Discovery of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of adaptor protein 2-associated kinase 1 for the treatment of pain.

Adaptor protein 2-associated kinase 1 (AAK1) is a member of the Ark1/Prk1 family of serine/threonine kinases and plays a role in modulating receptor endocytosis. AAK1 was identified as a potential therapeutic target for the treatment of neuropathic pain when it was shown that AAK1 knock out (KO) mice had a normal response to the acute pain phase of the mouse formalin model, but a reduced response to the persistent pain phase. Herein we report our early work investigating a series of pyrrolo[2,1-f][1,2,4]triazines as part of our efforts to recapitulate this KO phenotype with a potent, small molecule inhibitor of AAK1. The synthesis, structure-activity relationships (SAR), and in vivo evaluation of these AAK1 inhibitors is described.

Mechanistic investigation of liver injury induced by BMS-932481, an experimental ɣ-secretase modulator.

BMS-932481 was designed to modulate ɣ-secretase activity to produce shorter and less amyloidogenic peptides, potentially averting liabilities associated with complete enzymatic inhibition. Although it demonstrated the intended pharmacology in the clinic, BMS-932481 unexpectedly caused drug-induced liver injury (DILI) in a multiple ascending dose study characterized by dose- and exposure-dependence, delayed onset manifestation, and a high incidence of hepatocellular damage. Retrospective studies investigating the disposition and probable mechanisms of toxicity of BMS-932481 are presented here. These included a mass balance study in bile-duct-cannulated rats and a metabolite profiling study in human hepatocytes, which together demonstrated oxidative metabolism followed by biliary elimination as the primary means of disposition. Additionally, minimal protein covalent binding in hepatocytes and lack of bioactivation products excluded reactive metabolite formation as a probable toxicological mechanism. However, BMS-932481 and 3 major oxidative metabolites were found to inhibit the bile salt export pump (BSEP) and multidrug resistance protein 4 (MRP4) in vitro. Considering human plasma concentrations, the IC50 values against these efflux transporters were clinically meaningful, particularly in the high dose cohort. Active uptake into human hepatocytes in vitro suggested the potential for hepatic levels of BMS-932481 to be elevated further above plasma concentrations, enhancing DILI risk. Conversely, measures of mitochondrial functional decline in hepatocytes treated with BMS-932481 were minimal or modest, suggesting limited contributions to DILI. Collectively, these findings suggested that repeat administration of BMS-932481 likely resulted in high hepatic concentrations of BMS-932481 and its metabolites, which disrupted bile acid transport via BSEP and MRP4, elevating serum biomarkers of liver injury.

Ion-Scale Magnetic Flux Rope Generated From Electron-Scale Magnetopause Current Sheet: Magnetospheric Multiscale Observations.

We present in-depth analysis of three southward-moving meso-scale (ion-to magnetohydrodynamic-scale) flux transfer events (FTEs) and subsequent crossing of a reconnecting magnetopause current sheet (MPCS), which were observed on 8 December 2015 by the Magnetospheric Multiscale spacecraft in the subsolar region under southward and duskward magnetosheath magnetic field conditions. We aim to understand the generation mechanism of ion-scale magnetic flux ropes (ISFRs) and to reveal causal relationship among magnetic field structures, electromagnetic energy conversion, and kinetic processes in magnetic reconnection layers. Results from magnetic field reconstruction methods are consistent with a flux rope with a length of about one ion inertial length growing from an electron-scale current sheet (ECS) in the MPCS, supporting the idea that ISFRs can be generated through secondary reconnection in an ECS. Grad-Shafranov reconstruction applied to the three FTEs shows that the FTEs had axial orientations similar to that of the ISFR. This suggests that these FTEs also formed through the same secondary reconnection process, rather than multiple X-line reconnection at spatially separated locations. Four-spacecraft observations of electron pitch-angle distributions and energy conversion rate j·E'=j·E+ve×B suggest that the ISFR had three-dimensional magnetic topology and secondary reconnection was patchy or bursty. Previously reported positive and negative values of j·E', with magnitudes much larger than expected for typical MP reconnection, were seen in both magnetosheath and magnetospheric separatrix regions of the ISFR. Many of them coexisted with bi-directional electron beams and intense electric field fluctuations around the electron gyrofrequency, consistent with their origin in separatrix activities.

Magnetic Field Annihilation in a Magnetotail Electron Diffusion Region With Electron-Scale Magnetic Island.

