Search

Acute kidney injury (AKI) is a common clinical syndrome associated with adverse short- and long-term sequelae. Renal tubular epithelial cell (RTEC) dysfunction and cell death are among the key pathological features of AKI. Diverse systemic and localized stress conditions such as sepsis, rhabdomyolysis, cardiac surgery, and nephrotoxic drugs can trigger RTEC dysfunction. Through an unbiased RNA inhibition screen, we recently identified cyclin-dependent kinase-like 5 (Cdkl5), also known as serine/threonine kinase-9, as a critical regulator of RTEC dysfunction associated with nephrotoxic and ischemia-associated AKI. In the present study, we examined the role of Cdkl5 in rhabdomyolysis-associated AKI. Using activation-specific antibodies and kinase assays, we found that Cdkl5 is activated in RTECs early during the development of rhabdomyolysis-associated AKI. Furthermore, we found that RTEC-specific Cdkl5 gene ablation mitigates rhabdomyolysis-associated renal impairment. In addition, the small-molecule kinase inhibitor AST-487 alleviated rhabdomyolysis-associated AKI in a Cdkl5-dependent manner. Mechanistically, we demonstrated that Cdkl5 phosphorylates the transcriptional regulator sex-determining region Y box 9 (Sox9) and suppresses its protective function under stress conditions. On the basis of these results, we propose that, by suppressing the protective Sox9-directed transcriptional program, Cdkl5 contributes to rhabdomyolysis-associated renal impairment. All together, the present study identified Cdkl5 as a critical stress-induced kinase that drives RTEC dysfunction and kidney injury linked with distinct etiologies.

Treating acute kidney injury (AKI) represents an important unmet medical need both in terms of the seriousness of this medical problem and the number of patients. There is also a large untapped market opportunity in treating AKI. Over the years, there has been much effort in search of therapeutics with minimal success. However, over the same time period, new understanding of the underlying pathobiology and molecular mechanisms of kidney injury have undoubtedly helped the search for new therapeutics. Along this line, carbon monoxide (CO) has emerged as a promising therapeutic agent because of its demonstrated cytoprotective, and immunomodulatory effects. CO has also been shown to sensitize cancer, but not normal cells, to chemotherapy. This is particularly important in treating cisplatin-induced AKI, a common clinical problem that develops in patients receiving cisplatin therapies for a number of different solid organ malignancies. This review will examine and make the case that CO be developed into a therapeutic agent against AKI.

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.

AKI is a complex clinical condition associated with high mortality, morbidity, and health care costs. Despite improvements in methodology and design of clinical trials, and advances in understanding the underlying pathophysiology of rodent AKI, no pharmacologic agent exists for the prevention or treatment of AKI in humans. To address the barriers that affect successful clinical translation of drug targets identified and validated in preclinical animal models of AKI in this patient population, the National Institute of Diabetes and Digestive and Kidney Diseases convened the “AKI Outcomes: Overcoming Barriers in AKI” workshop on February 10–12, 2015. The workshop used a reverse translational medicine approach to identify steps necessary to achieve clinical success. During the workshop, breakout groups were charged first to design feasible, phase 2, proof-of-concept clinical trials for delayed transplant graft function, prevention of AKI (primary prevention), and treatment of AKI (secondary prevention and recovery). Breakout groups then were responsible for identification of preclinical animal models that would replicate the pathophysiology of the phase 2 proof-of-concept patient population, including primary and secondary end points. Breakout groups identified considerable gaps in knowledge regarding human AKI, our understanding of the pathophysiology of AKI in preclinical animal models, and the fidelity of cellular and molecular targets that have been evaluated preclinically to provide information regarding human AKI of various etiologies. The workshop concluded with attendees defining a new path forward to a better understanding of the etiology, pathology, and pathophysiology of human AKI.

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.

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.

