Enhanced Electrocatalytic Nitrate Reduction to Ammonia Using Functionalized Multi-Walled Carbon Nanotube-Supported Cobalt Catalyst.

Ammonia (NH3) is vital in modern agriculture and industry as a potential energy carrier. The electrocatalytic reduction of nitrate (NO3-) to ammonia under ambient conditions offers a sustainable alternative to the energy-intensive Haber-Bosch process. However, achieving high selectivity in this conversion poses significant challenges due to the multi-step electron and proton transfer processes and the low proton adsorption capacity of transition metal electrocatalysts. Herein, we introduce a novel approach by employing functionalized multi-walled carbon nanotubes (MWCNTs) as carriers for active cobalt catalysts. The exceptional conductivity of MWCNTs significantly reduces charge transfer resistance. Their unique hollow structure increases the electrochemical active surface area of the electrocatalyst. Additionally, the one-dimensional hollow tube structure and graphite-like layers within MWCNTs enhance adsorption properties, thus mitigating the diffusion of intermediate and stabilizing active cobalt species during nitrate reduction reaction (NitRR). Using the MWCNT-supported cobalt catalyst, we achieved a notable NH3 yield rate of 4.03 mg h-1 cm-2 and a high Faradaic efficiency of 84.72% in 0.1 M KOH with 0.1 M NO3-. This study demonstrates the potential of MWCNTs as advanced carriers in constructing electrocatalysts for efficient nitrate reduction.

The interface-mediated electron structure tuning of RuOx-Co3O4 nano-particles for efficient electrocatalytic nitrate reduction.

The energy-intensive processes for the industrial production of ammonia necessitates the development of new methods to be proposed that will aid in reducing the global energy consumption. Specifically, the electrocatalytic nitrate reduction reaction (NO3RR) to produce ammonia is more thermodynamically feasible than the electrocatalytic nitrogen reduction reaction (NRR). However, it is hindered by a low catalytic activity due to its complex reaction pathways. Herein, we synthesized a novel electrocatalyst, RuOx-Co3O4 nanoparticles, with abundant interfaces, which exhibited an enhanced catalytic activity for efficient ammonia synthesis. This catalyst delivered a partial current density of 65.8 mA cm-2 for NH3 production, a faradaic efficiency (FE) of 89.7%, and a superior ammonia yield rate of up to 210.5 µmol h-1 cm-2 at -0.6 V vs. RHE. X-ray photoelectron and Raman spectroscopy revealed that the formed interfacial Ru-O-Co bond can decorate the electronic structures of the active sites and accelerate the absorption of NO3-, thus promoting the production of ammonia.

Intranasal soluble ACE2 improves survival and prevents brain SARS-CoV-2 infection.

A soluble ACE2 protein bioengineered for long duration of action and high affinity to SARS-CoV-2 was administered either intranasally (IN) or intraperitoneally (IP) to SARS-CoV-2-inoculated k18hACE2 mice. This decoy protein (ACE2 618-DDC-ABD) was given either IN or IP, pre- and post-inoculation, or IN, IP, or IN + IP but only post-inoculation. Survival by day 5 was 0% in untreated mice, 40% in the IP-pre, and 90% in the IN-pre group. In the IN-pre group, brain histopathology was essentially normal and lung histopathology significantly improved. Consistent with this, brain SARS-CoV-2 titers were undetectable and lung titers reduced in the IN-pre group. When ACE2 618-DDC-ABD was administered only post-inoculation, survival was 30% in the IN + IP, 20% in the IN, and 20% in the IP group. We conclude that ACE2 618-DDC-ABD results in markedly improved survival and provides organ protection when given intranasally as compared with when given either systemically or after viral inoculation, and that lowering brain titers is a critical determinant of survival and organ protection.

Fecal ammonium in mice with CKD: gastrointestinal sequestration by sodium zirconium cyclosilicate.

