Validation of the Self-Reported Psoriasis Treatment Convenience Scale (PTCS).

INTRODUCTION: Adherence to topical treatments for psoriasis is reported to be poor. One key contributing factor is the inconvenience associated with formulations that may be greasy, time consuming to apply, and slow to absorb. There is a paucity of patient-reported outcome measures that evaluate psoriasis patients' perceptions of treatment convenience. The Psoriasis Treatment Convenience Scale (PTCS) was therefore developed and validated. METHODS: Following a literature review of issues relating to convenience of topical treatments, important items were identified and a draft version of the PTCS was developed and underwent content validity testing (n = 20). The revised scale was included in a clinical trial of topical therapy (n = 794; NCT03308799), and psychometric testing was performed. RESULTS: The final questionnaire included five core items and one overall satisfaction question. In psychometric testing, the scale demonstrated stability across trial population, and good validity, reliability, and sensitivity. CONCLUSION: The PTCS is a new, reliable, sensitive, validated tool for the assessment of patient-reported treatment convenience. Use of the PTCS will facilitate evaluation of convenience as part of the clinical development of topical therapies, and thus may help to improve patient adherence and, therefore, treatment outcomes.

Ultrastructural identification of developing proximal tubules based on three-dimensional reconstruction.

BACKGROUND: The cellular mechanisms involved in the development of proximal tubules are not only associated with morphogenesis in fetal life, but also with restoration of damaged tubules in adulthood. Knowledge about morphological features of cell differentiation and proliferation along the developing tubule is insufficient, which hinders identification of the cellular origin. OBJECTIVES: This study aimed to investigate ultrastructures of the proximal tubule at different stages of nephrogenesis. METHODS: Electron microscopy was used and guided by computer-assisted tubular tracing to identify the cellular structures. RESULTS: Renal vesicles and S-shaped bodies revealed more proliferative features, such as densely-packed fusiform-shaped cells with numerous protein-producing organelles than membrane specializations typical for mature tubules. At the capillary-loop stage the proximal tubules demonstrated all characteristics of the mature tubules, but not as developed, including shorter but densely packed microvilli, fewer lateral processes with cell-cell contacts, lower basal membrane infoldings, and lower mitochondrial volume density. However, they exhibited an elaborated endocytic system above the nucleus, indicating a membrane transport is being established. Abundant free- and endoplasmic reticulum-adhered ribosomes and Golgi complexes reflected active protein synthesis for cell growth and proliferation. Interestingly, electron dense cells were occasionally intermixed with electron lucent cells characterized by various organelles in less cytosol and a larger nucleus with abundant euchromatin, which is a feature of active proliferation. CONCLUSIONS: These ultrastructures indicate that the morphogenesis of the developing proximal tubule corresponds to the gradually established physiological activities. The two different cellular electron densities may suggest distinctive differentiation of the cells along the tubule.

A well-developed endolysosomal system reflects protein reabsorption in segment 1 and 2 of rat proximal tubules.

Proteinuria is a well-established marker and predictor of kidney disease. The receptors megalin and cubilin reabsorb filtered proteins and thereby proteinuria is avoided. It is unknown if all segments of the proximal tubule are involved in clearing the filtrate or if there exists a reserve capacity in case of increased glomerular protein filtration. To determine this, we performed serial sectioning of rat kidney and used stereology to quantify the endolysosomal system of the three segments of cortical and juxtamedullary nephrons by electron microscopy. Immunohistochemistry was applied to analyze the adaptor protein Dab2, which assists in megalin mediated endocytosis, megalin, and endocytic uptake of two endogenous megalin ligands; retinol binding protein and ß2-microglobulin at exact tubular positions. Proteinuric rats (puromycin-treated) and mice (podocin knock-out) were analyzed to clarify the response of the tubule to increased protein filtration. We found that the endolysosomal system was most prominent in segment 1 and 2, whereas segment 3 was less developed. The depth of ligand uptake varied among nephrons, but it descended into segment 2 although uptake was lower than in segment 1 and it was never observed in segment 3. This was supported by prominent expression of Dab2 in segment 1 and 2. When protein filtration increased, segment 3 was included in the reabsorption process in proteinuric animals. Thus, segment 1 and 2 are responsible for clearing the filtrate for protein during normal physiological conditions, but the tubule exhibits plasticity and is able to include segment 3 under proteinuric stress.