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

More than 350 mutations in the type-2 BMP (bone morphogenetic protein) receptor, BMPR2, have been identified in patients with heritable pulmonary arterial hypertension (HPAH). However, only 30% of BMPR2 mutation carriers develop PAH, and we cannot predict which of these carriers will develop clinical disease. One possibility is that the nature of the BMPR2 mutation affects disease severity. This hypothesis has been difficult to test clinically, given the rarity of HPAH and the complexity of the confounding genetic and environmental risk factors. To test this hypothesis, therefore, we evaluated the susceptibility to experimental pulmonary hypertension (PH) of mice carrying different HPAH-associated Bmpr2 mutations on otherwise identical genetic backgrounds. Mice with Bmpr2ΔEx4–5 mutations (Bmpr2+/−), in which the mutant protein is not expressed, develop less severe PH in response to hypoxia or hypoxia with vascular endothelial growth factor receptor inhibition than mice with an extracellular-domain Bmpr2ΔEx2 mutation (Bmpr2ΔEx2/+), in which the mutant protein is expressed. This was associated with a marked decrease in stabilizing phosphorylation of threonine 495 endothelial nitric oxide synthase (pThr495 eNOS) in Bmpr2ΔEx2/+ compared to wild-type and Bmpr2+/− mouse lungs. These findings provide the first experimental evidence that BMPR2 mutation types influence the severity of HPAH and suggest that patients with BMPR2 mutations who express mutant BMPR2 proteins by escaping non-sense-mediated messenger RNA decay (NMD− mutations) will develop more severe disease than HPAH patients with NMD+ mutations who do not express BMPR2 mutant proteins. Since decreased levels of pThr495 eNOS are associated with increased eNOS uncoupling, our data also suggest that this effect may result from defects in eNOS function.

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH) and is a significant cause of morbidity and mortality in other forms of pulmonary hypertension. There are no approved therapies directed at preserving RV function. F-series and E-series isoprostanes are increased in heart failure and PAH, correlate to the severity of disease, and can signal through the thromboxane-prostanoid (TP) receptor, with effects from vasoconstriction to fibrosis. The goal of these studies was to determine whether blockade of the TP receptor with the antagonist CPI211 was beneficial therapeutically in PAH-induced RV dysfunction. Mice with RV dysfunction due to pressure overload by pulmonary artery banding (PAB) were given vehicle or CPI211. Two weeks after PAB, CPI211-treated mice were protected from fibrosis with pressure overload. Gene expression arrays and immunoblotting, quantitative histology and morphometry, and flow cytometric analysis were used to determine the mechanism of CPI211 protection. TP receptor inhibition caused a near normalization of fibrotic area, prevented cellular hypertrophy while allowing increased RV mass, increased expression of antifibrotic thrombospondin-4, and blocked induction of the profibrotic transforming growth factor β (TGF-β) pathway. A thromboxane synthase inhibitor or low-dose aspirin failed to replicate these results, which suggests that a ligand other than thromboxane mediates fibrosis through the TP receptor after pressure overload. This study suggests that TP receptor antagonism may improve RV adaptation in situations of pressure overload by decreasing fibrosis and TGF-β signaling.

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.

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 most patients presenting with septic shock and developing AKI had evidence of kidney damage at the time of, or within 24 hours 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 sepsis-associated AKI (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 (PTBA), a small molecule histone deacetylase inhibitor, that enhances renal recovery and reduces fibrosis in both zebrafish and mouse models of AKI.

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Despite extensive research, no therapeutic interventions have been shown to prevent AKI, accelerate recovery of AKI, or reduce progression of AKI to CKD in patients. This failure in translation has led investigators to speculate that the animal models being used do not predict therapeutic responses in humans. Although this issue continues to be debated, an important concern that has not been addressed is whether improvements in preclinical study design can be identified that might also increase the likelihood of translating basic AKI research into clinical practice using the current models. In this review, we have taken an evidence-based approach to identify common weaknesses in study design and reporting in preclinical AKI research that may contribute to the poor translatability of the findings. We focused on use of N-acetylcysteine or sodium bicarbonate for the prevention of contrast-induced AKI and use of erythropoietin for the prevention of AKI, two therapeutic approaches that have been extensively studied in clinical trials. On the basis of our findings, we identified five areas for improvement in preclinical study design and reporting. These suggested and preliminary guidelines may help improve the quality of preclinical research for AKI drug development.

