Macrophages and fibroblasts in foreign body reactions: How mechanical cues drive cell functions?

Biomaterials, when implanted in the human body, can induce a series of cell- and cytokine-related reactions termed foreign body reactions (FBRs). In the progression of FBRs, macrophages regulate inflammation and healing by polarizing to either a pro-inflammatory or pro-healing phenotype and recruit fibroblasts by secreting cytokines. Stimulated by the biomaterials, fibrotic capsule is formed eventually. The implant, along with its newly formed capsule, introduces various mechanical cues that influence cellular functions. Mechanosensing proteins, such as integrins or ion channels, transduce extracellular mechanical signals into cytoplasm biochemical signals in response to mechanical stimuli. Consequently, the morphology, migration mode, function, and polarization state of the cells are affected. Modulated by different intracellular signaling pathways and their crosstalk, the expression of fibrotic genes increases with fibroblast activation and fibroblast to myofibroblast transition under stiff or force stimuli. However, summarized in most current studies, the outcomes of macrophage polarization in the effect of different mechanical cues are inconsistent. The underlying mechanisms should be investigated with more advanced technology and considering more interfering aspects. Further research is needed to determine how to modulate the progression of fibrotic capsule formation in FBR artificially.

The Role of Autophagy in Lamellar Body Formation and Surfactant Production in Type 2 Alveolar Epithelial Cells.

The lamellar body (LB), a concentric structure loaded with surfactant proteins and phospholipids, is an organelle specific to type 2 alveolar epithelial cells (AT2). However, the origin of LBs has not been fully elucidated. We have previously reported that autophagy regulates Weibel-Palade bodies (WPBs) formation, and here we demonstrated that autophagy is involved in LB maturation, another lysosome-related organelle. We found that during development, LBs were transformed from autophagic vacuoles containing cytoplasmic contents such as glycogen. Fusion between LBs and autophagosomes was observed in wild-type neonate mice. Moreover, the markers of autophagic activity, microtubule-associated protein 1 light chain 3B (LC3B), largely co-localized on the limiting membrane of the LB. Both autophagy-related gene 7 (Atg7) global knockout and conditional Atg7 knockdown in AT2 cells in mice led to defects in LB maturation and surfactant protein B production. Additionally, changes in autophagic activity altered LB formation and surfactant protein B production. Taken together, these results suggest that autophagy plays a critical role in the regulation of LB formation during development and the maintenance of LB homeostasis during adulthood.

Involvement of YTHDF1 in renal fibrosis progression via up-regulating YAP.

Renal fibrosis is a progressive, fatal renal disease characterized by the aberrant accumulation of myofibroblasts that produce excess extracellular matrix (ECM) in the renal interstitium and glomeruli. Yes-associated protein (YAP) has been regarded as a crucial modulator in myofibroblast transformation, but its upstream regulator remains a mystery. In the present study investigating the participation of m6A methylation during renal fibrosis through bioinformatics analysis, we identified YTHDF1, a modulator of m6A methylation, as a key contributor for renal fibrosis because it was highly expressed in human fibrotic kidneys and had a significant correction with YAP. Their co-localization in human fibrotic kidneys was additionally shown by immunofluorescence. We then found that YTHDF1 was also up-regulated in fibrotic mouse kidneys induced by unilateral ureteral obstruction (UUO), high-dose folic acid administration, or the unilateral ischemia-reperfusion injury, further supporting a causal role of YTHDF1 during renal fibrosis. Consistent with this notion, YTHDF1 knockdown alleviated the progression of renal fibrosis both in cultured cells induced by transforming growth factor-beta administration and in the UUO mouse model. Meanwhile, YAP was accordingly down-regulated when YTHDF1 was inhibited. Furthermore, the specific binding of YTHDF1 to YAP mRNA was detected using RNA Binding Protein Immunoprecipitation, and the up-regulation of fibrotic related molecules in cultured cells induced by YTHDF1 over-expression plasmid was attenuated by YAP siRNA. Taken together, our data highlight the potential utility of YTHDF1 as an indicator for renal fibrosis and suggest that YTHDF1 inhibition might be a promising therapeutic strategy to alleviate renal fibrosis via downregulating YAP.

Targeting Mechanosensitive Piezo1 Alleviated Renal Fibrosis Through p38MAPK-YAP Pathway.

