Elevated Free Water Research Articles

Blood-brain barrier disruption and increased free water are associated with worse cognitive performance in patients with chronic cerebrovascular disease

Progression of cerebral small vessel disease (CSVD) is associated with cognitive decline. Blood-brain barrier disruption (BBBD) and fluid extravasation to the interstitial space may contribute to progression of white matter hyperintensities (WMH). We hypothesized that increased free water (FW) would colocalize with BBBD and relate to cognitive performance. Patients with ischemic stroke/TIA at least 3 months prior and at least early confluent WMH were studied cross-sectionally with the Montreal Cognitive Assessment (MoCA), diffusion tensor imaging, and dynamic susceptibility contrast imaging. White matter (WM) was segmented into WMH, WMH penumbra, and normal appearing white matter (NAWM). Colocalization of elevated FW and BBBD and their associations MoCA performance were evaluated. 58 patients were included (mean age 69, 36 % female). Higher BBBD colocalized with elevated FW. Elevated FW in all white matter, NAWM, WMH penumbra, and WMH lesions was associated with lower MoCA score. Increased BBBD in all WM, NAWM, and WMH penumbra was associated with lower MoCA. In WMH penumbra, both elevated FW and increased BBBD were independently associated with lower MoCA. We found agreement between 2 different biomarkers implicated in the pathogenesis of CSVD that independently demonstrated association with cognitive performance when measured in the area of postulated disease activity.

Elevated extracellular free water in the brain predicts clinical improvement in first-episode psychosis.

Despite the diverse nature of clinical trajectories after a first-episode of psychosis, few baseline characteristics have been predictive of clinical improvement, and the neurobiological underpinnings of this heterogeneity remain largely unknown. Elevated extracellular free water (FW) in the brain is a diffusion imaging measure that has been consistently reported in different phases of psychosis that may indicate a neuroinflammatory state. Its predictive capacity in terms of clinical outcomes, however, is unknown. We used diffusion imaging to determine FW and tissue-specific fractional anisotropy (FA-t) in first-episode psychosis. Forty-seven participants were categorized as clinical "Improvers" (n=26) if they achieved a 20% decrease in total Brief Psychiatric Rating Scale (BPRS) score at 12 months. To determine the predictive capacity of FW and FA-t, these measures were introduced in a stepwise logistic regression model to predict clinical improvement. For those measures surviving the model, regional between-group differences were also investigated in cortical surface or white matter tracts as applicable. Higher gray matter (GM) FW (OR-CI 1.134 - 2.543) and FA-t (OR-CI: 0.905 - 2.038) both predicted Improver status. FW in GM also linearly correlated with BPRS total score at 12 months follow-up. Examining regional specificity, Improvers showed greater FW predominantly in temporal regions and higher FA-t values in several white matter tracts, including bilateral longitudinal superior fasciculus. Our results show that elevated FW in GM and FA-t predict further clinical improvement during the initial phases of psychosis. The potential roles of brain inflammatory processes in predicting clinical improvement are discussed.

Functional and free-water imaging in rapid eye movement behaviour disorder and Parkinson's disease.

It is established that one of the best predictors of a future diagnosis of Parkinson's disease is a current diagnosis of rapid eye movement behaviour disorder (RBD). In such patients, this provides a unique opportunity to study brain physiology and behavioural motor features of RBD that may precede early-stage Parkinson's disease. Based on prior work in early-stage Parkinson's disease, we aim to determine if the function of corticostriatal and cerebellar regions are impaired in RBD using task-based functional MRI and if structural changes can be detected within the caudate, putamen and substantia nigra in RBD using free-water imaging. To assess motor function, we measured performance on the Purdue Pegboard Test, which is affected in patients with RBD and Parkinson's disease. A cohort of 24 RBD, 39 early-stage Parkinson's disease and 25 controls were investigated. All participants were imaged at 3 Telsa. Individuals performed a unimanual grip force task during functional imaging. Participants also completed scales to assess cognition, sleep and motor symptoms. We found decreased functional activity in both RBD and Parkinson's disease within the motor cortex, caudate, putamen and thalamus compared with controls. There was elevated free-water-corrected fractional anisotropy in the putamen in RBD and Parkinson's disease and elevated free-water in the putamen and posterior substantia nigra in Parkinson's disease compared with controls. Participants with RBD and Parkinson's disease performed significantly worse on all tasks of the Purdue Pegboard Test compared with controls. The both hands task of the Purdue Pegboard Test was most sensitive in distinguishing between groups. A subgroup analysis of early-stage RBD (<2 years diagnosis) confirmed similar findings as those in the larger RBD group. These findings provide new evidence that the putamen is affected in early-stage RBD using both functional and free-water imaging. We also found evidence that the striatum, thalamus and motor cortex have reduced functional activity in early-stage RBD and Parkinson's disease. While the substantia nigra shows elevated free-water in Parkinson's disease, we did not observe this effect in early-stage RBD. These findings point to the corticostriatal and thalamocortical circuits being impaired in RBD patients.