The vertebrate kidney possesses the capacity to repair damaged nephrons, and this potential is conserved regardless of the complexity of species-specific kidneys. However, many aquatic vertebrates possess the ability to not only repair existing nephrons, but also generate new nephrons after injury. Adult zebrafish have the ability to recover from acute renal injury not only by replacing lost injured epithelial cells of endogenous nephrons, but by also generating de novo nephrons. This strong regeneration potential, along with other unique characteristics such as the high degree of genetic conservation with humans, the ease of harvesting externally fertilized, transparent embryos, the accessibility to larval and adult kidneys, and the ability to perform whole organism phenotypic small molecule screens, has positioned zebrafish as a unique vertebrate model to study kidney injury. In this review, we provide an overview of the contribution of zebrafish larvae/adult studies to the understanding of renal regeneration, diseases, and therapeutic discovery. Fil: Cirio, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: de Caestecker, Mark P.. Vanderbilt University; Estados Unidos Fil: Hukriede, Neil A.. University Of Pittsburgh; Estados Unidos

No therapies have been shown to improve outcomes in patients with acute kidney injury (AKI). Given the high morbidity and mortality associated with AKI this represents an important unmet medical need. A common feature of all of the therapeutic development efforts for AKI is that none were driven by target selection or preclinical modeling that was based primarily on human data. This is important when considering a heterogeneous and dynamic condition such as AKI, in which in the absence of more accurate molecular classifications, clinical cohorts are likely to include patients with different types of injury at different stages in the injury and repair continuum. The National Institutes of Health precision medicine initiative offers an opportunity to address this. By creating a molecular tissue atlas of AKI, defining patient subgroups, and identifying critical cells and pathways involved in human AKI, this initiative has the potential to transform our current approach to therapeutic discovery. In this review, we discuss the opportunities and challenges that this initiative presents, with a specific focus on AKI, what additional efforts will be needed to apply these discoveries to therapeutic development, and how we believe this effort might lead to the development of new therapeutics for subsets of patients with 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.

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/β-catenin and WT1 genes and performed immunostaining for markers of Wnt pathway activation (β-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 β-catenin in KWT (p = 0.013). All 15 KWT specimens were found to harbor wild-type CTNNB1/β-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.

The zebrafish has become a significant model system for studying renal organogenesis and disease, as well as for the quest for new therapeutics, because of the structural and functional simplicity of the embryonic kidney. Inroads to the nature and disease states of kidney-related ciliopathies and acute kidney injury (AKI) have been advanced by zebrafish studies. This model organism has been instrumental in the analysis of mutant gene function for human disease with respect to ciliopathies. Additionally, in the AKI field, recent work in the zebrafish has identified a bona fide adult zebrafish renal progenitor (stem) cell that is required for neo-nephrogenesis, both during the normal lifespan and in response to renal injury. Taken together, these studies solidify the zebrafish as a successful model system for studying the broad spectrum of ciliopathies and AKI that affect millions of humans worldwide, and point to a very promising future of zebrafish drug discovery. The emphasis of this review will be on the role of the zebrafish as a model for human kidney-related ciliopathies and AKI, and how our understanding of these complex pathologies is being furthered by this tiny teleost.

Late gestational placental insufficiency resulting in asymmetric intrauterine organ growth restriction (IUGR) is associated with an increased incidence of diabetes, cardiovascular and renal disease in adults. The molecular mechanisms mediating these defects are poorly understood. To explore this, we investigated the mechanisms leading to IUGR in Cited1 knockout mice, a genetic model of late gestational placental insufficiency. We show that loss of placental Cited1 leads to asymmetric IUGR with decreased liver, lung, and kidney sizes and preservation of fetal brain weight. IGF and insulin signaling regulate embryonic organ growth. IGF-I and IGF-II protein and mRNA expression are reduced in livers, lungs, and kidneys of embryonic d 18.5 embryos with IUGR. Decreased IGF-I is associated with reduced activating phosphorylation of the type 1 IGF receptor (pIGF-IR) in the kidney, whereas reduced IGF-II is associated with decreased phosphorylation of the insulin receptor (pIR) in the lung. In contrast, decreased pIR is associated with reduced IGF-I but not IGF-II in the liver. However, pancreatic β-cell mass and serum insulin levels are also decreased in mice with IUGR, suggesting that hepatic IR signaling may be regulated by alterations in fetal insulin production. These findings contrast with observations in IUGR fetal brains in which there is no change in IGF-IR/IR phosphorylation, and IGF-I and IGF-II expression is actually increased. In conclusion, IUGR disrupts normal fetal IGF and insulin production and is associated with organ-specific defects in IGF-IR and IR signaling that may regulate asymmetric IUGR in late gestational placental insufficiency.