Morphology of the initial nephron-collecting duct connection in mice using computerized 3D tracing and electron microscopy.

Recently, the cellular origin of the connecting tubule (CNT) has been genetically characterized. The CNT is a segment between two embryonically different structures, the collecting duct originating from ureteric bud (UB), and the nephron derived from the cap mesenchyme. However, the cellular detail at the initial connection is limited. The present study demonstrated that the initial connection was composed of cells which were closely associated with the renal vesicle (RV), the initial nephron, and connected with the basal epithelium of the terminal UB tip at discrete points. The identification of the RV and UB tip was based on tracing of tubules on serial epoxy sections at mouse embryonic day 17.5. The cells at the initial connection were characterized by 1) irregularly-shaped nuclei and cells with cytoplasmic processes, 2) electron dense nuclei, 3) abundant intercellular spaces, 4) extensive cell-cell contacts with cell junctions, often zonulae adherences and occasionally focal fusion of opposing plasma membranes, and 5) numerous mitochondria, densely packed rosette-like polyribosomes, and widespread rER in the cytoplasm. Moreover, the tracing revealed that a terminal UB tip frequently connected to two nephrons at different developing stages. The UB tips, the initial connections, and the distal tubules of the S-shaped bodies did not express Na+-Cl- cotransporter, H+-ATPase, or aquaporin 2, while they were expressed in immature CNT of the capillary-loop stage nephrons throughout the kidney development. Consequently, the cells at the initial connection exhibit the morphological features suggestive of energy demanding, protein producing, and intercellular communicating. The cell morphology together with transporter development indicates that these cells serve several functions during the development of the initial connection, and that these functions are different from the cells' final functions as transportation.

Morphologic and morphometric study on microvasculature of developing mouse kidneys.

A proper morphogenesis of the renal microvasculature is crucial not only for fulfilling the renal function but also to slow down the progression of chronic kidney disease in adulthood. However, the current description of the developing microvasculature is incomplete. The present study investigated the morphogenesis and volume densities of the renal microvasculature using computer-assisted tubular tracing, immunohistochemistry for CD34, and unbiased stereology. The earliest glomerular capillaries were observed at the lower cleft of the S-shaped nephrons, as simple loops connecting the afferent and efferent arterioles. In parallel with this, the peritubular capillaries were established. Noticeably, from early nephrogenesis on, the efferent arterioles of the early-formed glomeruli ran in close proximity to their own thick ascending limbs. In addition, the ascending vasa recta arising from the arcuate or interlobular veins also ran in close proximity to the thick descending limb. Thus, the tubules and vessels formed the typical countercurrent relation in the medulla. No loop bends were observed between descending and ascending vasa recta. The volume density of the cortical and medullary peritubular capillary increased 3.3- and 2.6-fold, respectively, from 2.34 (0.13) and 7.03 (0.09)% [means (SD)] at embryonic day 14.5 (E14.5) to 7.71 (0.44) and 18.27 (1.17)% at postnatal day 40 (P40). In contrast, the volume density of glomeruli changed only slightly during kidney development, from 4.61 (0.47)% at E14.5 to 6.07 (0.2)% at P7 to 4.19 (0.47)% at P40. These results reflect that the growth and formation of the renal microvasculature closely correspond to functional development of the tubules.

Temporal deletion of Aqp11 in mice is linked to the severity of cyst-like disease.