Urinary [Formula: see text] excretion is decreased in chronic kidney disease (CKD), but very little is known about fecal [Formula: see text] excretion. Sodium zirconium cyclosilicate (SZC) is a cation exchanger that selectively captures K+ in the gastrointestinal tract. We investigated if SZC can sequester [Formula: see text] in vivo and evaluated the effect of SZC on fecal [Formula: see text] in a mouse model of CKD. Mice with CKD induced by 5/6 kidney ablation were fed either a regular diet or a diet containing SZC (4 g/kg) and followed for 7 days. Fecal [Formula: see text] was measured before and after the addition of 50 meq KCl/L to release [Formula: see text] from SZC. [Formula: see text] sequestered in SZC in the gastrointestinal (GI) tract was estimated from the change in fecal [Formula: see text] observed when KCl was added to liberate the sequestered [Formula: see text]. In mice with CKD, fecal [Formula: see text] excretion was higher than in normal mice and also higher than urine [Formula: see text] excretion measured concurrently. Using data pooled from the SZC diet, the change in [Formula: see text] was 6.5 ± 0.6 compared with 0.6 ± 0.6 µmol/g on the normal diet (P < 0.0001). In conclusion, fecal [Formula: see text] excretion in CKD is increased and about sixfold higher than urine [Formula: see text] excretion, revealing an important route of elimination of [Formula: see text] present in the GI tract. SZC administration sequesters a substantial portion of [Formula: see text] in the GI tract, suggesting that the binding of [Formula: see text] offers therapeutic potential beyond its known primary action as a specific K+ binder.NEW & NOTEWORTHY Fecal [Formula: see text] excretion in chronic kidney disease is increased and about sixfold higher than urine [Formula: see text] excretion, revealing an important route of elimination of [Formula: see text] that is present in the gastrointestinal tract. Sodium zirconium cyclosilicate (SZC) administration sequesters a substantial portion of [Formula: see text], suggesting that binding of [Formula: see text] by SZC in the gastrointestinal tract offers therapeutic potential in chronic kidney disease and other clinical conditions beyond its known primary action of SZC as a specific K+ binder.

Superiority of intranasal over systemic administration of bioengineered soluble ACE2 for survival and brain protection against SARS-CoV-2 infection.

The present study was designed to investigate the effects of a soluble ACE2 protein termed ACE2 618-DDC-ABD, bioengineered to have long duration of action and high binding affinity to SARS-CoV-2, when administered either intranasally (IN) or intraperitoneally (IP) and before or after SARS-CoV-2 inoculation. K18hACE2 mice permissive for SARS-CoV-2 infection were inoculated with 2Ã-10 4 PFU wildtype SARS-CoV-2. In one protocol, ACE2 618-DDC-ABD was given either IN or IP, pre- and post-viral inoculation. In a second protocol, ACE2 618-DDC-ABD was given either IN, IP or IN+IP but only post-viral inoculation. In addition, A549 and Vero E6 cells were used to test neutralization of SARS-CoV-2 variants by ACE2 618-DDC-ABD at different concentrations. Survival by day 5 was 0% in infected untreated mice, and 40% in mice from the ACE2 618-DDC-ABD IP-pre treated group. By contrast, in the IN-pre group survival was 90%, histopathology of brain and kidney was essentially normal and markedly improved in the lungs. When ACE2 618-DDC-ABD was administered only post viral inoculation, survival was 30% in the IN+IP group, 20% in the IN and 0% in the IP group. Brain SARS-CoV-2 titers were high in all groups except for the IN-pre group where titers were undetectable in all mice. In cells permissive for SARS-CoV-2 infection, ACE2 618-DDC-ABD neutralized wildtype SARS-CoV-2 at high concentrations, whereas much lower concentrations neutralized omicron BA. 1. We conclude that ACE2 618-DDC-ABD provides much better survival and organ protection when administered intranasally than when given systemically or after viral inoculation and that lowering brain titers is a critical determinant of survival and organ protection.

Urinary Angiotensinogen in Patients With Type 1 Diabetes With Microalbuminuria: Gender Differences and Effect of Intensive Insulin Therapy.