Renal fibrosis is the most common pathological manifestation of a wide variety of chronic kidney disease. Increased extracellular matrix (ECM) secretion and enhanced microenvironment stiffening aggravate the progression of renal fibrosis. However, the related mechanisms remain unclear. Here, we evaluated the mechanism by which ECM stiffness aggravates renal fibrosis. In the present study, renal mesangial cells (MCs) were cultured on polyacrylamide hydrogels with different stiffness accurately detected by atomic force microscope (AFM), simulating the in vivo growth microenvironment of MCs in normal kidney and renal fibrosis. A series of in vitro knockdown and activation experiments were performed to establish the signaling pathway responsible for mechanics-induced MCs activation. In addition, an animal model of renal fibrosis was established in mice induced by unilateral ureteral obstruction (UUO). Lentiviral particles containing short hairpin RNA (sh RNA) targeting Piezo1 were used to explore the effect of Piezo1 knockdown on matrix stiffness-induced MCs activation and UUO-induced renal fibrosis. An in vitro experiment demonstrated that elevated ECM stiffness triggered the activation of Piezo1, which increased YAP nuclear translocation through the p38MAPK, and consequently led to increased ECM secretion. Furthermore, these consequences have been verified in the animal model of renal fibrosis induced by UUO and Piezo1 knockdown could alleviate UUO-induced fibrosis and improve renal function in vivo. Collectively, our results for the first time demonstrate enhanced matrix stiffness aggravates the progression of renal fibrosis through the Piezo1-p38MAPK-YAP pathway. Targeting mechanosensitive Piezo1 might be a potential therapeutic strategy for delaying the progression of renal fibrosis.

A-Lipoic Acid Alleviates Folic Acid-Induced Renal Damage Through Inhibition of Ferroptosis.

Folic acid (FA)-induced acute kidney injury (AKI) is characterized by the disturbance of redox homeostasis, resulting in massive tubular necrosis and inflammation. Α-lipoic acid (LA), as an antioxidant, has been reported to play an important role in renal protection, but the underlying mechanism remains poorly explored. The aim of this study is to investigate the protective effect of LA on FA-induced renal damage. Our findings showed that LA could ameliorate renal dysfunction and histopathologic damage induced by FA overdose injection. Moreover, FA injection induced severe inflammation, indicated by increased release of pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-1ß, as well as infiltration of macrophage, which can be alleviated by LA supplementation. In addition, LA not only reduced the cellular iron overload by upregulating the expressions of Ferritin and ferroportin (FPN), but also mitigated reactive oxygen species (ROS) accumulation and lipid peroxidation by increasing the levels of antioxidant glutathione (GSH) and glutathione peroxidase-4 (GPX4). More importantly, we found that LA supplementation could reduce the number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive tubular cells caused by FA, indicating that the tubular cell death mediated by ferroptosis may be inhibited. Further study demonstrated that LA supplementation could reverse the decreased expression of cystine/glutamate antiporter xCT (SLC7A11), which mediated GSH synthesis. What is more, mechanistic study indicated that p53 activation was involved in the inhibitory effect of SLC7A11 induced by FA administration, which could be suppressed by LA supplementation. Taken together, our findings indicated that LA played the protective effect on FA-induced renal damage mainly by inhibiting ferroptosis.

Automatic consecutive context perceived transformer GAN for serial sectioning image blind inpainting.

BACKGROUND AND OBJECTIVE: Serial sectioning is the routine method in histology study. In order to restore the defective images in section stack, and overcome the limitations of manual annotation of broken areas, we developed a fully automatic approach for locating and restoring the defective image stack. METHODS: We proposed a novel end-to-end framework named automatic consecutive context perceived transformer GAN (ACCP-GAN) for fully automatic serial sectioning image blind inpainting. The first stage network (auto-detection module) was designed to detect the broken areas and repair them roughly, then guided the second stage network (refined inpainting module) to generate these expected patches precisely; therefore, the segmentation part was integrated into restoring part. The transformer module (SPTransformer), based on self-attention mechanism, was introduced to make the refined inpainting module focus on the features from neighboring images to help in correcting inpainting results. Moreover, gated convolution was largely used to extract features from normal parts in the defective image. The framework was trained and validated on the N7 dataset (803 images), and the generalization ability of the model was tested on the E17 (701 images) and N5 (413 images) datasets, all of these images were collected for previous kidney study. RESULTS: N7 dataset was divided into training, validation, and test sets with a ratio of 6:2:2. Our model performed well in broken areas segmentation with the accuracy = 0.9995. The final restoration got the best performance with FSIM = 0.9478, MS-SSIM = 0.9592, PSNR = 29.7903, VIF = 0.8543, and FID = 47.2252 compared to the popular inpainting methods. The model was further tested on E15 and N5 datasets, and the generalization ability was satisfying. CONCLUSIONS: Our method could detect and restore the defective serial sectioning image stack automatically, even the broken patches were large on an individual image. The newly designed SPTransformer performed well in feature extraction. This method reduced the workload of manual annotation and improved the analysis or application of large scale sectioning image stack in histology research.

