In vitro assessment of the Kirpa Kit™ modified manual single lumen alternating micro-batch (mSLAMB) dialysis device.

BACKGROUND: Access to pediatric dialysis is challenged in low-resource settings due to high costs, scarcity of equipment, and the lack of qualified personnel availability. We demonstrated the manual single lumen alternating micro-batch (mSLAMB) device can remove small solutes in vitro without the need for electricity, batteries, or pumps. We developed a new version (Kirpa Kit™) to address some of the technical limitations of mSLAMB. Here, we compare the in vitro clearance performance and ease of use of the Kirpa Kit™ with that of prior mSLAMB configurations. METHODS: A mixture of expired packed red blood cells, 0.9% NaCl, urea, and heparin was used to test the efficiency of two mSLAMB configurations and the Kirpa Kit™ in removing potassium and urea. Clearance was evaluated by measuring percent reduction after 25-min sessions with each device. A survey was used to evaluate the ease of use of each configuration. RESULTS: The Kirpa Kit™ achieved a median urea reduction of 82.4% and potassium reduction of 82.1%, which were higher than those achieved with the best-performing mSLAMB configuration (urea 71.9%, potassium 75.4%). The Kirpa Kit™ was easier to use with a shorter perceived time of use than the mSLAMB. CONCLUSIONS: The Kirpa Kit™, evolution of mSLAMB, is easy to use and may have improved efficacy, making it an optimal candidate for in vivo testing.

Manual single lumen alternating micro-batch dialysis achieves reliable clearance via diffusion.

BACKGROUND: Acute kidney injury is a cause of preventable deaths in low resource settings due to lack of dialysis access and cost. A manual single lumen alternating micro-batch (mSLAMB) dialysis technique performs kidney replacement therapy using single lumen access, low-cost bags/tubing, intravenous fluids, and a filter without electricity, a battery, or a pump. We propose a protocol whereby mSLAMB can perform diffusive clearance simply and efficiently to bring dialysis to underserved populations. METHODS: Expired packed red blood cells mixed with crystalloid solution were spiked with urea and anticoagulated with heparin. A Static diffusion Technique (with short flushes of fluid before each filter pass) was compared to a Dynamic diffusion Technique (with fluid running through the filter during the forward pass) to assess urea and potassium clearance. Passive ultrafiltration was the difference between the 200 mL batch volume and volume returned to the blood bag per cycle. RESULTS: Five cycles achieved urea reduction ratios (URR) between 17-67% and potassium clearance of 18-60%, with higher percentages achieved from higher proportions of batch volume dialyzed to patient volume. Dynamic Technique increased clearance over the Static Technique. Passive ultrafiltration volumes were 2.5-10% of batch volume. CONCLUSION: mSLAMB dialysis performs diffusive clearance and passive ultrafiltration efficiently, while preserving resources and available manpower. IMPACT: mSLAMB is a dialysis technique that can perform efficient diffusive clearance and passive ultrafiltration without electricity, batteries, or a pump. With basic medical supplies and limited manpower, mSLAMB is a cost-effective means of providing emergency dialysis in low resource areas. We propose a basic algorithm for safe and cost-effective dialysis for people of different ages and sizes.

An IRF4-MYC-mTORC1 Integrated Pathway Controls Cell Growth and the Proliferative Capacity of Activated B Cells during B Cell Differentiation In Vivo.

Cell division is an essential component of B cell differentiation to Ab-secreting plasma cells, with critical reprogramming occurring during the initial stages of B cell activation. However, a complete understanding of the factors that coordinate early reprogramming events in vivo remain to be determined. In this study, we examined the initial reprogramming by IRF4 in activated B cells using an adoptive transfer system and mice with a B cell-specific deletion of IRF4. IRF4-deficient B cells responding to influenza, 4-hydroxy-3-nitrophenylacetyl-Ficoll, and LPS divided but stalled during the proliferative response. Gene expression profiling of IRF4-deficient B cells at discrete divisions revealed IRF4 was critical for inducing MYC target genes, oxidative phosphorylation, and glycolysis. Moreover, IRF4-deficient B cells maintained an inflammatory gene expression signature. Complementary chromatin accessibility analyses established a hierarchy of IRF4 activity and identified networks of dysregulated transcription factor families in IRF4-deficient B cells, including E-box binding bHLH family members. Indeed, B cells lacking IRF4 failed to fully induce Myc after stimulation and displayed aberrant cell cycle distribution. Furthermore, IRF4-deficient B cells showed reduced mTORC1 activity and failed to initiate the B cell activation unfolded protein response and grow in cell size. Myc overexpression in IRF4-deficient cells was sufficient to overcome the cell growth defect. Together, these data reveal an IRF4-MYC-mTORC1 relationship critical for controlling cell growth and the proliferative response during B cell differentiation.