We present observations in Earth's magnetotail by the Magnetospheric Multiscale spacecraft that are consistent with magnetic field annihilation, rather than magnetic topology change, causing fast magnetic-to-electron energy conversion in an electron-scale current sheet. Multi-spacecraft analysis for the magnetic field reconstruction shows that an electron-scale magnetic island was embedded in the observed electron diffusion region (EDR), suggesting an elongated shape of the EDR. Evidence for the annihilation was revealed in the form of the island growing at a rate much lower than expected for the standard X-type geometry of the EDR, which indicates that magnetic flux injected into the EDR was not ejected from the X-point or accumulated in the island, but was dissipated in the EDR. This energy conversion process is in contrast to that in the standard EDR of a reconnecting current sheet where the energy of antiparallel magnetic fields is mostly converted to electron bulk-flow energy. Fully kinetic simulation also demonstrates that an elongated EDR is subject to the formation of electron-scale magnetic islands in which fast but transient annihilation can occur. Consistent with the observations and simulation, theoretical analysis shows that fast magnetic diffusion can occur in an elongated EDR in the presence of nongyrotropic electron effects. We suggest that the annihilation in elongated EDRs may contribute to the dissipation of magnetic energy in a turbulent collisionless plasma.

Determining EMIC Wave Vector Properties Through Multi-Point Measurements: The Wave Curl Analysis.

Electromagnetic ion cyclotron (EMIC) waves play important roles in particle loss processes in the magnetosphere. Determining the evolution of EMIC waves as they propagate and how this evolution affects wave-particle interactions requires accurate knowledge of the wave vector, k. We present a technique using the curl of the wave magnetic field to determine k observationally, enabled by the unique configuration and instrumentation of the Magnetospheric MultiScale (MMS) spacecraft. The wave curl analysis is demonstrated for synthetic arbitrary electromagnetic waves with varying properties typical of observed EMIC waves. The method is also applied to an EMIC wave interval observed by MMS on October 28, 2015. The derived wave properties and k from the wave curl analysis for the observed EMIC wave are compared with the Waves in Homogenous, Anisotropic, Multi-component Plasma (WHAMP) wave dispersion solution and with results from other single- and multi-spacecraft techniques. We find good agreement between k from the wave curl analysis, k determined from other observational techniques, and k determined from WHAMP. Additionally, the variation of k due to the time and frequency intervals used in the wave curl analysis is explored. This exploration demonstrates that the method is robust when applied to a wave containing at least 3-4 wave periods and over a rather wide frequency range encompassing the peak wave emission. These results provide confidence that we are able to directly determine the wave vector properties using this multi-spacecraft method implementation, enabling systematic studies of EMIC wave k properties with MMS.

Reconstruction of the Electron Diffusion Region With Inertia and Compressibility Effects.

A method based on electron magnetohydrodynamics (EMHD) for the reconstruction of steady, two-dimensional plasma and magnetic field structures from data taken by a single spacecraft, first developed by Sonnerup et al. (2016), https://doi.org/10.1002/2016ja022430, is extended to accommodate inhomogeneity of the electron density and temperature, electron inertia effects, and guide magnetic field in and around the electron diffusion region (EDR), the central part of the magnetic reconnection region. The new method assumes that the electron density and temperature are constant along, but may vary across, the magnetic field lines. We present two models for the reconstruction of electron streamlines, one of which is not constrained by any specific formula for the electron pressure tensor term in the generalized Ohm's law that is responsible for electron unmagnetization in the EDR, and the other is a modification of the original model to include the inertia and compressibility effects. Benchmark tests using data from fully kinetic simulations show that our new method is applicable to both antiparallel and guide-field (component) reconnection, and the electron velocity field can be better reconstructed by including the inertia effects. The new EMHD reconstruction technique has been applied to an EDR of magnetotail reconnection encountered by the Magnetospheric Multiscale spacecraft on 11 July 2017, reported by Torbert et al. (2018), https://doi.org/10.1126/science.aat2998 and reconstructed with the original inertia-less version by Hasegawa et al. (2019), https://doi.org/10.1029/2018ja026051, which demonstrates that the new method better performs in recovering the electric field and electron streamlines than the original version.

Synthesis of functionalized derivatives of the gamma-secretase modulator BMS-932481 and identification of its major metabolite.

In an effort to improve physical properties by introducing polar functionality into the bicyclic pyrimidine gamma-secretase modulator (GSM) clinical candidate BMS-932481, we prepared several oxidative products of BMS-932481. Among the analogs that were prepared, the C-5 alcohol 3 was identified as the predominant metabolite of BMS-932481 found in rat and human liver microsomes. Alcohol 3 was determined to be chemically unstable, leading to the hypothesis that 3 may lead to the production of reactive species both in vitro and in vivo.

How Accurately Can We Measure the Reconnection Rate E M for the MMS Diffusion Region Event of 11 July 2017?