Transcallosal white matter and cortical gray matter variations in autistic adults ages 30-73 years: A bi-tensor free water imaging approach.

Background: Autism spectrum disorder (ASD) has long been recognized as a lifelong condition, but brain aging studies in autistic adults aged >30 years are limited. Free water, a novel brain imaging marker derived from diffusion MRI (dMRI), has shown promise in differentiating typical and pathological aging and monitoring brain degeneration. We aimed to examine free water and free water corrected dMRI measures to assess white and gray matter microstructure and their associations with age in autistic adults. Methods: Forty-three autistic adults ages 30-73 years and 43 age, sex, and IQ matched neurotypical controls participated in this cross-sectional study. We quantified fractional anisotropy (FA), free water, and free water-corrected FA (fwcFA) across 32 transcallosal white matter tracts and 94 gray matter areas in autistic adults and neurotypical controls. Follow-up analyses assessed age effect on dMRI metrics of the whole brain for both groups and the relationship between dMRI metrics and clinical measures of ASD in regions that significantly differentiated autistic adults from controls. Results: We found globally elevated free water in 24 transcallosal tracts in autistic adults. We identified negligible differences in dMRI metrics in gray matter between the two groups. Age-associated FA reductions and free water increases were featured in neurotypical controls; however, this brain aging profile was largely absent in autistic adults. Additionally, greater autism quotient (AQ) total raw score was associated with increased free water in the inferior frontal gyrus pars orbitalis and lateral orbital gyrus in autistic adults. Limitations: All autistic adults were cognitively capable individuals, minimizing the generalizability of the research findings across the spectrum. This study also involved a cross-sectional design, which limited inferences about the longitudinal microstructural changes of white and gray matter in ASD. Conclusions: We identified differential microstructural configurations between white and gray matter in autistic adults and that autistic individuals present more heterogeneous brain aging profiles compared to controls. Our clinical correlation analysis offered new evidence that elevated free water in some localized white matter tracts may critically contribute to autistic traits in ASD. Our findings underscored the importance of quantifying free water in dMRI studies of ASD.

Pulsed light treatment enhances starch hydrolysis and improves starch physicochemical properties of germinated brown rice.

Recently, germinated brown rice (GBR) has gained substantial attention as a functional food because of its nutritional attributes. Notably, pulsed light technology (PLT) has emerged as a promising tool for enhancing rice germination and, consequently, has improved the nutritional and functional qualities of GBR-derived products. However, further research is required to comprehensively understand the impact of PLT on GBR physicochemical properties. The present study aimed to investigate the stimulating effects of PLT on starch hydrolysis, starch structure and functional properties of GBR. The PLT substantially boosted α-amylase activity during brown rice germination, leading to a 10.9% reduction in total starch content and a 17.3% increase in reducing sugar content, accompanied by elevated free water levels. Structural analysis indicated no changes in starch crystalline types, whereas gelatinization temperature slightly increased. Pasting properties exhibited a significant drop in peak viscosity. Scanning electron microscopy showed surface erosion of starch granules with microstructural changes. Furthermore, correlation analysis established positive links between α-amylase activity, reducing sugar accumulation, starch structure and functional properties in GBR. The present study demonstrates that PLT enhanced the physicochemical properties of GBR starch, significantly improving the stability of GBR products, thereby contributing to expanded applicability of rice starch in the food industry. © 2023 Society of Chemical Industry.

Extracellular free water elevations are associated with brain volume and maternal cytokine response in a longitudinal nonhuman primate maternal immune activation model.