Increasing angiotensin-converting enzyme concentrations and absent angiotensin-converting enzyme activity are associated with adverse kidney outcomes in pediatric septic shock.

BACKGROUND: Sepsis-induced endothelial dysfunction is proposed to cause angiotensin-converting enzyme (ACE) dysfunction and renin-angiotensin-aldosterone system (RAAS) derangement, exacerbating vasodilatory shock and acute kidney injury (AKI). Few studies test this hypothesis directly, including none in children. We measured serum ACE concentrations and activity, and assessed their association with adverse kidney outcomes in pediatric septic shock. METHODS: A pilot study of 72 subjects aged 1 week-18 years from an existing multicenter, observational study. Serum ACE concentrations and activity were measured on Day 1; renin + prorenin concentrations were available from a previous study. The associations between individual RAAS components and a composite outcome (Day 1-7 severe persistent AKI, kidney replacement therapy use, or mortality) were assessed. RESULTS: 50/72 subjects (69%) had undetectable ACE activity (< 2.41 U/L) on Day 1 and 27/72 (38%) developed the composite outcome. Subjects with undetectable ACE activity had higher Day 1 renin + prorenin compared to those with activity (4533 vs. 2227 pg/ml, p = 0.017); ACE concentrations were no different between groups. Children with the composite outcome more commonly had undetectable ACE activity (85% vs. 65%, p = 0.025), and had higher Day 1 renin + prorenin (16,774 pg/ml vs. 3037 pg/ml, p < 0.001) and ACE concentrations (149 vs. 96 pg/ml, p = 0.019). On multivariable regression, increasing ACE concentrations (aOR 1.01, 95%CI 1.002-1.03, p = 0.015) and undetectable ACE activity (aOR 6.6, 95%CI 1.2-36.1, p = 0.031) retained associations with the composite outcome. CONCLUSIONS: ACE activity is diminished in pediatric septic shock, appears uncoupled from ACE concentrations, and is associated with adverse kidney outcomes. Further study is needed to validate these findings in larger cohorts.

Acute kidney injury biomarker olfactomedin 4 predicts furosemide responsiveness.

BACKGROUND: Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. Olfactomedin 4 (OLFM4), a secreted glycoprotein expressed in neutrophils and stressed epithelial cells, is upregulated in loop of Henle (LOH) cells following AKI. We hypothesized that urine OLFM4 (uOLFM4) will increase in patients with AKI and may predict furosemide responsiveness. METHODS: Urine from critically ill children was collected prospectively and tested for uOLFM4 concentrations with a Luminex immunoassay. Severe AKI was defined by KDIGO (stage 2/3) serum creatinine criteria. Furosemide responsiveness was defined as > 3 mL/kg/h of urine output in the 4 h after a 1 mg/kg IV furosemide dose administered as part of standard of care. RESULTS: Fifty-seven patients contributed 178 urine samples. Irrespective of sepsis status or AKI cause, uOLFM4 concentrations were higher in patients with AKI (221 ng/mL [IQR 93-425] vs. 36 ng/mL [IQR 15-115], p = 0.007). uOLFM4 concentrations were higher in patients unresponsive to furosemide (230 ng/mL [IQR 102-534] vs. 42 ng/mL [IQR 21-161], p = 0.04). Area under the receiver operating curve for association with furosemide responsiveness was 0.75 (95% CI, 0.60-0.90). CONCLUSIONS: AKI is associated with increased uOLFM4. Higher uOLFM4 is associated with a lack of response to furosemide. Further testing is warranted to determine whether uOLFM4 could identify patients most likely to benefit from earlier escalation from diuretics to kidney replacement therapy to maintain fluid balance. A higher resolution version of the Graphical abstract is available as Supplementary information.

Manual Single-Lumen Alternating Micro-Batch Device as Renal Replacement Therapy in Austere Environments.