Introduction: Angiotensinogen (AOG) is the precursor of peptides of the renin angiotensin system (RAS). Because insulin up-regulates transcriptional factors that normally repress kidney AOG synthesis, we evaluated urinary AOG (uAOG) in patients with type 1 diabetes (T1D) and microalbuminuria who are receiving either intensive or conventional insulin therapy. Methods: Urine samples from participants of the Diabetes Control and Complications Trial (DCCT) were used for the following: (i) uAOG/creatinine measurements in 103 patients with microalbuminuria and 103 patients with normoalbuminuria, matched for age, gender, disease duration, and allocation to insulin therapy; and (ii) uAOG/creatinine measurements from patients with microalbuminuria allocated to intensive insulin therapy (n = 58) or conventional insulin therapy (n = 41) after 3 years on each modality. Results: uAOG was higher in patients who started with microalbuminuria than in those with normoalbuminuria (6.65 vs. 4.0 ng/mg creatinine, P < 0.01). uAOG was higher in females than in males with microalbuminuria (11.7 vs. 5.4 ng/mg creatinine, P = 0.015). uAOG was lower in patients with microalbuminuria allocated to intensive insulin therapy than in conventional insulin therapy (3.98 vs. 7.42 ng/mg creatinine, P < 0.01). These differences in uAOG were observed though albumin excretion rate (AER) was not significantly different. Conclusion: In patients with T1D and microalbuminuria, uAOG is increased and varies with gender and the type of insulin therapy independently of AER. This suggests that AOG production is increased in females and it is decreased by intensive insulin therapy. The reduction in uAOG with intensive insulin therapy, by kidney RAS downregulation, may contribute to the known renoprotective action associated with intensive insulin and improved glycemic control.

A Novel Soluble ACE2 Protein Provides Lung and Kidney Protection in Mice Susceptible to Lethal SARS-CoV-2 Infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2) as a main receptor to enter target cells. The goal of this study was to demonstrate the preclinical efficacy of a novel soluble ACE2 protein with increased duration of action and binding capacity in a lethal mouse model of COVID-19. METHODS: A human soluble ACE2 variant fused with an albumin binding domain (ABD) was linked via a dimerization motif hinge-like 4-cysteine dodecapeptide (DDC) to improve binding capacity to SARS-CoV-2. This novel soluble ACE2 protein (ACE2-1-618-DDC-ABD) was then administered intranasally and intraperitoneally to mice before intranasal inoculation of SARS-CoV-2 and then for two additional days post viral inoculation. RESULTS: Untreated animals became severely ill, and all had to be humanely euthanized by day 6 or 7 and had pulmonary alveolar hemorrhage with mononuclear infiltrates. In contrast, all but one mouse infected with a lethal dose of SARS-CoV-2 that received ACE2-1-618-DDC-ABD survived. In the animals inoculated with SARS-CoV-2 that were untreated, viral titers were high in the lungs and brain, but viral titers were absent in the kidneys. Some untreated animals, however, had variable degrees of kidney proximal tubular injury as shown by attenuation of the proximal tubular brush border and increased NGAL and TUNEL staining. Viral titers in the lung and brain were reduced or nondetectable in mice that received ACE2-1-618-DDC-ABD, and the animals developed only moderate disease as assessed by a near-normal clinical score, minimal weight loss, and improved lung and kidney injury. CONCLUSIONS: This study demonstrates the preclinical efficacy of a novel soluble ACE2 protein, termed ACE2-1-618-DDC-ABD, in a lethal mouse model of SARS-CoV-2 infection that develops severe lung injury and variable degrees of moderate kidney proximal tubular injury.

Predator-Prey Interactions between Nonnative Juvenile Largemouth Bass (Micropterus salmoides) and Local Candidate Prey Species in the Pearl River Delta: Predation Capacity, Preference and Growth Performance.

