeutic development. However, the traditionally siloed, preclinical investigator-driven approaches that have been used to evaluate AKI therapeutics to date rarely account for the limitations of the model systems used and have given rise to false expectations of clinical efficacy in patients with different AKI pathophysiologies. To address this problem, there is a need to develop more flexible and integrated approaches, involving teams of investigators with expertise in a range of different model systems, working closely with clinical investigators, to develop robust preclinical evidence to support more focused interventions in patients with AKI.

Renal Cortex, Medulla and Pelvicaliceal System Segmentation on Arterial Phase CT Images with Random Patch-based Networks.

Renal segmentation on contrast-enhanced computed tomography (CT) provides distinct spatial context and morphology. Current studies for renal segmentations are highly dependent on manual efforts, which are time-consuming and tedious. Hence, developing an automatic framework for the segmentation of renal cortex, medulla and pelvicalyceal system is an important quantitative assessment of renal morphometry. Recent innovations in deep methods have driven performance toward levels for which clinical translation is appealing. However, the segmentation of renal structures can be challenging due to the limited field-of-view (FOV) and variability among patients. In this paper, we propose a method to automatically label the renal cortex, the medulla and pelvicalyceal system. First, we retrieved 45 clinically-acquired deidentified arterial phase CT scans (45 patients, 90 kidneys) without diagnosis codes (ICD-9) involving kidney abnormalities. Second, an interpreter performed manual segmentation to pelvis, medulla and cortex slice-by-slice on all retrieved subjects under expert supervision. Finally, we proposed a patch-based deep neural networks to automatically segment renal structures. Compared to the automatic baseline algorithm (3D U-Net) and conventional hierarchical method (3D U-Net Hierarchy), our proposed method achieves improvement of 0.7968 to 0.6749 (3D U-Net), 0.7482 (3D U-Net Hierarchy) in terms of mean Dice scores across three classes (p-value < 0.001, paired t-tests between our method and 3D U-Net Hierarchy). In summary, the proposed algorithm provides a precise and efficient method for labeling renal structures.

Construction of a Multi-Phase Contrast Computed Tomography Kidney Atlas.

The Human BioMolecular Atlas Program (HuBMAP) seeks to create a molecular atlas at the cellular level of the human body to spur interdisciplinary innovations across spatial and temporal scales. While the preponderance of effort is allocated towards cellular and molecular scale mapping, differentiating and contextualizing findings within tissues, organs and systems are essential for the HuBMAP efforts. The kidney is an initial organ target of HuBMAP, and constructing a framework (or atlas) for integrating information across scales is needed for visualizing and integrating information. However, there is no abdominal atlas currently available in the public domain. Substantial variation in healthy kidneys exists with sex, body size, and imaging protocols. With the integration of clinical archives for secondary research use, we are able to build atlases based on a diverse population and clinically relevant protocols. In this study, we created a computed tomography (CT) phase-specific atlas for the abdomen, which is optimized for the kidney organ. A two-stage registration pipeline was used by registering extracted abdominal volume of interest from body part regression, to a high-resolution CT. Affine and non-rigid registration were performed to all scans hierarchically. To generate and evaluate the atlas, multiphase CT scans of 500 control subjects (age: 15 - 50, 250 males, 250 females) are registered to the atlas target through the complete pipeline. The abdominal body and kidney registration are shown to be stable with the variance map computed from the result average template. Both left and right kidneys are substantially localized in the high-resolution target space, which successfully demonstrated the sharp details of its anatomical characteristics across each phase. We illustrated the applicability of the atlas template for integrating across normal kidney variation from 64 cm3 to 302 cm3.

Capillary rarefaction is more closely associated with CKD progression after cisplatin, rhabdomyolysis, and ischemia-reperfusion-induced AKI than renal fibrosis.

Acute kidney injury (AKI) is a strong independent predictor of mortality and often results in incomplete recovery of renal function, leading to progressive chronic kidney disease (CKD). Many clinical trials have been conducted on the basis of promising preclinical data, but no therapeutic interventions have been shown to improve long-term outcomes after AKI. This is partly due to the failure of preclinical studies to accurately model clinically relevant injury and long-term outcomes on CKD progression. Here, we evaluated the long-term effects of AKI on CKD progression in three animal models reflecting diverse etiologies of AKI: repeat-dose cisplatin, rhabdomyolysis, and ischemia-reperfusion injury. Using transdermal measurement of glomerular filtration rate as a clinically relevant measure of kidney function and quantification of peritubular capillary density to measure capillary rarefaction, we showed that repeat-dose cisplatin caused capillary rarefaction and decreased renal function in mice without a significant increase in interstitial fibrosis, whereas rhabdomyolysis-induced AKI led to severe interstitial fibrosis, but renal function and peritubular capillary density were preserved. Furthermore, long-term experiments in mice with unilateral ischemia-reperfusion injury showed that restoration of renal function 12 wk after a contralateral nephrectomy was associated with increasing fibrosis, but a reversal of capillary rarefaction was seen at 4 wk. These data demonstrate that clear dissociation between kidney function and fibrosis in these models of AKI to CKD progression and suggest that peritubular capillary rarefaction is more strongly associated with CKD progression than renal fibrosis.