INTRODUCTION: Electrolyte derangements, acidosis, and volume overload remain life-threatening emergencies in people with acute kidney injury in austere environments. A single-lumen alternating micro-batch (SLAMB) dialysis technique was designed to perform renal replacement therapy using a single-lumen access, low-cost disposable bags and tubing, widely available premade fluids, and a dialysis filter. A manual variation (mSLAMB) works without electricity, battery, or a pump. We modeled mSLAMB dialysis and predicted it could achieve adequate small solute clearance, blood flow rates, and ultrafiltration accuracy. METHODS: A 25- to 30-kg pediatric patient's blood volume was simulated by a 2-L bag of expired blood and spiked with 5 g of urea initially, then with 1-2 g between experiments. Experiments had 8 cycles totaling prescription volumes of 800-2,400 mL and were conducted with different ratios of hemofiltration fluid to blood volume. Concentrations of urea and potassium, final effluent volumes, and cycle duration were measured at the end of each cycle to determine clearance, ultrafiltration accuracy, and blood flow rates. RESULTS: Each cycle lasted 70-145 s. Experiments achieved a mean urea reduction ratio of 27.4 ± 7.1% and a mean potassium reduction of 23.4 ± 9.3%. The largest urea and potassium reduction percentage occurred with the first cycle. Increased hemofiltration fluid to blood volume ratio did not increase clearance. Mean (+/- standard deviation) blood flow ranged from 79.7 +/- 4.4 mL/min to 90.8 +/- 6.5 mL/min and increased with larger batch volume and height difference between reservoirs. Ultrafiltration accuracy ranged from 0 to 2.4% per cycle. DISCUSSION: mSLAMB dialysis is a simple, manual, cost-effective mode of dialysis capable of providing clearance and accurate ultrafiltration. With further refinement of technique, we believe this can be a potentially lifesaving treatment in austere conditions and low-resource settings.

Cell-Free DNA, Neutrophil extracellular traps (NETs), and Endothelial Injury in Coronavirus Disease 2019- (COVID-19-) Associated Acute Kidney Injury.

Introduction: Neutrophil extracellular traps (NETs) release (i.e., NETosis) has been recently implicated in the pathomechanism underlying severe end-organ damage in Coronavirus Disease 2019 (COVID-19) and could present a novel therapeutic target. We aimed to determine whether circulating levels of cell-free DNA (cfDNA), a surrogate for NETosis, may be associated with the development of acute kidney injury (AKI), a major contributor to poor outcomes and mortality in COVID-19. Methods: Blood samples were collected prospectively from adult patients infected with SARS-CoV-2 presenting to the emergency department (ED). Circulating levels of cfDNA were quantified from patients' serum. Further assessment of correlations between cfDNA levels and markers of AKI (i.e., serum creatinine (SCr), cystatin C, neutrophil gelatinase-associated lipocalin (NGAL)), biomarkers of thrombotic microangiopathy and of inflammation in patients' serum was performed. Results: Fifty-one COVID-19 patients were enrolled. cfDNA levels were found to be significantly higher in those who developed severe AKI (p < 0.001) and those needing renal replacement therapy (p = 0.020). cfDNA positively correlated with ED SCr, NGAL, cystatin C, neutrophil count, neutrophil-to-lymphocyte ratio, C3a, C5a, Scb5-9, IL-6, IL-8, IL-10, TNF-α, LDH, CRP, ferritin, and fibrinogen and negatively correlated with ADAMTS13/von-Willebrand factor ratio and lymphocyte count. In a multivariate logistic regression, a one-unit increase in cfDNA value was associated with 4.6% increased odds of severe AKI (OR = 1.046; p = 0.040). Finally, cfDNA significantly correlated with established NETs components, myeloperoxidase, and neutrophil elastase. Conclusion: Intravascular NETosis could be an important contributing factor in the development of microthrombosis and COVID-19-associated AKI. Further research is urgently needed to understand the role of NETosis in COVID-19 and evaluate therapeutic avenues for targeting this process.

The anti-inflammatory cytokine response characterized by elevated interleukin-10 is a stronger predictor of severe disease and poor outcomes than the pro-inflammatory cytokine response in coronavirus disease 2019 (COVID-19).