An ontogenetic dietary shift is crucial for the survival and growth of piscivorous largemouth bass (LB). However, there is much to learn about the predator-prey interaction during the switching process. We carried out a series of indoor experiments to examine the predation capacity, predation preference, and growth performance of exotic juvenile LB feeding on candidate prey species in the Pearl River Delta. The widely distributed oriental river prawn (Macrobranchium nipponense), barcheek goby (Ctenogobius giurinus), western mosquitofish (Gambusia affinis), silver carp (Hypophthalmichthys molitrix), and mud carp (Cirrhinus molitorella), with relatively similar total lengths, were selected as potential prey based on their availability and habitat use. Our results show that predation capacity and preference varied quantitatively and qualitatively among prey species. The number of oriental river prawns killed was significantly less than that of fish species, comparing the 1st hour with the 24th hour (p < 0.01). The feeding rhythm of LB varied significantly from crayfish to fish. Numerically, Jacobs' selection index reinforced LB's special preference for predating G. affinis. Although there were obvious variations in predation capacity and feed selection, no statistically significant growth differences were detected among LB groups feeding on live M. nipponense, G. affinis, H. molitrix, and C. molitorella (p < 0.05). These findings suggest that the successful ontogenetic dietary shift of juvenile LB may depend on the availability and vulnerability of local fish species. Further study on the reproductive phenology of potential fish prey may help to predict LB's establishment.

A Novel Soluble ACE2 Variant with Prolonged Duration of Action Neutralizes SARS-CoV-2 Infection in Human Kidney Organoids.

BACKGROUND: There is an urgent need for approaches to prevent and treat SARS-CoV-2 infection. Administration of soluble ACE2 protein acting as a decoy to bind to SARS-CoV-2 should limit viral uptake mediated by binding to membrane-bound full-length ACE2, and further therapeutic benefit should result from ensuring enzymatic ACE2 activity to affected organs in patients with COVID-19. METHODS: A short variant of human soluble ACE2 protein consisting of 618 amino acids (hACE2 1-618) was generated and fused with an albumin binding domain (ABD) using an artificial gene encoding ABDCon, with improved albumin binding affinity. Human kidney organoids were used for infectivity studies of SARS-CoV-2 in a BSL-3 facility to examine the neutralizing effect of these novel ACE2 variants. RESULTS: Whereas plasma ACE2 activity of the naked ACE2 1-618 and ACE2 1-740 lasted about 8 hours, the ACE2 1-618-ABD resulted in substantial activity at 96 hours, and it was still biologically active 3 days after injection. Human kidney organoids express ACE2 and TMPRSS2, and when infected with SARS-CoV-2, our modified long-acting ACE2 variant neutralized infection. CONCLUSIONS: This novel ACE2 1-618-ABD can neutralize SARS-CoV-2 infectivity in human kidney organoids, and its prolonged duration of action should ensure improved efficacy to prevent viral escape and dosing convenience.

Knockout of aminopeptidase A in mice causes functional alterations and morphological glomerular basement membrane changes in the kidneys.

Aminopeptidase A is one of the most potent enzymes within the renin-angiotensin system in terms of angiotensin II degradation. Here, we examined whether there is a kidney phenotype and any compensatory changes in other renin angiotensin system enzymes involved in the metabolism of angiotensin II associated with aminopeptidase A deficiency. Kidneys harvested from aminopeptidase A knockout mice were examined by light and electron microscopy, immunohistochemistry and immunofluorescence. Kidney angiotensin II levels and the ability of renin angiotensin system enzymes in the glomerulus to degrade angiotensin II ex vivo, their activities, protein and mRNA levels in kidney lysates were evaluated. Knockout mice had increased blood pressure and mild glomerular mesangial expansion without significant albuminuria. By electron microscopy, knockout mice exhibited a mild increase of the mesangial matrix, moderate thickening of the glomerular basement membrane but a striking appearance of knob-like structures. These knobs were seen in both male and female mice and persisted after the treatment of hypertension. In isolated glomeruli from knockout mice, the level of angiotensin II was more than three-fold higher as compared to wild type control mice. In kidney lysates from knockout mice angiotensin converting enzyme activity, protein and mRNA levels were markedly decreased possibly as a compensatory mechanism to reduce angiotensin II formation. Thus, our findings support a role for aminopeptidase A in the maintenance of glomerular structure and intra-kidney homeostasis of angiotensin peptides.

Isolation and genomic characterization of a pathogenic Providencia rettgeri strain G0519 in turtle Trachemys scripta.