Aquaporin 11 (AQP11) is a channel protein with unknown biological function that is expressed in multiple tissues, including the kidney proximal tubule (PT) epithelium. Constitutive deletion of Aqp11 in mice (Aqp11-/-) results in early postnatal vacuolization in the PT and development of apparent cysts at 2 wk of age. Electron microscopy of adult Aqp11-/- mouse PT cells revealed a dilated rough endoplasmic reticulum. These changes may cause renal failure and premature death. This study examined 1) whether postnatal deletion of Aqp11 affects PT injury and cyst formation, 2) the temporal role of Aqp11 deletion on cyst development, and 3) the nature of apparent cysts. Tamoxifen-inducible Aqp11-/- mice were generated (Ti-Aqp11-/-). Deletion of Aqp11 at postnatal days (P) P2, P4, P6, P8, and P12 was investigated. Deranged renal development, especially in kidney cortex, PT cell vacuolization, and apparent tubular cysts developed only in mice where Aqp11 gene disruption was induced until P8. Aqp11 gene deletion from P12 onward did not result in a clear deficiency in renal development, PT injury, or cyst formation. Intraperitoneal injection of biotinylated-dextran (10 kDa) into adult mice resulted in extensive endocytic dextran uptake in both cystic Aqp11-/- and control PT epithelium, respectively. This suggests that apparent cysts are not membrane-enclosed structures but represent PT dilations. We conclude that Aqp11-/- mice develop cyst-like dilated proximal tubules without documented cysts at time of death.

Nephron morphometry in mice and rats using tomographic microscopy.

The aim was to quantify the glomerular capillary surface area, the segmental tubular radius, length, and area of single nephrons in mouse and rat kidneys. Multiple 2.5-µm-thick serial Epon sections were obtained from three mouse and three rat kidneys for three-dimensional reconstruction of the nephron tubules. Micrographs were aligned for each kidney, and 359 nephrons were traced and their segments localized. Thirty mouse and thirty rat nephrons were selected for further investigation. The luminal radius of each segment was determined by two methods. The luminal surface area was estimated from the radius and length of each segment. High-resolution micrographs were recorded for five rat glomeruli, and the capillary surface area determined. The capillary volume and surface area were corrected for glomerular shrinkage. A positive correlation was found between glomerular capillary area and proximal tubule area. The thickest part of the nephron, i.e., the proximal tubule, was followed by the thinnest part of the nephron, i.e., the descending thin limb, and the diameters of the seven identified nephron segments share the same rank in the two species. The radius and length measurements from mouse and rat nephrons generally share the same pattern; rat tubular radius-to-mouse tubular radius ratio ≈ 1.47, and rat tubular length-to-mouse tubular length ratio ≈ 2.29, suggesting relatively longer tubules in the rat. The detailed tables of mouse and rat glomerular capillary area and segmental radius, length, and area values may be used to enhance understanding of the associated physiology, including existing steady-state models of the urine-concentrating mechanism.

Efficacy of Liraglutide for Weight Loss Among Patients With Type 2 Diabetes: The SCALE Diabetes Randomized Clinical Trial.

IMPORTANCE: Weight loss of 5% to 10% can improve type 2 diabetes and related comorbidities. Few safe, effective weight-management drugs are currently available. OBJECTIVE: To investigate efficacy and safety of liraglutide vs placebo for weight management in adults with overweight or obesity and type 2 diabetes. DESIGN, SETTING, AND PARTICIPANTS: Fifty-six-week randomized (2:1:1), double-blind, placebo-controlled, parallel-group trial with 12-week observational off-drug follow-up period. The study was conducted at 126 sites in 9 countries between June 2011 and January 2013. Of 1361 participants assessed for eligibility, 846 were randomized. Inclusion criteria were body mass index of 27.0 or greater, age 18 years or older, taking 0 to 3 oral hypoglycemic agents (metformin, thiazolidinedione, sulfonylurea) with stable body weight, and glycated hemoglobin level 7.0% to 10.0%. INTERVENTIONS: Once-daily, subcutaneous liraglutide (3.0 mg) (n = 423), liraglutide (1.8 mg) (n = 211), or placebo (n = 212), all as adjunct to 500 kcal/d dietary deficit and increased physical activity (≥150 min/wk). MAIN OUTCOMES AND MEASURES: Three coprimary end points: relative change in weight, proportion of participants losing 5% or more, or more than 10%, of baseline weight at week 56. RESULTS: Baseline weight was 105.7 kg with liraglutide (3.0-mg dose), 105.8 kg with liraglutide (1.8-mg dose), and 106.5 kg with placebo. Weight loss was 6.0% (6.4 kg) with liraglutide (3.0-mg dose), 4.7% (5.0 kg) with liraglutide (1.8-mg dose), and 2.0% (2.2 kg) with placebo (estimated difference for liraglutide [3.0 mg] vs placebo, -4.00% [95% CI, -5.10% to -2.90%]; liraglutide [1.8 mg] vs placebo, -2.71% [95% CI, -4.00% to -1.42%]; P < .001 for both). Weight loss of 5% or greater occurred in 54.3% with liraglutide (3.0 mg) and 40.4% with liraglutide (1.8 mg) vs 21.4% with placebo (estimated difference for liraglutide [3.0 mg] vs placebo, 32.9% [95% CI, 24.6% to 41.2%]; for liraglutide [1.8 mg] vs placebo, 19.0% [95% CI, 9.1% to 28.8%]; P < .001 for both). Weight loss greater than 10% occurred in 25.2% with liraglutide (3.0 mg) and 15.9% with liraglutide (1.8 mg) vs 6.7% with placebo (estimated difference for liraglutide [3.0 mg] vs placebo, 18.5% [95% CI, 12.7% to 24.4%], P < .001; for liraglutide [1.8 mg] vs placebo, 9.3% [95% CI, 2.7% to 15.8%], P = .006). More gastrointestinal disorders were reported with liraglutide (3.0 mg) vs liraglutide (1.8 mg) and placebo. No pancreatitis was reported. CONCLUSIONS AND RELEVANCE: Among overweight and obese participants with type 2 diabetes, use of subcutaneous liraglutide (3.0 mg) daily, compared with placebo, resulted in weight loss over 56 weeks. Further studies are needed to evaluate longer-term efficacy and safety. TRIAL REGISTRATION: clinicaltrials.gov Identifier:NCT01272232.