Maternal infection has emerged as an important environmental risk factor for neurodevelopmental disorders, including schizophrenia and autism spectrum disorders. Animal model systems of maternal immune activation (MIA) suggest that the maternal immune response plays a significant role in the offspring's neurodevelopment and behavioral outcomes. Extracellular free water is a measure of freely diffusing water in the brain that may be associated with neuroinflammation and impacted by MIA. The present study evaluates the brain diffusion characteristics of male rhesus monkeys (Macaca mulatta) born to MIA-exposed dams (n = 14) treated with a modified form of the viral mimic polyinosinic:polycytidylic acid at the end of the first trimester. Control dams received saline injections at the end of the first trimester (n = 10) or were untreated (n = 4). Offspring underwent diffusion MRI scans at 6, 12, 24, 36, and 45 months. Offspring born to MIA-exposed dams showed significantly increased extracellular free water in cingulate cortex gray matter starting as early as 6 months of age and persisting through 45 months. In addition, offspring gray matter free water in this region was significantly correlated with the magnitude of the maternal IL-6 response in the MIA-exposed dams. Significant correlations between brain volume and extracellular free water in the MIA-exposed offspring also indicate converging, multimodal evidence of the impact of MIA on brain development. These findings provide strong evidence for the construct validity of the nonhuman primate MIA model as a system of relevance for investigating the pathophysiology of human neurodevelopmental psychiatric disorders. Elevated free water in individuals exposed to immune activation in utero could represent an early marker of a perturbed or vulnerable neurodevelopmental trajectory.

Free water and iron content in the substantia nigra at different stages of Parkinson's disease

PurposeAbnormalities in free water (FW) and susceptibility values exist in the substantia nigra (SN) of patients with Parkinson’s disease (PD), but their role in characterizing the disease processes remains uncertain. This study investigated these values at various SN locations and stages of PD, and their relationship with clinical symptoms. MethodFW and quantitative susceptibility mapping (QSM) values were evaluated in the anterior and posterior SN, along with swallow-tail-sign (STS) ratings, in patients with PD (early-stage: n = 39; middle-to-advanced-stage: n = 97) and healthy controls (n = 82). The correlation between these indices and motor and non-motor symptoms, and their capability to distinguish PD from healthy controls, were also examined. ResultsIncreased FW in the anterior and posterior SN and increased QSM values in the posterior SN were observed in both early-stage and middle-to-advanced-stage PD patients (p < 0.05). However, there was no significant difference in FW, QSM values, or STS ratings among patients at different stages. FW and QSM values correlated with motor symptoms in middle-to-advanced-stage patients (p < 0.05), while STS ratings were associated with non-motor symptoms (p < 0.05). Additionally, combining FW and QSM values in the posterior SN with STS ratings in logistic regression showed better performance in distinguishing PD (area under curve = 0.931) compared to using STS ratings alone (area under curve = 0.880). ConclusionsFindings suggest elevated FW and iron content in PD at different stages, with dissociation in SN location between the two indices. Elevated signals are related to the motor symptom severity in middle-to-advanced-stage patients, and may have the potential for PD diagnosis and symptom assessment.

Free water in T2 FLAIR white matter hyperintensity lesions

AbstractBackgroundWhite matter (WM) free water (FW) is likely associated with cerebral small vessel disease (CSVD). FW is the fraction of unconstrained water within an image voxel, which can be estimated from diffusion‐weighted images. T2‐weighted Fluid‐Attenuated Inversion Recovery (FLAIR) white matter hyperintensity (WMH) is a widely used index to assess the damages caused by CSVD. It is critical to characterize how FW content is altered in WMH lesions. In this work, we proposed a data processing framework to assess FW distributions in WMH and normal‐appearing WM as well as in different WM fiber tracts.MethodSingle‐shell diffusion‐weighted image (SS‐DWI) and T2 FLAIR image data of 133 cognitively normal (CN) adults (Table 1) were obtained from the ADNI dataset (http://adni.loni.usc.edu). FW maps were generated from SS‐DWI images using the DIPY software package (https://dipy.org/). WMH lesions were identified with the lesion segmentation toolbox implemented in SPM. We co‐registered FW and WMH maps of individual subjects in the MNI space. Then we derived group WM fiber tracts using the tract‐based spatial statistics (TBSS). Finally, we examined the localization of WMH and FW in the major WM tracts.ResultFigure 1 shows the FW map, FLAIR image, and WMH lesion from a representative subject. Figure 2 shows group average and individual FW distributions in WMH and normal‐appearing WM. Two sample T‐Test found that FW content in WMH is significantly higher than that in normal‐appearing WM (mean FW and standard deviation FW in WMH are 0.430 and 0.112, mean FW and standard deviation FW in normal‐appearing WM are 0.203 and 0.06, P &lt; 1×10‐30). TBSS based analysis showed that: 1) WMH lesions are mainly located in the corona radiata, posterior thalamic radiation, tapetum, superior fronto‐occipital fasciculus, superior longitudinal fasciculus, corpus callosum, and sagittal stratum; 2) Compared with mean FW in normal‐appearing WM, elevated FW content is mainly at the fornix, tapetum, cingulum hippocampus, superior fronto‐occipital fasciculus, corpus callosum, sagittal stratum, and posterior thalamic radiation.ConclusionWe developed an image processing pipeline to estimate FW changes in WMH lesions and normal‐appearing WM. Free water content is elevated in WMH lesions, which is likely driven by CSVD.