We investigate the accuracy with which the reconnection electric field E M can be determined from in situ plasma data. We study the magnetotail electron diffusion region observed by National Aeronautics and Space Administration's Magnetospheric Multiscale (MMS) on 11 July 2017 at 22:34 UT and focus on the very large errors in E M that result from errors in an L M N boundary normal coordinate system. We determine several L M N coordinates for this MMS event using several different methods. We use these M axes to estimate E M. We find some consensus that the reconnection rate was roughly E M = 3.2 ± 0.6 mV/m, which corresponds to a normalized reconnection rate of 0.18 ± 0.035. Minimum variance analysis of the electron velocity (MVA-v e), MVA of E, minimization of Faraday residue, and an adjusted version of the maximum directional derivative of the magnetic field (MDD-B) technique all produce reasonably similar coordinate axes. We use virtual MMS data from a particle-in-cell simulation of this event to estimate the errors in the coordinate axes and reconnection rate associated with MVA-v e and MDD-B. The L and M directions are most reliably determined by MVA-v e when the spacecraft observes a clear electron jet reversal. When the magnetic field data have errors as small as 0.5% of the background field strength, the M direction obtained by MDD-B technique may be off by as much as 35°. The normal direction is most accurately obtained by MDD-B. Overall, we find that these techniques were able to identify E M from the virtual data within error bars ≥20%.

Robust Translation of γ-Secretase Modulator Pharmacology across Preclinical Species and Human Subjects.

The amyloid-ß peptide (Aß)-in particular, the 42-amino acid form, Aß1-42-is thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Thus, several therapeutic modalities aiming to inhibit Aß synthesis or increase the clearance of Aß have entered clinical trials, including γ-secretase inhibitors, anti-Aß antibodies, and amyloid-ß precursor protein cleaving enzyme inhibitors. A unique class of small molecules, γ-secretase modulators (GSMs), selectively reduce Aß1-42 production, and may also decrease Aß1-40 while simultaneously increasing one or more shorter Aß peptides, such as Aß1-38 and Aß1-37. GSMs are particularly attractive because they do not alter the total amount of Aß peptides produced by γ-secretase activity; they spare the processing of other γ-secretase substrates, such as Notch; and they do not cause accumulation of the potentially toxic processing intermediate, ß-C-terminal fragment. This report describes the translation of pharmacological activity across species for two novel GSMs, (S)-7-(4-fluorophenyl)-N2-(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481) and (S,Z)-17-(4-chloro-2-fluorophenyl)-34-(3-methyl-1H-1,2,4-triazol-1-yl)-16,17-dihydro-15H-4-oxa-2,9-diaza-1(2,4)-cyclopenta[d]pyrimidina-3(1,3)-benzenacyclononaphan-6-ene (BMS-986133). These GSMs are highly potent in vitro, exhibit dose- and time-dependent activity in vivo, and have consistent levels of pharmacological effect across rats, dogs, monkeys, and human subjects. In rats, the two GSMs exhibit similar pharmacokinetics/pharmacodynamics between the brain and cerebrospinal fluid. In all species, GSM treatment decreased Aß1-42 and Aß1-40 levels while increasing Aß1-38 and Aß1-37 by a corresponding amount. Thus, the GSM mechanism and central activity translate across preclinical species and humans, thereby validating this therapeutic modality for potential utility in AD.

Synthesis of quaternary aryl phosphonium salts: photoredox-mediated phosphine arylation.

We report a synthesis method for the construction of quaternary aryl phoshonium salts at ambient temperature. The regiospecific reaction involves the coupling of phosphines with aryl radicals derived from diaryliodonium salts under photoredox conditions.

Synthesis of malhamensilipin A exploiting iterative epoxidation/chlorination: experimental and computational analysis of epoxide-derived chloronium ions.

We report a 12-step catalytic enantioselective formal synthesis of malhamensilipin A (3) and diastereoisomeric analogues from (E)-2-undecenal. The convergent synthesis relied upon iterative epoxidation and phosphorus(v)-mediated deoxydichlorination reactions as well a titanium-mediated epoxide-opening to construct the C11-C16 stereohexad. The latter transformation occurred with very high levels of stereoretention regardless of the C13 configuration of the parent epoxide, implicating anchimeric assistance of either the γ- or δ-chlorine atoms, and the formation of chloretanium or chlorolanium ions, respectively. A computational analysis of the chloronium ion intermediates provided support for the involvement of chlorolanium ions, whereas the potential chloretanium ions were found to be less likely intermediates on the basis of their greater carbocationic character.

Identification and Preclinical Pharmacology of the γ-Secretase Modulator BMS-869780.