Providencia rettgeri infection has occurred occasionally in aquaculture, but is rare in turtles. Here, a pathogenic P. rettgeri strain G0519 was isolated from a diseased slider turtle (Trachemys scripta) in China, and qPCR assay was established for the RTX toxin (rtxD) gene. Histopathological examination showed that many inflammatory cells were infiltrated into heart, liver and intestine, as well as the necrosis of liver, kidney and spleen. The genome consisted of one circular chromosome (4.493 Mb) and one plasmid (18.8 kb), and predicted to contain 4170 and 19 protein-coding genes, respectively. Multiple pathogenic and virulence factors (e.g., fimbria, adhesion, invasion, toxin, hemolysin, chemotaxis, secretion system), multidrug-resistant genes (e.g., ampC, per-1, oxa-1, sul1, tetR) and a novel genomic resistance island PRI519 were identified. Comparative genome analysis revealed the closest relationship was with P. rettgeri, and with P. heimbachae closer than with other Providencia spp. To our knowledge, this was first report on genomic characterization of multidrug-resistant pathogenic P. rettgeri in cultured turtles.

Ang II (Angiotensin II) Conversion to Angiotensin-(1-7) in the Circulation Is POP (Prolyloligopeptidase)-Dependent and ACE2 (Angiotensin-Converting Enzyme 2)-Independent.

The Ang II (Angiotensin II)-Angiotensin-(1-7) axis of the Renin Angiotensin System encompasses 3 enzymes that form Angiotensin-(1-7) [Ang-(1-7)] directly from Ang II: ACE2 (angiotensin-converting enzyme 2), PRCP (prolylcarboxypeptidase), and POP (prolyloligopeptidase). We investigated their relative contribution to Ang-(1-7) formation in vivo and also ex vivo in serum, lungs, and kidneys using models of genetic ablation coupled with pharmacological inhibitors. In wild-type (WT) mice, infusion of Ang II resulted in a rapid increase of plasma Ang-(1-7). In ACE2-/-/PRCP-/- mice, Ang II infusion resulted in a similar increase in Ang-(1-7) as in WT (563±48 versus 537±70 fmol/mL, respectively), showing that the bulk of Ang-(1-7) formation in circulation is essentially independent of ACE2 and PRCP. By contrast, a POP inhibitor, Z-Pro-Prolinal reduced the rise in plasma Ang-(1-7) after infusing Ang II to control WT mice. In POP-/- mice, the increase in Ang-(1-7) was also blunted as compared with WT mice (309±46 and 472±28 fmol/mL, respectively P=0.01), and moreover, the rate of recovery from acute Ang II-induced hypertension was delayed (P=0.016). In ex vivo studies, POP inhibition with ZZP reduced Ang-(1-7) formation from Ang II markedly in serum and in lung lysates. By contrast, in kidney lysates, the absence of ACE2, but not POP, obliterated Ang-(1-7) formation from added Ang II. We conclude that POP is the main enzyme responsible for Ang II conversion to Ang-(1-7) in the circulation and in the lungs, whereas Ang-(1-7) formation in the kidney is mainly ACE2-dependent.

Urinary angiotensinogen antedates the development of stage 3 CKD in patients with type 1 diabetes mellitus.