The evolution of white matter microstructural changes after mild traumatic brain injury: A longitudinal DTI and NODDI study.

Neuroimaging biomarkers that can detect white matter (WM) pathology after mild traumatic brain injury (mTBI) and predict long-term outcome are needed to improve care and develop therapies. We used diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) to investigate WM microstructure cross-sectionally and longitudinally after mTBI and correlate these with neuropsychological performance. Cross-sectionally, early decreases of fractional anisotropy and increases of mean diffusivity corresponded to WM regions with elevated free water fraction on NODDI. This elevated free water was more extensive in the patient subgroup reporting more early postconcussive symptoms. The longer-term longitudinal WM changes consisted of declining neurite density on NODDI, suggesting axonal degeneration from diffuse axonal injury for which NODDI is more sensitive than DTI. Therefore, NODDI is a more sensitive and specific biomarker than DTI for WM microstructural changes due to mTBI that merits further study for mTBI diagnosis, prognosis, and treatment monitoring.

Extracellular free water and glutathione in first-episode psychosis-a multimodal investigation of an inflammatory model for psychosis.

Evidence has been accumulating for an immune-based component to the etiology of psychotic disorders. Advancements in diffusion magnetic resonance imaging (MRI) have enabled estimation of extracellular free water (FW), a putative biomarker of neuroinflammation. Furthermore, inflammatory processes may be associated with altered brain levels of metabolites, such as glutathione (GSH). Consequently, we sought to test the hypotheses that FW is increased and associated with decreased GSH in patients with first episode schizophrenia (SZ) compared to healthy controls (HC). SZ (n=36) and HC (n=40) subjects underwent a multi-shell diffusion MRI scan on a Siemens 3T scanner. 1H-MR spectroscopy data were acquired using a GSH-optimized MEGA-PRESS editing sequence and GSH/creatine ratios were calculated for DLPFC (SZ: n=33, HC: n=37) and visual cortex (SZ: n=29, HC: n=35) voxels. Symptoms and functioning were measured using the SANS, SAPS, BPRS and GSF/GRF. SZ demonstrated significantly elevated FW in whole-brain gray (p=.001) but not white matter (p=.060). There was no significant difference between groups in GSH in either voxel. However, there was a significant negative correlation between DLPFC GSH and both whole-brain and DLPFC-specific gray matter FW in SZ (r=−.48 and −.47, respectively; both p<.05), while this relationship was nonsignificant in HC and in both groups in visual cortex. These data illustrate an important relationship between a metabolite known to be important for immune function – GSH – and the diffusion extracellular FW measure, which provides additional support for these measures as neuroinflammatory biomarkers that could potentially provide tractable treatment targets to guide pharmacological intervention.

SA82. Extracellular Free Water and Glutathione in First-Episode Psychosis: A Multimodal Investigation of an Inflammatory Model for Psychosis