OBJECTIVES: Severe coronavirus disease 2019 (COVID-19) is associated with a dysregulated immune state. While research has focused on the hyperinflammation, little research has been performed on the compensatory anti-inflammatory response. The aim of this study was to evaluate the anti-inflammatory cytokine response to COVID-19, by assessing interleukin-10 (IL-10) and IL-10/lymphocyte count ratio and their association with outcomes. METHODS: Adult patients presenting to the emergency department (ED) with laboratory-confirmed COVID-19 were recruited. The primary endpoint was maximum COVID-19 severity within 30 days of index ED visit. RESULTS: A total of 52 COVID-19 patients were enrolled. IL-10 and IL-10/lymphocyte count were significantly higher in patients with severe disease (p<0.05), as well as in those who developed severe acute kidney injury (AKI) and new positive bacterial cultures (all p≤0.01). In multivariable analysis, a one-unit increase in IL-10 and IL-10/lymphocyte count were associated with 42% (p=0.031) and 32% (p=0.013) increased odds, respectively, of severe COVID-19. When standardized to a one-unit standard deviations scale, an increase in the IL-10 was a stronger predictor of maximum 30-day severity and severe AKI than increases in IL-6 or IL-8. CONCLUSIONS: The hyperinflammatory response to COVID-19 is accompanied by a simultaneous anti-inflammatory response, which is associated with poor outcomes and may increase the risk of new positive bacterial cultures. IL-10 and IL-10/lymphocyte count at ED presentation were independent predictors of COVID-19 severity. Moreover, elevated IL-10 was more strongly associated with outcomes than pro-inflammatory IL-6 or IL-8. The anti-inflammatory response in COVID-19 requires further investigation to enable more precise immunomodulatory therapy against SARS-CoV-2.

Serum ACE activity and plasma ACE concentration in patients with SARS-CoV-2 infection.

Significant controversy has arisen over the role of the renin-angiotensin-aldosterone system (RAAS) in COVID-19 pathophysiology. In this prospective, observational study, we evaluated plasma angiotensin converting enzyme (ACE) concentration and serum ACE activity in 52 adults with laboratory-confirmed SARS-CoV-2 infection and 27 non-COVID-19 sick controls. No significant differences were observed in ACE activity in COVID-19 patients versus non-COVID-19 sick controls (41.1 [interquartile range (IQR): 23.0-55.2] vs. 42.9 [IQR 13.6-74.2] U/L, p = .649, respectively). Similarly, no differences were observed in ACE concentration in COVID-19 patients versus non-COVID-19 sick controls (108.4 [IQR: 95.8-142.2] vs. 133.8 [IQR: 100.2-173.7] µg/L, p = .059, respectively). Neither ACE activity (p = .751), nor ACE concentration (p = .283) was associated with COVID-19 severity. Moreover, neither ACE activity, nor ACE concentration was correlated with any inflammatory biomarkers.

Antenatal betamethasone attenuates the angiotensin-(1-7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells.

We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na+) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to angiotensin-(1-7) [Ang-(1-7)]. The present study determined the Na+ uptake and nitric oxide (NO) response to low-dose Ang-(1-7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as percentage of basal uptake or area under the curve for Na+ or percentage of control for NO. Male Beta RPTC exhibited greater Na+ uptake than male vehicle cells (433 ± 28 vs. 330 ± 26%; P < 0.05); however, Beta exposure had no effect on Na+ uptake in the female cells (255 ± 16 vs. 255 ± 14%; P > 0.05). Ang-(1-7) significantly inhibited Na+ uptake in RPTC from vehicle male (214 ± 11%) and from both vehicle (190 ± 14%) and Beta (209 ± 11%) females but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1-7) in male but not female RPTC. Both the Na+ and NO responses to Ang-(1-7) were blocked by Mas receptor antagonist d-Ala7-Ang-(1-7). We conclude that the tubular Ang-(1-7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.

Angiotensinogen import in isolated proximal tubules: evidence for mitochondrial trafficking and uptake.

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled 125I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; n = 4). Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min-1·mg protein-1; n = 3). Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; P < 0.05, n = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.

Antenatal steroid exposure and heart rate variability in adolescents born with very low birth weight.