We examined if urinary angiotensinogen (uAOG), a marker of intrarenal renin-angiotensin system activity, antedates stage 3 chronic kidney disease (CKD) using samples from participants in the Diabetes Control and Complications Trial (DCCT) and later in the Epidemiology of Diabetes Intervention and Complications (EDIC) trial. In a nested case-control design, cases were matched at the outcome visit (eGFR less than 60, 21-59 mL/min per 1.73 m2 ) on age, gender, and diabetes duration, with controls: eGFR (95, 75-119, mL/min per 1.73 m2 .) Additionally, in an exploratory analysis progressive renal decline (PRD), defined as eGFR loss >3.5 mL/min per 1.73m2 /year, was evaluated using only data from EDIC because no progressions were observed during DCCT. At the EDIC visit, which antedated the GFR outcome visit by 2 years (range 1-7years) the median uAOG/creatinine was markedly higher in cases than in controls (13.9 vs. 3.8 ng/mg P = 0.003) whereas at the DCCT visit, which antedated the GFR outcome by 17 to 20 years it was not (2.75 vs. 3.16 ng/mg, respectively). The Odds Ratio for uAOG and CKD stage 3 development was significant after adjusting for eGFR, HbA1c, and systolic blood pressure 1.82 (1.00-3.29) but no longer significant when Albumin Excretion Ratio (AER) was included 1.21 (0.65-2.24).In the PRD analysis, uAOG/creatinine was sixfold higher in participants who experienced PRD than in those who did not (26 vs. 4.0 ng/mg, P = 0.003). The Odds Ratio for uAOG and PRD was significant after adjusting for eGFR, HbA1c, and systolic blood pressure 2.48 (1.46-4.22) but no longer significant when AER was included 1.32 (0.76-2.30). In people with type1 diabetes, a robust increase in uAOG antedates the development of stage 3 CKD but is not superior to AER in predicting this renal outcome. Increased uAOG moreover is associated with PRD, an index of progression to End Stage Kidney Disease (ESKD).

Urinary Renin in Patients and Mice With Diabetic Kidney Disease.

In patients with diabetic kidney disease (DKD), plasma renin activity is usually decreased, but there is limited information on urinary renin and its origin. Urinary renin was evaluated in samples from patients with longstanding type I diabetes mellitus and mice with streptozotocin-induced diabetes mellitus. Renin-reporter mouse model (Ren1d-Cre;mT/mG) was made diabetic with streptozotocin to examine whether the distribution of cells of the renin lineage was altered in a chronic diabetic environment. Active renin was increased in urine samples from patients with DKD (n=36), compared with those without DKD (n=38; 3.2 versus 1.3 pg/mg creatinine; P<0.001). In mice with streptozotocin-induced diabetes mellitus, urine renin was also increased compared with nondiabetic controls. By immunohistochemistry, in mice with streptozotocin-induced diabetes mellitus, juxtaglomerular apparatus and proximal tubular renin staining were reduced, whereas collecting tubule staining, by contrast, was increased. To examine the role of filtration and tubular reabsorption on urinary renin, mice were either infused with either mouse or human recombinant renin and lysine (a blocker of proximal tubular protein reabsorption). Infusion of either form of renin together with lysine markedly increased urinary renin such that it was no longer different between nondiabetic and diabetic mice. Megalin mRNA was reduced in the kidney cortex of streptozotocin-treated mice (0.70±0.09 versus 1.01±0.04 in controls, P=0.01) consistent with impaired tubular reabsorption. In Ren1d-Cre;mT/mG with streptozotocin-induced diabetes mellitus, the distribution of renin lineage cells within the kidney was similar to nondiabetic renin-reporter mice. No evidence for migration of cells of renin linage to the collecting duct in diabetic mice could be found. Renin mRNA in microdissected collecting ducts from streptozotocin-treated mice, moreover, was not significantly different than in controls, whereas in kidney cortex, largely reflecting juxtaglomerular apparatus renin, it was significantly reduced. In conclusion, in urine from patients with type 1 diabetes mellitus and DKD and from mice with streptozotocin-induced diabetes mellitus, renin is elevated. This cannot be attributed to production from cells of the renin lineage migrating to the collecting duct in a chronic hyperglycemic environment. Rather, the elevated levels of urinary renin found in DKD are best attributed to altered glomerular filteration and impaired proximal tubular reabsorption.

Apelinergic system in the kidney: implications for diabetic kidney disease.