Background: Evidence has been accumulating for an immune-based component to the etiology of psychotic disorders. Advancements in diffusion magnetic resonance imaging (MRI) have enabled estimation of extracellular free water (FW), a putative biomarker of neuroinflammation. Furthermore, there is evidence that neuroimmune activation may alter brain levels of metabolites (e.g., glutathione [GSH]) that can be measured noninvasively with proton magnetic resonance spectroscopy (1H-MRS). Consequently, we sought to test the hypothesis that patients with first-episode psychosis (FEP) have increased FW and decreased GSH when compared to healthy controls (HC). Methods: FEP (n = 32) participants were identified based on an SCID (DSM-IV) diagnosis of Schizophrenia, Schizoaffective Disorder, Schizophreniform Disorder, or Bipolar Disorder with psychotic features. HC (n=27) had no family history of a psychotic disorder and no current or lifetime Axis 1 psychiatric disorder. All participants underwent a diffusion MRI scan on a Siemens 3T scanner in which multiple b-value shells were acquired. MPRAGE images were segmented and aligned to the diffusion-weighted images to compute mean FW values for gray and white matter whole-brain masks. 1H-MRS was acquired using a GSH-optimized MEGA-PRESS editing sequence and GSH–creatine ratios were calculated for voxels within the DLPFC and visual cortex. The addition of visual cortex GSH analyses represent new data that has not previously been presented and allow for identification of regionally specific GSH differences. Results: FEP demonstrated significantly elevated FW in whole-brain gray (P < .05) with no difference in white matter (P = .06). There were no significant group differences in GSH within the DLPFC (P = .1) or visual cortex (P = .41). There was a significant negative correlation between DLPFC GSH and both gray and white matter FW in FEP (r = −.35 and −.4, respectively; both P < .05). No relationships emerged between visual cortex GSH levels and either gray or white matter FW in the FEP sample. Finally, no relationships were identified between GSH and FW in the HC sample. Conclusion: These data provide compelling convergent evidence for the presence of neuroinflammatory processes in FEP. The inverse relationship between GSH and FW implies a common linkage to neuroinflammatory processes that may be present in FEP. Importantly, GSH measurements may be complicated by a genetic polymorphism that affects GSH synthesis, which will need to be accounted for in future studies. Nevertheless, these data suggest that FW and GSH show promise as neuroinflammatory biomarkers and could potentially provide tractable treatment targets for pharmacological intervention.

In vivo imaging reveals impaired connectivity across cortical and subcortical networks in a mouse model of DYT1 dystonia

Developing in vivo functional and structural neuroimaging assays in Dyt1 ΔGAG heterozygous knock-in (Dyt1 KI) mice provide insight into the pathophysiology underlying DYT1 dystonia. In the current study, we examined in vivo functional connectivity of large-scale cortical and subcortical networks in Dyt1 KI mice and wild-type (WT) controls using resting-state functional magnetic resonance imaging (MRI) and an independent component analysis. In addition, using diffusion MRI we examined how structural integrity across the basal ganglia and cerebellum directly relates to impairments in functional connectivity. Compared to WT mice, Dyt1 KI mice revealed increased functional connectivity across the striatum, thalamus, and somatosensory cortex; and reduced functional connectivity in the motor and cerebellar cortices. Further, Dyt1 KI mice demonstrated elevated free-water (FW) in the striatum and cerebellum compared to WT mice, and increased FW was correlated with impairments in functional connectivity across basal ganglia, cerebellum, and sensorimotor cortex. The current study provides the first in vivo MRI-based evidence in support of the hypothesis that the deletion of a 3-base pair (ΔGAG) sequence in the Dyt1 gene encoding torsinA has network level effects on in vivo functional connectivity and microstructural integrity across the sensorimotor cortex, basal ganglia, and cerebellum.

Sensory and motor cortex function contributes to symptom severity in spinocerebellar ataxia type 6.

Spinocerebellar ataxia type 6 (SCA6) is a genetic disease that causes degeneration of Purkinje cells, and recent evidence points to degeneration of Betz cells in the motor cortex. The relation between functional activity of motor cortex and symptom severity during a hand-grip motor control in vivo has not yet been investigated. This study explored both functional changes in the sensorimotor cortex and cerebellar regions and structural alterations in the cerebellum for SCA6 patients as compared to age-matched healthy controls using a multimodal imaging approach (task-based fMRI, task-based functional connectivity, and free-water diffusion MRI). Further, we tested their relation with the severity of ataxia symptoms. SCA6 patients had reduced functional activity in the sensorimotor cortex, supplementary motor area (SMA), cerebellar vermis, and cerebellar lobules I-VI (corrected P<0.05). Reduced task-based functional connectivity between cortical motor regions (i.e., primary motor cortex and SMA) and cerebellar regions (i.e., vermis and lobules I-VI) was found in SCA6 (corrected P<0.05). SCA6 had elevated free-water values throughout the cerebellum as compared with controls (corrected P<0.05). Importantly, reduced functional activity in the sensorimotor cortex and SMA and increased free-water in the superior cerebellar peduncle and cerebellar lobule V were related to more severe symptoms in SCA6 (all pairs: R 2≥0.4 and corrected P<0.05). Current results demonstrate that impaired functional activity in sensorimotor cortex and SMA and elevated free-water of lobule V and superior cerebellar peduncle are both related to symptom severity, and may provide candidate biomarkers for SCA6.