BACKGROUND: Reduced heart rate variability (HRV) suggests autonomic imbalance in the control of heart rate and is associated with unfavorable cardiometabolic outcomes. We examined whether antenatal corticosteroid (ANCS) exposure had long-term programming effects on HRV in adolescents born with very low birth weight (VLBW). METHODS: Follow-up study of a cohort of VLBW 14-y olds born between 1992 and 1996 with 50% exposed to ANCS. HRV in both the time and frequency domains using Nevrokard Software was determined from a 5-min electrocardiogram tracing. RESULTS: HRV data from 89 (35 male, 53 non-black) exposed (ANCS+) and 77 (28 male, 29 non-black) unexposed (ANCS-) adolescents were analyzed. HRV did not differ between ANCS+ and ANCS- black participants. However, in non-black participants, a significant interaction between ANCS and sex was observed, with ANCS- females having significantly greater HRV than ANCS+ females and males, and ANCS- males for both time and frequency domain variables. CONCLUSION: Among non-black adolescents born with VLBW, ANCS exposure is associated with reduced HRV with apparent sex-specificity. Reduced HRV has been associated with development of adverse cardiometabolic outcomes, thus supporting the need to monitor these outcomes in VLBW adolescents as they mature.

Antenatal corticosteroids and the renin-angiotensin-aldosterone system in adolescents born preterm.

BACKGROUND: Antenatal corticosteroid (ANCS) treatment hastens fetal lung maturity and improves survival of premature infants, but the long-term effects of ANCS are not well-described. Animal models suggest that ANCS increases the risk of cardiovascular disease through programmed changes in the renin-angiotensin (Ang)-aldosterone system (RAAS). We hypothesized that ANCS exposure alters the RAAS in adolescents born prematurely. METHODS: A cohort of 173 adolescents born prematurely was evaluated, of whom 92 were exposed to ANCS. We measured plasma and urine Ang II and Ang-(1-7) and calculated Ang II/Ang-(1-7) ratios. We used general linear regression models to estimate the difference in the RAAS between the ANCS-exposed and unexposed groups, adjusting for confounding variables. RESULTS: In unadjusted analyses, and after adjustment for sex, race, and maternal hypertension, ANCS exposure was associated with increased urinary Ang II/Ang-(1-7) (estimate 0.27 (95% CI 0.03, 0.5), P = 0.03), increased plasma Ang-(1-7) (0.66 (0.26, 1.07), P = 0.002), and decreased plasma Ang II/Ang-(1-7) (-0.48 (-0.91, -0.06), P = 0.03). CONCLUSION: These alterations indicate an imbalance in the urinary RAAS, promoting the actions of Ang II at the expense of Ang-(1-7), which over time may increase the risk of renal inflammation and fibrosis and ultimately hypertension and renal disease.

Evidence for a mitochondrial angiotensin-(1-7) system in the kidney.

Evidence for an intracellular renin-angiotensin system (RAS) in various cell organelles now includes the endoplasmic reticulum, nucleus, and mitochondria (Mito). Indeed, angiotensin (ANG) AT1 and AT2 receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively, in previous studies. We undertook a biochemical analysis of the Mito RAS from male and female sheep kidney cortex. Mito were isolated by differential centrifugation followed by a discontinuous Percoll gradient and were coenriched in Mito membrane markers VDAC and ATP synthase, but not ß-actin or cathepsin B. Two distinct renin antibodies identified a 37-kDa protein band in Mito; angiotensinogen (Aogen) conversion was abolished by the inhibitor aliskiren. Mito Aogen was detected by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the ANG I N terminus. ANG peptides were quantified by three direct RIAs; mitochondrial ANG II and ANG-(1-7) contents were higher compared with ANG I (23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; P < 0.01, n = 3). 125I-ANG I metabolism primarily revealed the formation of 125I-ANG-(1-7) in Mito that reflects the endopeptidases neprilysin and thimet oligopeptidase. Last, immunoblot studies utilizing the ANG-(1-7)/Mas receptor antibody revealed the protein in isolated Mito from sheep renal cortex. Collectively, the current data demonstrate that Mito actively metabolize the RAS precursor protein Aogen, suggesting that ANG-(1-7) may be generated within Mito to establish an intramitochondrial RAS tone and contribute to renal mitochondrial function.

Budget impact of incorporating one instillation of hexaminolevulinate hydrochloride blue-light cytoscopy in transurethral bladder tumour resection for patients with non-muscle-invasive bladder cancer in Sweden.