Towards Automated Three-Dimensional Tracking of Nephrons through Stacked Histological Image Sets.

An automated approach for tracking individual nephrons through three-dimensional histological image sets of mouse and rat kidneys is presented. In a previous study, the available images were tracked manually through the image sets in order to explore renal microarchitecture. The purpose of the current research is to reduce the time and effort required to manually trace nephrons by creating an automated, intelligent system as a standard tool for such datasets. The algorithm is robust enough to isolate closely packed nephrons and track their convoluted paths despite a number of nonideal, interfering conditions such as local image distortions, artefacts, and interstitial tissue interference. The system comprises image preprocessing, feature extraction, and a custom graph-based tracking algorithm, which is validated by a rule base and a machine learning algorithm. A study of a selection of automatically tracked nephrons, when compared with manual tracking, yields a 95% tracking accuracy for structures in the cortex, while those in the medulla have lower accuracy due to narrower diameter and higher density. Limited manual intervention is introduced to improve tracking, enabling full nephron paths to be obtained with an average of 17 manual corrections per mouse nephron and 58 manual corrections per rat nephron.

A reference-free method for brightness compensation and contrast enhancement of micrographs of serial sections.

Three-dimensional (3D) reconstruction of an organ or tissue from a stack of histologic serial sections provides valuable morphological information. The procedure includes section preparation of the organ or tissue, micrographs acquisition, image registration, 3D reconstruction, and visualization. However, the brightness and contrast through the image stack may not be consistent due to imperfections in the staining procedure, which may cause difficulties in micro-structure identification using virtual sections, region segmentation, automatic target tracing, etc. In the present study, a reference-free method, Sequential Histogram Fitting Algorithm (SHFA), is therefore developed for adjusting the severe and irregular variance of brightness and contrast within the image stack. To apply the SHFA, the gray value histograms of individual images are first calculated over the entire image stack and a set of landmark gray values are chosen. Then the histograms are transformed so that there are no abrupt changes in progressing through the stack. Finally, the pixel gray values of the original images are transformed into the desired ones based on the relationship between the original and the transformed histograms. The SHFA is tested on an image stacks from mouse kidney sections stained with toluidine blue, and captured by a slide scanner. As results, the images through the entire stack reveal homogenous brightness and consistent contrast. In addition, subtle color differences in the tissue are well preserved so that the morphological details can be recognized, even in virtual sections. In conclusion, compared with the existing histogram-based methods, the present study provides a practical method suitable for compensating brightness, and improving contrast of images derived from a large number of serial sections of biological organ.

Non-rigid landmark-based large-scale image registration in 3-D reconstruction of mouse and rat kidney nephrons.