Bone morphogenetic protein (BMP) signaling has been linked to the development of pulmonary hypertension (PH). Inhibitors of differentiation (ID) proteins (ID1–4) are a family of basic helix-loop-helix transcription factors that are downstream targets of the BMP signaling pathway, but the role that ID proteins play in the development of PH is unknown. To address this, we evaluated pulmonary expression of ID proteins in a mouse model of hypoxia-induced PH. There is selective induction of ID1 and ID3 expression in hypoxic pulmonary vascular smooth muscle cells (VSMCs) in vivo, and ID1 and ID3 expression are increased by hypoxia in cultured pulmonary VSMCs in a BMP-dependent fashion. ID4 protein is barely detectable in the mouse lung, and while ID2 is induced in hypoxic peripheral VSMCs in vivo, it is not increased by hypoxia or BMP signaling in cultured pulmonary VSMCs. In addition, the PH response to chronic hypoxia is indistinguishable between wild type and Id1 null mice. This is associated with a compensatory increase in ID3 but not ID2 expression in pulmonary VSMCs of Id1 null mice. These findings indicate that ID1 is dispensable for mounting a normal pulmonary vascular response to hypoxia, but suggest that ID3 may compensate for loss of ID1 expression in pulmonary VSMCs. Taken together, these findings indicate that ID1 and ID3 expression are regulated in a BMP-dependent fashion in hypoxic pulmonary VSMCs, and that ID1 and ID3 may play a cooperative role in regulating BMP-dependent VSMC responses to chronic hypoxia.

Genetic and functional studies indicate that common components of the Bone Morphogenetic Protein (BMP) signaling pathway play critical roles in regulating vascular development in the embryo, and in promoting vascular homeostasis and disease in the adult. However, discrepancies between in vitro and in vivo findings, and distinct functional properties of the BMP signaling pathway in different vascular beds have led to controversies in the field that have been difficult to reconcile. This review attempts to clarify some of these issues by providing an up to date overview of the biology and genetics of BMP signaling relevant to the intact vasculature.

The bone morphogenetic protein (BMP) type 2 receptor ligand, Bmp2, is upregulated in the peripheral pulmonary vasculature during hypoxia-induced pulmonary hypertension (PH). This contrasts with the expression of Bmp4, which is expressed in respiratory epithelia throughout the lung. Unlike heterozygous null Bmp4 mice ( Bmp4LacZ/+), which are protected from the development of hypoxic PH, mice that are heterozygous null for Bmp2 ( Bmp2+/−) develop more severe hypoxic PH than their wild-type littermates. This is associated with reduced endothelial nitric oxide synthase (eNOS) expression and activity in the pulmonary vasculature of hypoxic Bmp2+/−but not Bmp4LacZ/+mutant mice. Furthermore, exogenous BMP2 upregulates eNOS expression and activity in intrapulmonary artery and pulmonary endothelial cell preparations, indicating that eNOS is a target of Bmp2 signaling in the pulmonary vasculature. Together, these data demonstrate that Bmp2 and Bmp4 exert opposing roles in hypoxic PH and suggest that the protective effects of Bmp2 are mediated by increasing eNOS expression and activity in the hypoxic pulmonary vasculature.

A number of studies have shown that placental insufficiency affects embryonic patterning of the kidney and leads to a decreased number of functioning nephrons in adulthood; however, there is circumstantial evidence that placental insufficiency may also affect renal medullary growth, which could account for cases of unexplained renal medullary dysplasia and for abnormalities in renal function among infants who had experienced intrauterine growth retardation. We observed that mice with late gestational placental insufficiency associated with genetic loss of Cited1 expression in the placenta had renal medullary dysplasia. This was not caused by lower urinary tract obstruction or by defects in branching of the ureteric bud during early nephrogenesis but was associated with decreased tissue oxygenation and increased apoptosis in the expanding renal medulla. Loss of placental Cited1 was required for Cited1 mutants to develop renal dysplasia, and this was not dependent on alterations in embryonic Cited1 expression. Taken together, these findings suggest that renal medullary dysplasia in Cited1 mutant mice is a direct consequence of decreased tissue oxygenation resulting from placental insufficiency.