OBJECTIVES: To explore the cost impact on Swedish healthcare of incorporating one instillation of hexaminolevulinate hydrochloride (HAL) blue-light cystoscopy into transurethral resection of bladder tumour (TURBT) in patients with suspected new or recurrent non-muscle-invasive bladder cancer (NMIBC). MATERIALS AND METHODS: A decision tree model was built based on European Association of Urology guidelines for the treatment and management of NMIBC. Input data were compiled from two recent studies comparing recurrence rates of bladder cancer in patients undergoing TURBT with either the current standard of care (SOC) of white-light cystoscopy, or with the SOC and HAL blue-light cystoscopy. Using these published data with clinical cost data for surgical and outpatient procedures and pharmaceutical costs, the model reported on the clinical and economic differences associated with the two treatment options. RESULTS: This model demonstrates the significant clinical benefits likely to be observed through the incorporation of HAL blue-light cystoscopy for TURBT in terms of reductions in recurrences of bladder cancer. Analysis of economic outputs of the model found that the use of one instillation of HAL for TURBT in all Swedish patients with NMIBC is likely to be cost-neutral or cost-saving over 5 years relative to the current SOC of white-light cystoscopy. CONCLUSIONS: The results of this analysis provide additional health economic rationale for the incorporation of a single instillation of HAL blue-light cystoscopy for TURBT in the treatment of patients with NMIBC in Sweden.

Antenatal glucocorticoid treatment alters Na+ uptake in renal proximal tubule cells from adult offspring in a sex-specific manner.

We have shown a sex-specific effect of fetal programming on Na(+) excretion in adult sheep. The site of this effect in the kidney is unknown. Therefore, we tested the hypothesis that renal proximal tubule cells (RPTCs) from adult male sheep exposed to betamethasone (Beta) before birth have greater Na(+) uptake than do RPTCs from vehicle-exposed male sheep and that RPTCs from female sheep similarly exposed are not influenced by antenatal Beta. In isolated RPTCs from 1- to 1.5-yr-old male and female sheep, we measured Na(+) uptake under basal conditions and after stimulation with ANG II. To gain insight into the mechanisms involved, we also measured nitric oxide (NO) levels, ANG II receptor mRNA levels, and expression of Na(+)/H(+) exchanger 3. Basal Na(+) uptake increased more in cells from Beta-exposed male sheep than in cells from vehicle-exposed male sheep (400% vs. 300%, P < 0.00001). ANG II-stimulated Na(+) uptake was also greater in cells from Beta-exposed males. Beta exposure did not increase Na(+) uptake by RPTCs from female sheep. NO production was suppressed more by ANG II in RPTCs from Beta-exposed males than in RPTCs from either vehicle-exposed male or female sheep. Our data suggest that one site of the sex-specific effect of Beta-induced fetal programming in the kidney is the RPTC and that the enhanced Na(+) uptake induced by antenatal Beta in male RPTCs may be related to the suppression of NO in these cells.

An angiotensin-(1-7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme.

Angiotensin 1-7 [ANG-(1-7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min(-1)·mg(-1)) compared with the tubules (96 ± 12 fmol·min(-1)·mg(-1)) and cortex (107 ± 9 fmol·min(-1)·mg(-1)). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone.

Corticosterone suppresses vasotocin-enhanced clasping behavior in male rough-skinned newts by novel mechanisms interfering with V1a receptor availability and receptor-mediated endocytosis.

In rough-skinned newts, Taricha granulosa, exposure to an acute stressor results in the rapid release of corticosterone (CORT), which suppresses the ability of vasotocin (VT) to enhance clasping behavior. CORT also suppresses VT-induced spontaneous activity and sensory responsiveness of clasp-controlling neurons in the rostromedial reticular formation (Rf). The cellular mechanisms underlying this interaction remain unclear. We hypothesized that CORT blocks VT-enhanced clasping by interfering with V1a receptor availability and/or VT-induced endocytosis. We administered a physiologically active fluorescent VT conjugated to Oregon Green (VT-OG) to the fourth ventricle 9 min after an intraperitoneal injection of CORT (0, 10, 40 µg/0.1mL amphibian Ringers). The brains were collected 30 min post-VT-OG, fixed, and imaged with confocal microscopy. CORT diminished the number of endocytosed vesicles, percent area containing VT-OG, sum intensity of VT-OG, and the amount of VT-V1a within each vesicle; indicating that CORT was interfering with V1a receptor availability and VT-V1a receptor-mediated endocytosis. CORT actions were brain location-specific and season-dependent in a manner that is consistent with the natural and context-dependent expression of clasping behavior. Furthermore, the sensitivity of the Rf to CORT was much higher in animals during the breeding season, arguing for ethologically appropriate seasonal variation in CORT's ability to prevent VT-induced endocytosis. Our data are consistent with the time course and interaction effects of CORT and VT on clasping behavior and neurophysiology. CORT interference with VT-induced endocytosis may be a common mechanism employed by hormones across taxa for mediating rapid context- and season-specific behavioral responses.