BACKGROUND: Serial histological sections are suffering from mechanical distortions that disturb the reconstruction of 3-D objects. We have corrected such artifacts with a non-rigid landmark-based method that respects the original geometry in the tissue block. The method is exemplified on a large scale in the registration of semi-thin serial sections of the mouse and rat kidneys, and has been tested on FFPE-sections. AIM: In this study of mouse and rat kidneys, we have measured and characterized the deformations introduced in the preparation of 2.5-µm-thick Epon sections and then eliminated them by a landmark-based non-rigid transformation (NRT). METHODS: We obtained 2.5-µm-thick serial Epon sections from three mouse kidneys and three rat kidneys for 3-D reconstruction of the nephron tubules. First, the images from 3000 serial mouse and 13,000 serial rat sections underwent a classic rigid registration (CRR), and the distortions were measured and indexed. The section images underwent a further NRT in order to compensate for the deformations. The NRT used is a classic interactive landmark-based approach. The quality of the NRT was verified by comparing the geometry of the transformed images with corresponding block images. RESULTS: After CRR, the 2.5-µm-thick sections had a linear deformation of up to 2%, the tubular lengths were overestimated with up to 1.5×, and it was most difficult to trace the tubules from section to section. After the additional NRT, the geometry of the images reflected the original geometry in the block, the tubular lengths were no longer overestimated, and the NRT highly facilitated the tracing of the tubular system. CONCLUSIONS: NRT has facilitated the tracing of the tubular system in kidneys, a tracing, which would otherwise have been most difficult to perform. NRT has yielded substantial new knowledge to segmental and spatial nephron organization in the mouse and rat kidneys.

Management and interpretation of heterogeneous observational data: using insulin-like growth factor-I data from the NordiNet® International Outcome Study.

OBJECTIVE: The NordiNet® International Outcome Study (IOS), a large-scale, non-interventional, multi-centre, real-world study of Norditropin® treatment, registers insulin-like growth factor-I (IGF-I) values, as measured by different assays. This paper considers the potential biases introduced by using a single IGF-I reference data set in analysing NordiNet® IOS data. DESIGN: To evaluate possible biases from different IGF-I assays used across NordiNet® IOS, a mixed-effect linear model was fitted to IGF-I data (analyses on log-transformed data). Pre-growth hormone treatment (pre-GHT) IGF-I values were assumed to depend on diagnosis, sex and age. During GHT, a treatment-effect dependent on these factors was added. Differences between assays were assumed multiplicative on the original scale. Individual measurements were scaled to a common level (Nichols Advantage) giving adjusted IGF-I standard deviation score (SDS) values. RESULTS: In total, 49 495 IGF-I measurements were available from 9481 paediatric patients. Mixed-effect linear modelling showed a systematic difference between IGF-I levels measured by different assays. Differences were minimised when assessing change in IGF-I SDS from the start of GHT to 1-year follow-up. This applied to values adjusted for actual-assay used and for unadjusted delta IGF-I SDS values. Largest differences between unadjusted change in IGF-I SDS values were: for growth hormone deficiency 0.1 (girls) and 0.3 (boys); for small-for-gestational age 0.1; and for Turner syndrome 0.2. Similar magnitude differences were seen for data with unknown assay. CONCLUSIONS: Analysis and modelling suggest the current approach to IGF-I data collection and analyses in the NordiNet® IOS is sound: in a large cohort without assay-used information, potential bias is minimised by analysing changes in IGF-I SDS.

Three-dimensional reconstruction of the rat nephron.

This study gives a three-dimensional (3D) structural analysis of rat nephrons and their connections to collecting ducts. Approximately 4,500 2.5-µm-thick serial sections from the renal surface to the papillary tip were obtained from each of 3 kidneys of Wistar rats. Digital images were recorded and aligned into three image stacks and traced from image to image. Short-loop nephrons (SLNs), long-loop nephrons (LLNs), and collecting ducts (CDs) were reconstructed in 3D. We identified a well-defined boundary between the outer stripe and the inner stripe of the outer medulla corresponding to the transition of descending thick limbs to descending thin limbs and between the inner stripe and the inner medulla, i.e., the transition of ascending thin limbs into ascending thick limbs of LLNs. In all nephrons, a mosaic pattern of proximal tubule (PT) cells and descending thin limb (DTL) cells was observed at the transition between the PT and the DTL. The course of the LLNs revealed tortuous proximal "straight" tubules and winding of the DTLs within the outer half of the inner stripe. The localization of loop bends of SLNs in the inner stripe of the outer medulla and the bends of LLNs in the inner medulla reflected the localization of their glomeruli; i.e., the deeper the glomerulus, the deeper the bend. Each CD drained approximately three to six nephrons with a different pattern than previously established in mice. This information will provide a basis for evaluation of structural changes within nephrons as a result of physiological or pharmaceutical intervention.