Patients with familial pulmonary arterial hypertension inherit heterozygous mutations of the type 2 bone morphogenetic protein (BMP) receptor BMPR2. To explore the cellular mechanisms of this disease, we evaluated the pulmonary vascular responses to chronic hypoxia in mice carrying heterozygous hypomorphic Bmpr2 mutations ( Bmpr2ΔEx2/+). These mice develop more severe pulmonary hypertension after prolonged exposure to hypoxia without an associated increase in pulmonary vascular remodeling or proliferation compared with wild-type mice. This is associated with defective endothelial-dependent vasodilatation and enhanced vasoconstriction in isolated intrapulmonary artery preparations. In addition, there is a selective decrease in hypoxia-induced, BMP-dependent, endothelial nitric oxide synthase expression and Smad signaling in the intact lungs and in cultured pulmonary microvascular endothelial cells from Bmpr2ΔEx2/+ mutant mice. These findings indicate that the pulmonary endothelium is a target of abnormal BMP signaling in Bmpr2ΔEx2/+ mutant mice and suggest that endothelial dysfunction contributes to their increased susceptibility to hypoxic pulmonary hypertension.

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.

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.

Acute kidney injury (AKI) is a serious complication, commonly occurring in the critically ill population, with devastating short- and long-term consequences. Despite standardization of the definition and staging of AKI, early recognition remains challenging given that serum creatinine level is a marker, albeit imperfect, of kidney function and not kidney injury. Furthermore, the delay in increase in serum creatinine level after loss of glomerular filtration also prevents timely detection of decreased kidney function in patients with AKI. During the past decade, numerous clinical investigations have evaluated the utility of several biomarkers in the early diagnosis and risk stratification of AKI. In 2014, the US Food and Drug Administration approved the marketing of a test based on the combination of urine concentrations of tissue inhibitor of metalloproteinase 2 and insulin-like growth factor binding protein 7 ([TIMP-2] × [IGFBP7]) to determine whether certain critically ill patients are at risk for developing moderate to severe AKI. The optimal role of this biomarker in the diagnosis, management, and prognosis of AKI in different clinical settings requires further clarification. In this perspective, we summarize the biological actions of these 2 cell-cycle arrest biomarkers and present important considerations regarding the clinical application, interpretation, and limitations of this novel test for the early detection of AKI.

We show that 1 of the type II bone morphogenetic protein (BMP) receptor ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated in hypoxia-induced pulmonary hypertension (PH). Furthermore, heterozygous null Bmp4 lacZ/+ mice are protected from the development of hypoxia-induced PH, vascular smooth muscle cell proliferation, and vascular remodeling. This is associated with a reduction in hypoxia-induced Smad1/5/8 phosphorylation and Id1 expression in the pulmonary vasculature. In addition, pulmonary microvascular endothelial cells secrete BMP4 in response to hypoxia and promote proliferation and migration of vascular smooth muscle cells in a BMP4-dependent fashion. These findings indicate that BMP4 plays a dominant role in regulating BMP signaling in the hypoxic pulmonary vasculature and suggest that endothelium-derived BMP4 plays a direct, paracrine role in promoting smooth muscle proliferation and remodeling in hypoxic PH.