Sex-specific effect of antenatal betamethasone exposure on renal oxidative stress induced by angiotensins in adult sheep.

Prenatal glucocorticoid administration in clinically relevant doses reduces nephron number and renal function in adulthood and is associated with hypertension. Nephron loss in early life may predispose the kidney to other insults later but whether sex influences increases in renal susceptibility is unclear. Therefore, we determined, in male and female adult sheep, whether antenatal glucocorticoid (betamethasone) exposure increased 8-isoprostane (marker of oxidative stress) and protein excretion after acute nephron reduction and intrarenal infusions of angiotensin peptides. We also examined whether renal proximal tubule cells (PTCs) could contribute to alterations in 8-isoprostane excretion in a sex-specific fashion. In vivo, ANG II significantly increased 8-isoprostane excretion by 49% and protein excretion by 44% in male betamethasone- but not in female betamethasone- or vehicle-treated sheep. ANG-(1-7) decreased 8-isoprostane excretion but did not affect protein excretion in either group. In vitro, ANG II stimulated 8-isoprostane release from PTCs of male but not female betamethasone-treated sheep. Male betamethasone-exposed sheep had increased p47 phox abundance in the renal cortex while superoxide dismutase (SOD) activity was increased only in females. We conclude that antenatal glucocorticoid exposure enhances the susceptibility of the kidney to oxidative stress induced by ANG II in a sex-specific fashion and the renal proximal tubule is one target of the sex-specific effects of antenatal steroids. ANG-(1-7) may mitigate the impact of prenatal glucocorticoids on the kidney. P47 phox activation may be responsible for the increased oxidative stress and proteinuria in males. The protection from renal oxidative stress in females is associated with increased SOD activity.

Evidence for an angiotensin-(1-7) neuropeptidase expressed in the brain medulla and CSF of sheep.

Angiotensin-(1-7) [Ang-(1-7)] is an alternative product of the brain renin-angiotensin system that exhibits central actions to lower blood pressure and improve baroreflex sensitivity. We previously identified a peptidase that metabolizes Ang-(1-7) to the inactive metabolite product Ang-(1-4) in CSF of adult sheep. This study purified the peptidase 1445-fold from sheep brain medulla and characterized this activity. The peptidase was sensitive to the chelating agents o-phenanthroline and EDTA, as well as the mercury compound p-chloromercuribenzoic acid (PCMB). Selective inhibitors to angiotensin-converting enzyme, neprilysin, neurolysin, and thimet oligopeptidase did not attenuate activity; however, the metallopeptidase agent JMV-390 was a potent inhibitor of Ang-(1-7) hydrolysis (Ki = 0.8 nM). Kinetic studies using (125) I-labeled Ang-(1-7), Ang II, and Ang I revealed comparable apparent Km values (2.6, 2.8, and 4.3 µM, respectively), but a higher apparent Vmax for Ang-(1-7) (72 vs. 30 and 6 nmol/min/mg, respectively; p < 0.01). HPLC analysis of the activity confirmed the processing of unlabeled Ang-(1-7) to Ang-(1-4) by the peptidase, but revealed < 5% hydrolysis of Ang II or Ang I, and no hydrolysis of neurotensin, bradykinin or apelin-13. The unique characteristics of the purified neuropeptidase may portend a novel pathway to influence actions of Ang-(1-7) within the brain. Angiotensin-(1-7) actions are mediated by the AT7 /Mas receptor and include reduced blood pressure, decreased oxidative stress, enhanced baroreflex sensitivity, and increased nitric oxide (NO). Ang-(1-7) is directly formed from Ang I by neprilysin (NEP). We identify a new pathway for Ang-(1-7) metabolism in the brain distinct from angiotensin-converting enzyme-dependent hydrolysis. The Ang-(1-7) endopeptidase (A7-EP) degrades the peptide to Ang-(1-4) and may influence central Ang-(1-7) tone.