Free-water imaging in Parkinson's disease and atypical parkinsonism.

Conventional single tensor diffusion analysis models have provided mixed findings in the substantia nigra of Parkinson's disease, but recent work using a bi-tensor analysis model has shown more promising results. Using a bi-tensor model, free-water values were found to be increased in the posterior substantia nigra of Parkinson's disease compared with controls at a single site and in a multi-site cohort. Further, free-water increased longitudinally over 1 year in the posterior substantia nigra of Parkinson's disease. Here, we test the hypothesis that other parkinsonian disorders such as multiple system atrophy and progressive supranuclear palsy have elevated free-water in the substantia nigra. Equally important, however, is whether the bi-tensor diffusion model is able to detect alterations in other brain regions beyond the substantia nigra in Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy and to accurately distinguish between these diseases. Free-water and free-water-corrected fractional anisotropy maps were compared across 72 individuals in the basal ganglia, midbrain, thalamus, dentate nucleus, cerebellar peduncles, cerebellar vermis and lobules V and VI, and corpus callosum. Compared with controls, free-water was increased in the anterior and posterior substantia nigra of Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy. Despite no other changes in Parkinson's disease, we observed elevated free-water in all regions except the dentate nucleus, subthalamic nucleus, and corpus callosum of multiple system atrophy, and in all regions examined for progressive supranuclear palsy. Compared with controls, free-water-corrected fractional anisotropy values were increased for multiple system atrophy in the putamen and caudate, and increased for progressive supranuclear palsy in the putamen, caudate, thalamus, and vermis, and decreased in the superior cerebellar peduncle and corpus callosum. For all disease group comparisons, the support vector machine 10-fold cross-validation area under the curve was between 0.93-1.00 and there was high sensitivity and specificity. The regions and diffusion measures selected by the model varied across comparisons and are consistent with pathological studies. In conclusion, the current study used a novel bi-tensor diffusion analysis model to indicate that all forms of parkinsonism had elevated free-water in the substantia nigra. Beyond the substantia nigra, both multiple system atrophy and progressive supranuclear palsy, but not Parkinson's disease, showed a broad network of elevated free-water and altered free-water corrected fractional anisotropy that included the basal ganglia, thalamus, and cerebellum. These findings may be helpful in the differential diagnosis of parkinsonian disorders, and thereby facilitate the development and assessment of targeted therapies.

Downregulation of aquaporin-2 parallels changes in renal water excretion in unilateral ureteral obstruction.

In bilateral ureteral obstruction, both aquaporin-2 (AQP2) levels and urinary concentrating capacity are markedly reduced. However, the mechanisms involved in AQP2 downregulation are unknown. In rats with unilateral ureteral obstruction (UUO) the relative role of intrarenal and systemic factors can be evaluated. Semiquantitative immunoblotting revealed a marked decrease in AQP2 in obstructed kidneys to 23 +/- 7% (n = 9) of sham levels. This downregulation persisted 24 h after release of UUO. Furthermore, there was a significant but less extensive downregulation of AQP2 in the nonobstructed kidneys to 75 +/- 7% (n = 9) of sham levels. Consistent with impairment of collecting duct water reabsorption, free water clearance was greatly elevated in the obstructed kidneys (-2 +/- 1 microliter-min-1.kg-1, determined immediately after release) and only moderately elevated in nonobstructed kidneys (-44 +/- 5 microliters.min-1.kg-1) compared with sham-operated controls (-59 +/- 3 microliters.min-1.kg-1). Also AQP2 mRNA levels were reduced in obstructed kidneys. Immunocytochemistry confirmed the marked decrease in AQP2 expression in obstructed kidneys. In nonobstructed kidneys AQP2 was predominantly found in intracellular vesicles, which together with the reduced expression and elevated free water clearance strongly suggests a role of AQP2 in the observed compensatory diuresis from nonobstructed kidneys. The much lower AQP2 protein and mRNA levels in obstructed vs. nonobstructed kidneys are consistent with intrarenal factors playing a major role for downregulation of AQP2.