Spatial organization of the vascular bundle and the interbundle region: three-dimensional reconstruction at the inner stripe of the outer medulla in the mouse kidney.

The vascular bundle (VB) is a complex structure that resides in the inner stripe of the outer medulla. At present, the tubulovascular spatial organization of the VB, which is crucial for the formation of the osmolarity gradient and for solute transport, is still under debate. In this study, we used computer-assisted digital tracing combined with aquaporin-1 immunohistochemistry to reconstruct all tubules and vessels in the VB of the mouse kidney. We found, first, that the descending and ascending vasa recta travelled exclusively through the VB. The ascending vasa recta received no tributaries (no branches) along their entire path in the medulla and were not connected with the capillary plexus in the interbundle region. Second, a specific group of the descending vasa recta were closely accompanied by the longest ascending vasa recta, which connected only to the capillary plexus at the tip of the papilla. Third, the descending thin limbs of all short-looped nephrons travelled exclusively through the outer part of the VB. The loops of these nephrons (both descending and ascending parts) were distributed in a regular pattern based on their length. Finally, the thick ascending limbs of all long-looped nephrons were located at the margin of the VB (except a few within the VB), which formed a layer separating the VB from the interbundle region. In conclusion, our three-dimensional analysis of the VB strongly suggest a lateral osmolarity heterogeneity across the inner stripe of the outer medulla, which might work as a driving force for water and solute transport.

Direct physical contact between intercalated cells in the distal convoluted tubule and the afferent arteriole in mouse kidneys.

Recent physiological studies in the kidney proposed the existence of a secondary feedback mechanism termed 'crosstalk' localized after the macula densa. This newly discovered crosstalk contact between the nephron tubule and its own afferent arteriole may potentially revolutionize our understanding of renal vascular resistance and electrolyte regulation. However, the nature of such a crosstalk mechanism is still debated due to a lack of direct and comprehensive morphological evidence. Its exact location along the nephron, its prevalence among the different types of nephrons, and the type of cells involved are yet unknown. To address these issues, computer assisted 3-dimensional nephron tracing was applied in combination with direct immunohistochemistry on plastic sections and electron microscopy. 'Random' contacts in the cortex were identified by the tracing and excluded. We investigated a total of 168 nephrons from all cortical regions. The results demonstrated that the crosstalk contact existed, and that it was only present in certain nephrons (90% of the short-looped and 75% of the long-looped nephrons). The crosstalk contacts always occurred at a specific position--the last 10% of the distal convoluted tubule. Importantly, we demonstrated, for the first time, that the cells found in the tubule wall at the contact site were always type nonA-nonB intercalated cells. In conclusion, the present work confirmed the existence of a post macula densa physical crosstalk contact.

Interactive 3D computer model of the human corneolimbal region: crypts, projections and stem cells.

PURPOSE: This study aims to clarify the existence of and to map the localization of different proposed stem cell niches in the corneal limbal region. MATERIALS AND METHODS: One human eye was cut into 2200 consecutive sections. Every other section was stained with haematoxylin and eosin, digitized at low and high magnification, aligned, 3D reconstructed and visualized using interactive 3D visualization software. The visualization software has interactive tools that make free rotations in all directions possible and makes it possible to create virtual sections independent of the original cutting plan. In all, one low-magnification and 24 high-magnification interactive 3D models were created. Immunohistochemistry against stem cell markers p63 and ΔNp63α was performed as a supplement to the 3D models. RESULTS: Using the interactive 3D models, we identified three types of stem cell niches in the limbal region: limbal epithelial crypts (LECs), limbal crypts (LCs) and focal stromal projections (FSPs). In all, eight LECs, 25 LCs and 105 FSPs were identified in the limbal region. The LECs, LCs and FSPs were predominantly located in the superior limbal region with seven LECs, 19 LCs and 93 FSPs in the superior limbal region and one LEC, six LCs and 12 FSPs in the inferior limbal region. Only few LECs, LCs and FSPs were localized nasally and temporally. CONCLUSION: Interactive 3D models are a powerful tool that may help to shed more light on the existence and spatial localization of the different stem cell niches (LECs, LCs and FSPs) in the corneal limbal region.

