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.

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.

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.

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.

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.

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.

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.

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.

Retinoic Acid Signaling Coordinates Macrophage-Dependent Injury and Repair after AKI.

Retinoic acid (RA) has been used therapeutically to reduce injury and fibrosis in models of AKI, but little is known about the regulation of this pathway and what role it has in regulating injury and repair after AKI. In these studies, we show that RA signaling is activated in mouse and zebrafish models of AKI, and that these responses limit the extent of injury and promote normal repair. These effects were mediated through a novel mechanism by which RA signaling coordinated the dynamic equilibrium of inflammatory M1 spectrum versus alternatively activated M2 spectrum macrophages. Our data suggest that locally synthesized RA represses proinflammatory macrophages, thereby reducing macrophage-dependent injury post-AKI, and activates RA signaling in injured tubular epithelium, which in turn promotes alternatively activated M2 spectrum macrophages. Because RA signaling has an essential role in kidney development but is repressed in the adult, these findings provide evidence of an embryonic signaling pathway that is reactivated after AKI and involved in reducing injury and enhancing repair.

Heterozygous null bone morphogenetic protein receptor type 2 mutations promote SRC kinase-dependent caveolar trafficking defects and endothelial dysfunction in pulmonary arterial hypertension.

Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2(+/-)) similar to those found in the majority of HPAH patients. We show that Bmpr2(+/-) PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2(+/-) PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2(+/-) PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2(+/-) PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients.

Bridging translation for acute kidney injury with better preclinical modeling of human disease.

The current lack of effective therapeutics for patients with acute kidney injury (AKI) represents an important and unmet medical need. Given the importance of the clinical problem, it is time for us to take a few steps back and reexamine current practices. The focus of this review is to explore the extent to which failure of therapeutic translation from animal studies to human studies stems from deficiencies in the preclinical models of AKI. We will evaluate whether the preclinical models of AKI that are commonly used recapitulate the known pathophysiologies of AKI that are being modeled in humans, focusing on four common scenarios that are studied in clinical therapeutic intervention trials: cardiac surgery-induced AKI; contrast-induced AKI; cisplatin-induced AKI; and sepsis associated AKI. Based on our observations, we have identified a number of common limitations in current preclinical modeling of AKI that could be addressed. In the long term, we suggest that progress in developing better preclinical models of AKI will depend on developing a better understanding of human AKI. To this this end, we suggest that there is a need to develop greater in-depth molecular analyses of kidney biopsy tissues coupled with improved clinical and molecular classification of patients with AKI.

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.

Delayed treatment with PTBA analogs reduces postinjury renal fibrosis after kidney injury.

No therapies have been shown to accelerate recovery or prevent fibrosis after acute kidney injury (AKI). In part, this is because most therapeutic candidates have to be given at the time of injury and the diagnosis of AKI is usually made too late for drugs to be efficacious. Strategies to enhance post-AKI repair represent an attractive approach to address this. Using a phenotypic screen in zebrafish, we identified 4-(phenylthio)butanoic acid (PTBA), which promotes proliferation of embryonic kidney progenitor cells (EKPCs), and the PTBA methyl ester UPHD25, which also increases postinjury repair in ischemia-reperfusion and aristolochic acid-induced AKI in mice. In these studies, a new panel of PTBA analogs was evaluated. Initial screening was performed in zebrafish EKPC assays followed by survival assays in a gentamicin-induced AKI larvae zebrafish model. Using this approach, we identified UPHD186, which in contrast to UPHD25, accelerates recovery and reduces fibrosis when administered several days after ischemia-reperfusion AKI and reduces fibrosis after unilateral ureteric obstruction in mice. UPHD25 and 186 are efficiently metabolized to the active analog PTBA in liver and kidney microsome assays, indicating both compounds may act as PTBA prodrugs in vivo. UPHD186 persists longer in the circulation than UPHD25, suggesting that sustained levels of UPHD186 may increase efficacy by acting as a reservoir for renal metabolism to PTBA. These findings validate use of zebrafish EKPC and AKI assays as a drug discovery strategy for molecules that reduce fibrosis in multiple AKI models and can be administered days after initiation of injury.

A PTBA small molecule enhances recovery and reduces postinjury fibrosis after aristolochic acid-induced kidney injury.