Cell-free hemoglobin augments acute kidney injury during experimental sepsis.

Acute kidney injury is a common complication of severe sepsis and contributes to high mortality. The molecular mechanisms of acute kidney injury during sepsis are not fully understood. Because hemoproteins, including myoglobin and hemoglobin, are known to mediate kidney injury during rhabdomyolysis, we hypothesized that cell-free hemoglobin (CFH) would exacerbate acute kidney injury during sepsis. Sepsis was induced in mice by intraperitoneal injection of cecal slurry (CS). To mimic elevated levels of CFH observed during human sepsis, mice also received a retroorbital injection of CFH or dextrose control. Four groups of mice were analyzed: sham treated (sham), CFH alone, CS alone, and CS + CFH. The addition of CFH to CS reduced 48-h survival compared with CS alone (67% vs. 97%, P = 0.001) and increased the severity of illness. After 24 and 48 h, CS + CFH mice had a reduced glomerular filtration rate from baseline, whereas sham, CFH, and CS mice maintained baseline glomerular filtration rate. Biomarkers of acute kidney injury, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), were markedly elevated in CS+CFH compared with CS (8-fold for NGAL and 2.4-fold for KIM-1, P < 0.002 for each) after 48 h. Histological examination showed a trend toward increased tubular injury in CS + CFH-exposed kidneys compared with CS-exposed kidneys. However, there were similar levels of renal oxidative injury and apoptosis in the CS + CFH group compared with the CS group. Kidney levels of multiple proinflammatory cytokines were similar between CS and CS + CFH groups. Human renal tubule cells (HK-2) exposed to CFH demonstrated increased cytotoxicity. Together, these results show that CFH exacerbates acute kidney injury in a mouse model of experimental sepsis, potentially through increased renal tubular injury.

Long-term outcomes in mouse models of ischemia-reperfusion-induced acute kidney injury.

Severe acute kidney injury has a high mortality and is a risk factor for progressive chronic kidney disease. None of the potential therapies that have been identified in preclinical studies have successfully improved clinical outcomes. This failure is partly because animal models rarely reflect the complexity of human disease: most preclinical studies are short term and are commonly performed in healthy, young, male mice. Therapies that are effective in preclinical models that share common clinical features seen in patients with acute kidney injury, including genetic diversity, different sexes, and comorbidities, and evaluate long-term outcomes are more likely to predict success in the clinic. Here, we evaluated susceptibility to chronic kidney disease after ischemia-reperfusion injury with delayed nephrectomy by monitoring long-term functional and histological responses to injury. We defined conditions required to induce long-term postinjury renal dysfunction and fibrosis without increased mortality in a reproducible way and evaluate effect of mouse strains, sexes, and preexisting diabetes on these responses.

The Extracellular Matrix Receptor Discoidin Domain Receptor 1 Regulates Collagen Transcription by Translocating to the Nucleus.

BACKGROUND: The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus via a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with transcription factors. METHODS: To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation. RESULTS: We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and ß-actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. CONCLUSIONS: These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.

Sepsis-Associated Acute Kidney Injury: A Problem Deserving of New Solutions.

Sepsis-associated acute kidney injury (S-AKI) significantly worsens patient prognosis, and recent evidence suggests that the injury process begins early and may be sustained by therapies used to treat the sepsis (e.g., fluids resuscitation, antibiotics). While efforts to develop less-injurious treatments are making progress, some degree of secondary injury is to be expected. So too is the inevitable nature of organ injury, which is often present at the time the patient seeks medical attention. We recently found that most patients presenting with septic shock and developing AKI had evidence of kidney damage at the time of, or within 24 h of their admission. In such patients, prevention is not a viable option, as injury has already occurred by the time of presentation. Since S-AKI patients are at increased risk of developing chronic kidney disease, a fundamental target for interventions in S-AKI is to prevent fibrosis (maladaptive repair) while stimulating regeneration (proliferation of viable epithelial cells). Using a pathway-agnostic, proliferation-based phenotypic assay, we discovered phenylthiobutanoic acid, a small molecule histone deacetylase inhibitor, that enhances renal recovery and reduces fibrosis in both zebrafish and mouse models of AKI.

Low-grade albuminuria in pulmonary arterial hypertension.