The bioactive peptides of the apelinergic system and its receptor APJ have been shown to play a protective role in experimental cardiovascular and diabetic kidney disease (DKD). Mechanisms of this renoprotective effect remain to be elucidated. In this study, we examined the localization of APJ within the normal kidney and its kidney expression in the db/db model of DKD. The effect of hyperglycemia and angiotensin II on APJ was examined in cultured podocytes. In the glomerulus, APJ colocalized with podocyte but not endothelial cell markers. In podocytes stimulated with Pyr1 Apelin-13, a change in the phosphorylation status of the signaling proteins, AKT, ERK, and p70S6K, was observed with an increase 15 min after stimulation. Apelin-13 decreased activity of Caspase-3 in podocytes after high glucose treatment reflecting an antiapoptotic effect of APJ stimulation. In podocytes, APJ mRNA was downregulated in high glucose, when compared to normal glucose conditions and exposure to angiotensin II led to a further significant decrease in APJ mRNA. APJ and preproapelin mRNA levels in kidneys from db/db mice were markedly decreased along with decreased tubular APJ protein by western blotting and immunostaining when compared to db/m controls. In conclusion, the apelinergic system is decreased in kidneys from db/db mice. Within the glomerulus, APJ is mainly localized in podocytes and in this cell type its activation by Apelin-13 abolishes the proapoptotic effect of high glucose, suggesting a potential therapeutic role of apelin and emerging agonists with extended half-life for therapy of DKD.

Transcription Factor 21 Is Required for Branching Morphogenesis and Regulates the Gdnf-Axis in Kidney Development.

BACKGROUND: The mammalian kidney develops through reciprocal inductive signals between the metanephric mesenchyme and ureteric bud. Transcription factor 21 (Tcf21) is highly expressed in the metanephric mesenchyme, including Six2-expressing cap mesenchyme and Foxd1-expressing stromal mesenchyme. Tcf21 knockout mice die in the perinatal period from severe renal hypodysplasia. In humans, Tcf21 mRNA levels are reduced in renal tissue from human fetuses with renal dysplasia. The molecular mechanisms underlying these renal defects are not yet known. METHODS: Using a variety of techniques to assess kidney development and gene expression, we compared the phenotypes of wild-type mice, mice with germline deletion of the Tcf21 gene, mice with stromal mesenchyme-specific Tcf21 deletion, and mice with cap mesenchyme-specific Tcf21 deletion. RESULTS: Germline deletion of Tcf21 leads to impaired ureteric bud branching and is accompanied by downregulated expression of Gdnf-Ret-Wnt11, a key pathway required for branching morphogenesis. Selective removal of Tcf21 from the renal stroma is also associated with attenuation of the Gdnf signaling axis and leads to a defect in ureteric bud branching, a paucity of collecting ducts, and a defect in urine concentration capacity. In contrast, deletion of Tcf21 from the cap mesenchyme leads to abnormal glomerulogenesis and massive proteinuria, but no downregulation of Gdnf-Ret-Wnt11 or obvious defect in branching. CONCLUSIONS: Our findings indicate that Tcf21 has distinct roles in the cap mesenchyme and stromal mesenchyme compartments during kidney development and suggest that Tcf21 regulates key molecular pathways required for branching morphogenesis.

Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation.

Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that potently degrades angiotensin II to angiotensin 1-7. Previous studies showed that injection of the enzymatic ectodomain of recombinant ACE2 (rACE2) markedly increases circulatory levels of ACE2 activity, and effectively lowered blood pressure in angiotensin II-induced hypertension. However, due to the short plasma half-life of rACE2, its therapeutic potential for chronic use is limited. To circumvent this, we generated a chimeric fusion of rACE2 and the immunoglobulin fragment Fc segment to increase its plasma stability. This rACE2-Fc fusion protein retained full peptidase activity and exhibited greatly extended plasma half-life in mice, from less than two hours of the original rACE2, to over a week. A single 2.5 mg/kg injection of rACE2-Fc increased the overall angiotensin II-conversion activities in blood by up to 100-fold and enhanced blood pressure recovery from acute angiotensin II induced hypertension seven days after administration. To assess rACE2-Fc given weekly on cardiac protection, we performed studies in mice continuously infused with angiotensin II for 28 days and in a Renin transgenic mouse model of hypertension. The angiotensin II infused mice achieved sustained blood pressure control and reduced cardiac hypertrophy and fibrosis. In chronic hypertensive transgenic mice, weekly injections of rACE2-Fc effectively lowered plasma angiotensin II and blood pressure. Additionally, rACE2-Fc ameliorated albuminuria, and reduced kidney and cardiac fibrosis. Thus, our chimeric fusion strategy for rACE2-Fc is suitable for future development of new renin angiotensin system-based inhibition therapies.