Phenylthiobutanoic acids (PTBAs) are a new class of histone deacetylase (HDAC) inhibitors that accelerate recovery and reduce postinjury fibrosis after ischemia-reperfusion-induced acute kidney injury. However, unlike the more common scenario in which patients present with protracted and less clearly defined onset of renal injury, this model of acute kidney injury gives rise to a clearly defined injury that begins to resolve over a short period of time. In these studies, we show for the first time that treatment with the PTBA analog methyl-4-(phenylthio)butanoate (M4PTB) accelerates recovery and reduces postinjury fibrosis in a progressive model of acute kidney injury and renal fibrosis that occurs after aristolochic acid injection in mice. These effects are apparent when M4PTB treatment is delayed 4 days after the initiating injury and are associated with increased proliferation and decreased G2/M arrest of regenerating renal tubular epithelial cells. In addition, there is reduced peritubular macrophage infiltration and decreased expression of the macrophage chemokines CX3Cl1 and CCL2. Since macrophage infiltration plays a role in promoting kidney injury, and since renal tubular epithelial cells show defective repair and a marked increase in maladaptive G2/M arrest after aristolochic acid injury, these findings suggest M4PTB may be particularly beneficial in reducing injury and enhancing intrinsic cellular repair even when administered days after aristolochic acid ingestion.

Kidney regeneration: common themes from the embryo to the adult.

The vertebrate kidney has an inherent ability to regenerate following acute damage. Successful regeneration of the injured kidney requires the rapid replacement of damaged tubular epithelial cells and reconstitution of normal tubular function. Identifying the cells that participate in the regeneration process as well as the molecular mechanisms involved may reveal therapeutic targets for the treatment of kidney disease. Renal regeneration is associated with the expression of genetic pathways that are necessary for kidney organogenesis, suggesting that the regenerating tubular epithelium may be "reprogrammed" to a less-differentiated, progenitor state. This review will highlight data from various vertebrate models supporting the hypothesis that nephrogenic genes are reactivated as part of the process of kidney regeneration following acute kidney injury (AKI). Emphasis will be placed on the reactivation of developmental pathways and how our understanding of the resulting regeneration process may be enhanced by lessons learned in the embryonic kidney.

Histone deacetylase inhibitor enhances recovery after AKI.

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.

HDAC inhibitors in kidney development and disease.

The discovery that histone deacetylase inhibitors (HDACis) can attenuate acute kidney injury (AKI)-mediated damage and reduce fibrosis in kidney disease models has opened the possibility of utilizing HDACis as therapeutics for renal injury. Studies to date have made it abundantly clear that HDACi treatment results in a plethora of molecular changes, which are not always linked to histone acetylation, and that there is an essential need to understand the specific target(s) of any HDACi of interest. New lines of investigation are beginning to delve more deeply into target identification of specific HDACis and to address the relative toxicity of different HDACi classes. This review will focus on the utilization of HDACis during kidney organogenesis, injury, and disease, as well as on the development of these compounds as therapeutics.

Molecular characterization of Wilms' tumor from a resource-constrained region of sub-Saharan Africa.

Sub-Saharan African children have an increased incidence of Wilms' tumor (WT) and experience alarmingly poor outcomes. Although these outcomes are largely due to inadequate therapy, we hypothesized that WT from this region exhibits features of biological aggressiveness that may warrant broader implementation of high-risk therapeutic protocols. We evaluated 15 Kenyan WT (KWT) for features of aggressive disease (blastemal predominance and Ki67/cellular proliferation) and treatment resistance (anaplasia and p53 immunopositivity). To explore the additional biological features of KWT, we determined the mutational status of the CTNNB1/ß-catenin and WT1 genes and performed immunostaining for markers of Wnt pathway activation (ß-catenin) and nephronic progenitor cell self-renewal (WT1, CITED1 and SIX2). We characterized the proteome of KWT using imaging mass spectrometry (IMS). The results were compared to histology- and age-matched North American WT (NAWT) controls. For patients with KWT, blastemal predominance was noted in 53.3% and anaplasia in 13%. We detected increased loss to follow-up (p = 0.028), disease relapse (p = 0.044), mortality (p = 0.001) and nuclear unrest (p = 0.001) in patients with KWT compared to controls. KWT and NAWT showed similar Ki67/cellular proliferation. We detected an increased proportion of epithelial nuclear ß-catenin in KWT (p = 0.013). All 15 KWT specimens were found to harbor wild-type CTNNB1/ß-catenin, and one contained a WT1 nonsense mutation. WT1 was detected by immunostaining in 100% of KWT, CITED1 in 80% and SIX2 in 80%. IMS revealed a molecular signature unique to KWT that was distinct from NAWT. The African WT specimens appear to express markers of adverse clinical behavior and treatment resistance and may require alternative therapies or implementation of high-risk treatment protocols.

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.