Alzheimer's disease is the most prevalent cause of dementia and is associated with accumulation of amyloid-ß peptide (Aß), particularly the 42-amino acid Aß1-42, in the brain. Aß1-42 levels can be decreased by γ-secretase modulators (GSM), which are small molecules that modulate γ-secretase, an enzyme essential for Aß production. BMS-869780 is a potent GSM that decreased Aß1-42 and Aß1-40 and increased Aß1-37 and Aß1-38, without inhibiting overall levels of Aß peptides or other APP processing intermediates. BMS-869780 also did not inhibit Notch processing by γ-secretase and lowered brain Aß1-42 without evidence of Notch-related side effects in rats. Human pharmacokinetic (PK) parameters were predicted through allometric scaling of PK in rat, dog, and monkey and were combined with the rat pharmacodynamic (PD) parameters to predict the relationship between BMS-869780 dose, exposure and Aß1-42 levels in human. Off-target and safety margins were then based on comparisons to the predicted exposure required for robust Aß1-42 lowering. Because of insufficient safety predictions and the relatively high predicted human daily dose of 700 mg, further evaluation of BMS-869780 as a potential clinical candidate was discontinued. Nevertheless, BMS-869780 demonstrates the potential of the GSM approach for robust lowering of brain Aß1-42 without Notch-related side effects.

Pharmacodynamics of selective inhibition of γ-secretase by avagacestat.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid ß-peptide (Aß), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aß and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aß and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aß levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aß40 and Aß42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aß40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aß levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

Distribution of volumes of individual glomeruli in kidneys at autopsy: association with age, nephron number, birth weight and body mass index.

BACKGROUND: Glomerular hypertrophy occurs in a number of normal and pathological states. Glomerular volume in kidneys at autopsy is usually indirectly derived from estimates of total glomerular mass and nephron number, and provides only a single value per kidney, with no indication of the range of volumes of glomeruli within the kidney of any given subject. We review findings of the distribution of volumes of different glomeruli within subjects without kidney disease, and their correlations with age, nephron number, birth weight and body mass index (BMI). METHODS: The study describes findings from autopsy kidneys of selected adult white males from the Southeast USA who had unexpected deaths, and who did not have renal scarring or renal disease. Total glomerular (nephron) number and total glomerular volume were estimated using the disector/fractionator combination, and mean glomerular volume (Vglom) was derived. The volumes of 30 individual glomeruli (IGV) in each subject were determined using the disector/Cavalieri method. IGV values were compared by categories of age, nephron number, birth weight and BMI. RESULTS: There was substantial variation in IGV within subjects. Older age, lower nephron number, lower birth weight and gross obesity were associated with higher mean IGV and with greater IGV heterogeneity. High Vglom and high IGVs were associated with more glomerulosclerosis. However, amongst the generally modest numbers of sclerosed glomeruli, the pattern was uniformly of ischemic collapse of the glomerular tuft. There was no detectable focal segmental glomerular tuft injury. DISCUSSION: In this series of people without overt renal disease, greater age, nephron deficit, lower birth weight and obesity were marked by glomerular enlargement and greater glomerular volume heterogeneity within individuals.

In vitro-in vivo extrapolation of hepatic clearance involving active uptake: theoretical and experimental aspects.

The importance of hepatic uptake transporters in drug clearance is well recognized. The subject is reviewed with the intention of providing an overview of the concepts in order to link the increasing knowledge of transporter-mediated uptake into established models of hepatic clearance. In order to understand and quantify their impact, models of hepatic elimination that incorporate permeability barriers are required. Models that include both active and passive uptake into hepatocytes are discussed and simulations of the influence of active uptake and passive diffusion on hepatic clearance are presented. The advantages and weaknesses of a number of in vitro assays of hepatic uptake are described, and their ability to predict hepatic clearance is reviewed.

Reduced nephron number and glomerulomegaly in Australian Aborigines: a group at high risk for renal disease and hypertension.