A Fluorometric Method of Measuring Carboxypeptidase Activities for Angiotensin II and Apelin-13.

Degradation of the biologically potent octapeptide angiotensin Ang II-(1-8) is mediated by the activities of several peptidases. The conversion of Ang II to the septapeptide Ang-(1-7) is of particular interest as the latter also confers organ protection. The conversion is catalyzed by angiotensin-converting enzyme 2 and other enzymes that selectively cleave the peptide bond between the proline and the phenylalanine at the carboxyl terminus of Ang II. The contribution of various enzyme activities that collectively lead to the formation of Ang-(1-7) from Ang II, in both normal conditions and in disease states, remains only partially understood. This is largely due to the lack of a reliable and sensitive method to detect these converting activities in complex samples, such as blood and tissues. Here, we report a fluorometric method to measure carboxypeptidase activities that cleave the proline-phenylalanine dipeptide bond in Ang II. This method is also suitable for measuring the conversion of apelin-13. The assay detects the release of phenylalanine amino acid in a reaction with the yeast enzyme of phenylalanine ammonia lyase (PAL). When used in cell and mouse organs, the assay can robustly measure endogenous Ang II and apelin-13-converting activities involved in the renin-angiotensin and the apelinergic systems, respectively.

Prolylcarboxypeptidase deficiency is associated with increased blood pressure, glomerular lesions, and cardiac dysfunction independent of altered circulating and cardiac angiotensin II.

Prolylcarboxypeptidase (PRCP) is a carboxypeptidase that cleaves angiotensin II (AngII) forming Ang(1-7). The impact of genetic PRCP deficiency on AngII metabolism, blood pressure (BP), kidney histology, and cardiac phenotype was investigated in two lines of PRCP-deficient mice: KST302 derived in C57BL/6 background and GST090 derived in FVB/N background. The GST090 line had increased mean arterial pressure (MAP) (113.7 ± 2.07 vs. WT 105.0 ± 1.23 mmHg; p < 0.01) and left ventricular hypertrophy (LVH) (ratio of diastolic left ventricular posterior wall dimension to left ventricular diameter 0.239 ± 0.0163 vs. WT 0.193 ± 0.0049; p < 0.05). Mice in the KST302 line also had mild hypertension and LVH. Cardiac defects, increased glomerular size, and glomerular mesangial expansion were also observed. After infusion of AngII to mice in the KST302 line, both MAP and LVH increased, but the constitutive differences between the gene trap mice and controls were no longer observed. Plasma and cardiac AngII and Ang(1-7) were not significantly different between PRCP-deficient mice and controls. Thus, PRCP deficiency is associated with elevated blood pressure and cardiac alterations including LVH and cardiac defects independently of systemic or cardiac AngII and Ang(1-7). An ex vivo assay showed that recombinant PRCP, unlike recombinant ACE2, did not degrade AngII to form Ang(1-7) in plasma at pH 7.4. PRCP was localized in α-intercalated cells of the kidney collecting tubule. The low pH prevailing at this site and the acidic pH preference of PRCP suggest a role of this enzyme in regulating AngII degradation in the collecting tubule where this peptide increases sodium reabsorption and therfore BP. However, there are other potential mechanisms for increased BP in this model that need to be considered as well. PRCP converts AngII to Ang(1-7) but only at an acidic pH. Global PRCP deficiency causes heart and kidney alterations and a moderate rise in BP. PRCP is abundant in the kidney collecting tubules, where the prevailing pH is low. In collecting tubules, PRCP deficiency could result in impaired AngII degradation. Increased AngII at this nephron site stimulates Na reabsorption and increases BP. KEY MESSAGE: Prolylcarboxypeptidase (PRCP) converts AngII to Ang (1-7) but only at an acidic pH. Global PRCP deficiency causes heart and kidney alterations and a moderate rise in BP. PRCP is abundant in the kidney collecting tubules, where the prevailing pH is low. In collecting tubules, PRCP deficiency could result in impaired AngII degradation. Increased AngII at this nephron site stimulates Na reabsorption and increases BP.