Expression of Bcl-2 and Bax in mouse renal tubules during kidney development.

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated.

Supersampling in PET. A concept for improvement of the resolution of positron emission tomography.

At present, the maximum spatial resolution of state-of-the-art PET scanners is not better than 5 mm, whereas the maximum resolution obtained in the resulting images is approximately 10 mm. At least some of this difference is a result of an inherent, complex noise component that can be eliminated using "supersampling". The aim of the study was to apply the supersampling technique to PET image recordings to lower the noise and to improve the in-plane resolution of the recorded images. A standardized phantom was investigated with the supersampling technique. In addition to the standard CT and PET series, 14 further PET series were recorded with the phantom at exactly the same position, and 4 further series were recorded with the phantom placed in another well defined position. After merging corresponding images, the image noise index and the image resolution are both substantially improved. The suggested PET image recording and processing technique provides a substantial increase in PET image quality without exposing the patient to any additional radiation, and might therefore become a powerful tool for investigative as well as diagnostic purposes.

Aquaporin-1 is not expressed in descending thin limbs of short-loop nephrons.

In mammalian kidneys, aquaporin-1 is responsible for water reabsorption along the proximal tubule and is also thought to be involved in the concentration of urine that occurs in the medulla. It has been suggested, however, that aquaporin-1 is not expressed in the last part of the descending thin limbs of short loop nephrons in rats and mice, and its expression in this region in humans has not been studied. We examined the expression of aquaporin-1 and the urea transporter UT-A2 in serial sections of mouse nephrons in the inner stripe of the outer medulla using immunohistochemistry. In contrast to previous observations, we demonstrate a complete absence of aquaporin-1 along the entire length of descending thin limbs of 90% of short loop nephrons. Conversely, as expected, we identified aquaporin-1 in proximal tubules, descending thin limbs of long loop nephrons, and medullary descending vasa recta. We also observed this abrupt transition from aquaporin-1-positive proximal tubules to aquaporin-1-negative descending thin limbs of short loop nephrons in sections of human and rat kidneys. UT-A2 was restricted to the last 28% to 44% of the descending thin limbs of all short loop nephrons. Because the majority of nephrons are of the short loop variety, our findings suggest that the mechanisms of water transport in the descending thin limbs of short loop nephrons should be reevaluated. Likewise, the roles of aquaporin-1 and UT-A2 in the countercurrent multiplier and water conversation may need to be readdressed.

Three-dimensional reconstruction of the mouse nephron.

Renal function is crucially dependent on renal microstructure which provides the basis for the regulatory mechanisms that control the transport of water and solutes between filtrate and plasma and the urinary concentration. This study provides new, detailed information on mouse renal architecture, including the spatial course of the tubules, lengths of different segments of nephrons, histotopography of tubules and vascular bundles, and epithelial ultrastructure at well-defined positions along Henle's loop and the distal convolution of nephrons. Three-dimensional reconstruction of 200 nephrons and collecting ducts was performed on aligned digital images, obtained from 2.5-mum-thick serial sections of mouse kidneys. Important new findings were highlighted: (1) A tortuous course of the descending thin limbs of long-looped nephrons and a winding course of the thick ascending limbs of short-looped nephrons contributed to a 27% average increase in the lengths of the corresponding segments, (2) the thick-walled tubules incorporated in the central part of the vascular bundles in the inner stripe of the outer medulla were identified as thick ascending limbs of long-looped nephrons, and (3) three types of short-looped nephron bends were identified to relate to the length and the position of the nephron and its corresponding glomerulus. The ultrastructure of the tubule segments was identified and suggests important implications for renal transport mechanisms that should be considered when evaluating the segmental distribution of water and solute transporters within the normal and diseased kidney.