Aborigines in remote areas of Australia have much higher rates of renal disease, as well as hypertension and cardiovascular disease, than non-Aboriginal Australians. We compared kidney findings in Aboriginal and non-Aboriginal people in one remote region. Glomerular number and mean glomerular volume were estimated with the disector/fractionator combination in the right kidney of 19 Aborigines and 24 non-Aboriginal people undergoing forensic autopsy for sudden or unexpected death in the Top End of the Northern Territory. Aborigines had 30% fewer glomeruli than non-Aborigines--202,000 fewer glomeruli per kidney, or an estimated 404,000 fewer per person (P=0.036). Their mean glomerular volume was 27% larger (P=0.016). Glomerular number was significantly correlated with adult height, inferring a relationship with birthweight, which, on average, is much lower in Aboriginal than non-Aboriginal people. Aboriginal people with a history of hypertension had 30% fewer glomeruli than those without--250,000 fewer per kidney (P=0.03), or 500,000 fewer per person, and their mean glomerular volume was about 25% larger. The lower nephron number in Aboriginal people is compatible with their susceptibility to renal failure. The additional nephron deficit associated with hypertension is compatible with other reports. Lower nephron numbers are probably due in part to reduced nephron endowment, which is related to a suboptimal intrauterine environment. Compensatory glomerular hypertrophy in people with fewer nephrons, while minimizing loss of total filtering surface area, might be exacerbating nephron loss. Optimization of fetal growth should ultimately reduce the florid epidemic of renal disease, hypertension, and cardiovascular disease.

Hypertension, glomerular number, and birth weight in African Americans and white subjects in the southeastern United States.

Low nephron number has been related to low birth weight and hypertension. In the southeastern United States, the estimated prevalence of chronic kidney disease due to hypertension is five times greater for African Americans than white subjects. This study investigates the relationships between total glomerular number (Nglom), blood pressure, and birth weight in southeastern African Americans and white subjects. Stereological estimates of Nglom were obtained using the physical disector/fractionator technique on autopsy kidneys from 62 African American and 60 white subjects 30-65 years of age. By medical history and recorded blood pressures, 41 African Americans, and 24 white subjects were identified as hypertensive and 21 African Americans and 36 white subjects as normotensive. Mean arterial blood pressure (MAP) was obtained on 81 and birth weights on 63 subjects. For African Americans, relationships between MAP, Nglom, and birth weight were not significant. For white subjects, they were as follows: MAP and Nglom (r=-0.4551, P=0.0047); Nglom and birth weight (r=0.5730, P=0.0022); MAP and birth weight (r=-0.4228, P=0.0377). For African Americans, average Nglom of 961 840+/-292 750 for normotensive and 867 358+/-341 958 for hypertensive patients were not significantly different (P=0.285). For white subjects, average Nglom of 923 377+/-256 391 for normotensive and 754 319+/-329 506 for hypertensive patients were significantly different (P=0.03). The data indicate that low nephron number and possibly low birth weight may play a role in the development of hypertension in white subjects but not African Americans.

Dynamics of {beta}-amyloid reductions in brain, cerebrospinal fluid, and plasma of {beta}-amyloid precursor protein transgenic mice treated with a {gamma}-secretase inhibitor.

gamma-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer's disease (AD). gamma-Secretase inhibitors reduce brain beta-amyloid peptide (Abeta), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human beta-amyloid precursor protein (APP) are valuable models to examine the dynamics of Abeta changes with gamma-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a gamma-secretase inhibitor, showed dose- and time dependent reductions of Abeta in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Abeta levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Abeta in plaques showed reductions in CSF Abeta in the absence of measurable changes in plaque Abeta in the brain after up to 2 weeks of treatment. Hence, CSF Abeta levels were a valuable measure of gamma-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8(+) thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for gamma-secretase inhibitors to reduce brain Abeta without causing Notch-mediated toxicity.

Maternal glucocorticoid treatment programs alterations in the renin-angiotensin system of the ovine fetal kidney.

Ovine fetuses exposed to high concentrations of synthetic (dexamethasone, D) or naturally occurring glucocorticoids (cortisol, F) in utero during early gestation develop high blood pressure in adulthood. To investigate potential mechanisms involved, we examined the role of the renal renin-angiotensin system (RAS). Ewes were infused with isotonic saline (S, n = 11), D (n = 12, 0.48 mg/h), or F (n = 5, 5 mg/h) for 48 h between d 26 and 28 of gestation (term = 150 d). Ewes carrying twins (S, n = 5; D, n = 6; F, n = 5) were killed at 130 d of gestation. The mRNA levels for angiotensinogen, the AT(1) receptor and AT(2) receptor, were increased in the fetal kidneys after D treatment. Prenatal infusions of F produced similar effects on the AT(1) receptor. Single fetuses (S, n = 6; D, n = 6) were cannulated and infused with angiotensin II for 3 d beginning at 127 d of gestation. Basal blood pressure was similar in both groups and increased similarly with angiotensin II infusion. However, increases in urine flow and glomerular filtration rate were significantly reduced and kidney weights increased in the D-treated group. These results indicate that treatment with D very early in gestation causes significant alterations in the RAS in the fetal kidney 100 d later with functional consequences. Changes in the RAS in the developing kidney may be an important mechanism in the development of adult disease.