Low-grade albuminuria, determined by the urinary albumin to creatinine ratio, has been linked to systemic vascular dysfunction and is associated with cardiovascular mortality. Pulmonary arterial hypertension is related to mutations in the bone morphogenetic protein receptor type 2, pulmonary vascular dysfunction and is increasingly recognized as a systemic disease. In a total of 283 patients (two independent cohorts) diagnosed with pulmonary arterial hypertension, 18 unaffected BMPR2 mutation carriers and 68 healthy controls, spot urinary albumin to creatinine ratio and its relationship to demographic, functional, hemodynamic and outcome data were analyzed. Pulmonary arterial hypertension patients and unaffected BMPR2 mutation carriers had significantly elevated urinary albumin to creatinine ratios compared with healthy controls ( P < 0.01; P = 0.04). In pulmonary arterial hypertension patients, the urinary albumin to creatinine ratio was associated with older age, lower six-minute walking distance, elevated levels of C-reactive protein and hemoglobin A1c, but there was no correlation between the urinary albumin to creatinine ratio and hemodynamic variables. Pulmonary arterial hypertension patients with a urinary albumin to creatinine ratio above 10 µg/mg had significantly higher rates of poor outcome ( P < 0.001). This study shows that low-grade albuminuria is prevalent in pulmonary arterial hypertension patients and is associated with poor outcome. This study shows that albuminuria in pulmonary arterial hypertension is associated with systemic inflammation and insulin resistance.

BMPR2 mutations and endothelial dysfunction in pulmonary arterial hypertension (2017 Grover Conference Series).

Despite the discovery more than 15 years ago that patients with hereditary pulmonary arterial hypertension (HPAH) inherit BMP type 2 receptor ( BMPR2) mutations, it is still unclear how these mutations cause disease. In part, this is attributable to the rarity of HPAH and difficulty obtaining tissue samples from patients with early disease. However, in addition, limitations to the approaches used to study the effects of BMPR2 mutations on the pulmonary vasculature have restricted our ability to determine how individual mutations give rise to progressive pulmonary vascular pathology in HPAH. The importance of understanding the mechanisms by which BMPR2 mutations cause disease in patients with HPAH is underscored by evidence that there is reduced BMPR2 expression in patients with other, more common, non-hereditary form of PAH, and that restoration of BMPR2 expression reverses established disease in experimental models of pulmonary hypertension. In this paper, we focus on the effects on endothelial function. We discuss some of the controversies and challenges that have faced investigators exploring the role of BMPR2 mutations in HPAH, focusing specifically on the effects different BMPR2 mutation have on endothelial function, and whether there are qualitative differences between different BMPR2 mutations. We discuss evidence that BMPR2 signaling regulates a number of responses that may account for endothelial abnormalities in HPAH and summarize limitations of the models that are used to study these effects. Finally, we discuss evidence that BMPR2-dependent effects on endothelial metabolism provides a unifying explanation for the many of the BMPR2 mutation-dependent effects that have been described in patients with HPAH.

Correction: Deletion of Fibroblast Growth Factor Receptor 2 from the Peri-Wolffian Duct Stroma Leads to Ureteric Induction Abnormalities and Vesicoureteral Reflux.

[This corrects the article DOI: 10.1371/journal.pone.0056062.].

Pyridoxamine reduces postinjury fibrosis and improves functional recovery after acute kidney injury.

Acute kidney injury (AKI) is a common and independent risk factor for death and chronic kidney disease (CKD). Despite promising preclinical data, there is no evidence that antioxidants reduce the severity of injury, increase recovery, or prevent CKD in patients with AKI. Pyridoxamine (PM) is a structural analog of vitamin B6 that interferes with oxidative macromolecular damage via a number of different mechanisms and is in a phase 3 clinical efficacy trial to delay CKD progression in patients with diabetic kidney disease. Because oxidative stress is implicated as one of the main drivers of renal injury after AKI, the ability of PM to interfere with multiple aspects of oxidative damage may be favorable for AKI treatment. In these studies we therefore evaluated PM treatment in a mouse model of AKI. Pretreatment with PM caused a dose-dependent reduction in acute tubular injury, long-term postinjury fibrosis, as well as improved functional recovery after ischemia-reperfusion AKI (IR-AKI). This was associated with a dose-dependent reduction in the oxidative stress marker isofuran-to-F2-isoprostane ratio, indicating that PM reduces renal oxidative damage post-AKI. PM also reduced postinjury fibrosis when administered 24 h after the initiating injury, but this was not associated with improvement in functional recovery after IR-AKI. This is the first report showing that treatment with PM reduces short- and long-term injury, fibrosis, and renal functional recovery after IR-AKI. These preclinical findings suggest that PM, which has a favorable clinical safety profile, holds therapeutic promise for AKI and, most importantly, for prevention of adverse long-term outcomes after AKI.