See related article, pages 1323–1330 Serotonin (5-HT, 5-hydroxytryptamine) has long been recognized as one of the most potent naturally occurring pulmonary vasoconstrictors.1 It was first implicated in the pathogenesis of pulmonary arterial hypertension (PAH) after an outbreak of the disease in Switzerland in the 1960’s among patients taking aminorex fumarate, an appetite suppressant that inhibits serotonin uptake by platelets.2 Since that time further outbreaks of PAH have been identified in Europe and the USA associated with the use of fenfluramine-derivate anorexigens,3–5 eventually leading to their withdrawal from the world market in 1997. Although this was, at least in retrospect, a predictable tragedy, it has ironically opened avenues of research into the biology of serotonin signaling in PAH. As fenfluramine-derivatives are substrates for the serotonin transporter (5-HTT, SERT) proteins,6 this suggests that abnormal SERT expression or functional activity could play a role in the pathogenesis of PAH. There is now a body of evidence supporting this hypothesis that provides hope for the development of effective therapeutic strategies targeting specific components of this signaling pathway in patients with these diseases. Most of the serotonin produced in the body is secreted by enterochromaffin cells of the intestine into the portal circulation where it is partially metabolized by the liver. However, levels of free circulating serotonin are maintained in the low nanomolar range through energy-dependent SERT-mediated transport into platelets. This led some researchers to hypothesize that fenfluramines might cause PAH by increasing free plasma levels of serotonin. However, this hypothesis is inconsistent with the observation that chronic treatment with fenfluramine-derivatives if anything reduces plasma levels of serotonin.6 This suggests that other SERT-related effects promote PAH in susceptible patients. This is supported by the observation that patients with idiopathic PAH have increased frequency of the so called L-type polymorphism …

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. Fil: Cirio, Maria Cecilia. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: de Groh, Eric D.. University of Pittsburgh; Estados Unidos Fil: de Caestecker, Mark P.. Vanderbilt University; Estados Unidos Fil: Davidson, Alan J.. The University of Auckland; Nueva Zelanda Fil: Hukriede, Neil A.. University of Pittsburgh; Estados Unidos

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

Purpose Pax3cre-mediated deletion of fibroblast growth factor receptor 2 (Fgfr2) broadly in renal and urinary tract mesenchyme led to ureteric bud (UB) induction defects and vesicoureteral reflux (VUR), although the mechanisms were unclear. Here, we investigated whether Fgfr2 acts specifically in peri-Wolffian duct stroma (ST) to regulate UB induction and development of VUR and the mechanisms of Fgfr2 activity. Methods We conditionally deleted Fgfr2 in ST (Fgfr2ST−/−) using Tbx18cre mice. To look for ureteric bud induction defects in young embryos, we assessed length and apoptosis of common nephric ducts (CNDs). We performed 3D reconstructions and histological analyses of urinary tracts of embryos and postnatal mice and cystograms in postnatal mice to test for VUR. We performed in situ hybridization and real-time PCR in young embryos to determine mechanisms underlying UB induction defects. Results We confirmed that Fgfr2 is expressed in ST and that Fgfr2 was efficiently deleted in this tissue in Fgfr2ST−/− mice at embryonic day (E) 10.5. E11.5 Fgfr2ST−/− mice had randomized UB induction sites with approximately 1/3 arising too high and 1/3 too low from the Wolffian duct; however, apoptosis was unaltered in E12.5 mutant CNDs. While ureters were histologically normal, E15.5 Fgfr2ST−/− mice exhibit improper ureteral insertion sites into the bladder, consistent with the ureteric induction defects. While ureter and bladder histology appeared normal, postnatal day (P) 1 mutants had high rates of VUR versus controls (75% versus 3%, p = 0.001) and occasionally other defects including renal hypoplasia and duplex systems. P1 mutant mice also had improper ureteral bladder insertion sites and shortened intravesicular tunnel lengths that correlated with VUR. E10.5 Fgfr2ST−/− mice had decreases in Bmp4 mRNA in stromal tissues, suggesting a mechanism underlying the ureteric induction and VUR phenotypes. Conclusion Mutations in FGFR2 could possibly cause VUR in humans.

Recovery from acute kidney injury requires regeneration of tubule cells. Because calcineurin induces nuclear transport of NFATc proteins, whose expression pattern correlates with the nephron segments injured by calcineurin inhibitors, we hypothesized that NFATc1 plays a role in modifying epithelial regeneration after injury. To test this, we induced proximal tubular cell (PTC) injury in Balb/c mice and Nfatc1(+/-) mice with mercuric chloride; the PTCs of Nfatc1(+/-) mice demonstrated increased apoptosis, sustained injury, and delayed regeneration. To attenuate NFATc1 activity further, we injected cyclosporin A daily. Cyclosporin A-treated Nfatc1(+/-) mice demonstrated rapid and severe injury after administration of mercuric chloride, with increased serum creatinine, increased apoptosis, decreased PTC proliferation, and increased mortality compared with similarly treated wild-type mice. Using a novel NFATc1 transgenic line that reports activation of an NFATc1 enhancer domain critical for NFATc1 autoamplification, we demonstrated accentuated NFATc1 expression in a PTC subpopulation after mercuric chloride-induced injury. In addition, NFATc1-labeled, apoptosis-resistant PTCs proliferated to repair the damaged proximal tubule segment. These data provide evidence for a resident progenitor PTC population and suggest a role for NFATc1 in the regeneration of injured proximal tubules.

Early kidney development in mammals is characterized by reciprocal tissue interaction between the ureteric bud and the metanephric mesenchyme. The coordinated response to this interaction is regulated largely at the transcriptional level. Here, we investigate the expression and function of Cited1, a transcriptional cofactor that we have previously implicated in kidney development. We show that Cited1 is expressed in the metanephric mesenchyme after invasion of the ureteric bud and that its expression is limited to the cap mesenchyme, those cells that aggregate most tightly around the tip of the ureteric bud and give rise to nephronic epithelium of the adult kidney. Cited1 is down-regulated during the initial stages of epithelial conversion and is not expressed past this progenitor stage. Despite its unique expression pattern, deletion of Cited1 does not disrupt kidney development. We hypothesized that this finding was due to functional redundancy with other members of this gene family. The expression pattern of Cited2 overlaps that of Cited1, but its deletion, either alone or in combination with Cited1, does not disrupt epithelial differentiation of the metanephric mesenchyme. From these studies, we conclude that Cited1 and 2 are dynamically expressed during kidney development, but are not required for nephrogenesis. Developmental Dynamics 236:2321–2330, 2007. © 2007 Wiley-Liss, Inc.

Longstanding diabetes causes renal injury with early dropout of podocytes, albuminuria, glomerular and tubulointerstitial fibrosis, and progressive renal failure. The renal pathology seems to be driven, in part, by TGF-beta and is associated with a loss of renal bone morphogenic protein-7 (BMP-7) expression. Here, the hypothesis that maintenance of renal (especially podocyte) BMP-7 by transgenic expression reduces diabetic renal injury was tested. Diabetic mice that expressed the phosphoenolpyruvate carboxykinase promoter-driven BMP-7 transgene and nondiabetic, transgenic mice as well as diabetic and nondiabetic wild-type controls were studied for up to 1 yr. Transgenic expression of BMP-7 in glomerular podocytes and proximal tubules prevents podocyte dropout and reductions in nephrin levels in diabetic mice. Maintenance of BMP-7 also reduces glomerular fibrosis and interstitial collagen accumulation as well as collagen I and fibronectin expression. Diabetic wild-type mice develop progressive albuminuria, which is substantially reduced in transgenic mice. These effects of the BMP-7 transgene occur without changing renal TGF-beta levels. It is concluded that maintenance of renal BMP-7 during the evolution of diabetic nephropathy reduces diabetic renal injury, especially podocyte dropout. The findings also establish a role for endogenous glomerular BMP-7 as an autocrine regulator of podocyte integrity in vivo.

In a screen to identify factors that regulate the conversion of mesenchyme to epithelium during the early stages of nephrogenesis, it was found that the Smad4-interacting transcriptional cofactor, Cited1, is expressed in the condensed cap mesenchyme surrounding the tip of the ureteric bud (UB), is downregulated after differentiation into epithelia, and has the capacity to block UB branching and epithelial morphogenesis in cultured metanephroi. Cited1 represses Wnt/beta-catenin but activates Smad4-dependent transcription involved in TGF-beta and Bmp signaling. By modifying these pathways, Cited1 may coordinate cellular differentiation and survival signals that regulate nephronic patterning in the metanephros.

The close spatial apposition between podocytes, fenestrated endothelia, and mesangial cells within the glomerulus has led to speculation that paracrine growth factors secreted by these cells are required to maintain structural and functional integrity of glomerular permselectivity in adults. This

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.