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.

Ibudilast Attenuates Folic Acid-Induced Acute Kidney Injury by Blocking Pyroptosis Through TLR4-Mediated NF-κB and MAPK Signaling Pathways.

Folic acid (FA)-induced renal tubule damage, which is characterized by extensive inflammation, is a common model of acute kidney injury (AKI). Pyroptosis, a pro-inflammatory form of cell death due to the activation of inflammatory caspases, is involved in AKI progression. Ibudilast, a TLR4 antagonist, has been used in the clinic to exert an anti-inflammatory effect on asthma. However, researchers have not explored whether ibudilast exerts a protective effect on AKI by inhibiting inflammation. In the present study, ibudilast reversed FA-induced AKI in mice, as indicated by the reduced serum creatinine and urea nitrogen levels, and improved renal pathology, as well as the downregulation of kidney injury marker-1. In addition, ibudilast significantly increased the production of the anti-inflammatory factor IL-10 while suppressing the secretion of the pro-inflammatory cytokine TNF-α and macrophage infiltration. Moreover, in the injured kidney, ibudilast reduced the levels of both inflammasome markers (NLRP3) and pyroptosis-related proteins (caspase-1, IL1-ß, IL-18, and GSDMD cleavage), and decreased the number of TUNEL-positive cells. Further mechanistic studies showed that ibudilast administration inhibited the FA-induced upregulation of TLR4, blocked NF-κB nuclear translocation, and reduced the phosphorylation of NF-κB and IκBα, p38, ERK, and JNK. Thus, this study substantiates the protective effect of ibudilast on FA-induced AKI in mice and suggests that protection might be achieved by reducing pyroptosis and inflammation, likely through the inhibition of TLR4-mediated NF-κB and MAPK signaling pathways.

Role of tryptophan metabolism in cancers and therapeutic implications.

Tryptophan (Trp) metabolism is associated with diverse biological processes, including nerve conduction, inflammation, and the immune response. The majority of free Trp is broken down through the kynurenine (Kyn) pathway (KP), in which indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO) catalyze the rate-limiting step. Clinical studies have demonstrated that Trp metabolism promotes tumor progression due to modulation of the immunosuppressive microenvironment through multiple mechanisms. In this process, IDO-expressing dendritic cells (DCs) exhibit tolerogenic potential and orchestrate T cell immune responses. Various signaling molecules control IDO expression, initiating the immunoregulatory pathway of Trp catabolism. Based on these characteristics, KP enzymes and catabolites are emerging as significant prognostic indicators and potential therapeutic targets of cancer. The physiological and oncologic roles of Trp metabolism are briefly summarized here, along with great challenges for treatment strategies.

Roxadustat (FG-4592) Facilitates Recovery From Renal Damage by Ameliorating Mitochondrial Dysfunction Induced by Folic Acid.

Incomplete recovery from acute kidney injury induced by folic acid is a major risk factor for progression to chronic kidney disease. Mitochondrial dysfunction has been considered a crucial contributor to maladaptive repair in acute kidney injury. Treatment with FG-4592, an inhibitor of hypoxia inducible factor prolyl-hydroxylase, is emerging as a new approach to attenuate renal damage; however, the underlying mechanism has not been fully elucidated. The current research demonstrated the protective effect of FG-4592 against renal dysfunction and histopathological damage on the 7th day after FA administration. FG-4592 accelerated tubular repair by promoting tubular cell regeneration, as indicated by increased proliferation of cell nuclear antigen-positive tubular cells, and facilitated structural integrity, as reflected by up-regulation of the epithelial inter-cellular tight junction molecule occludin-1 and the adherens junction molecule E-cadherin. Furthermore, FG-4592 ameliorated tubular functional recovery by restoring the function-related proteins aquaporin1, aquaporin2, and sodium chloride cotransporter. Specifically, FG-4592 pretreatment inhibited hypoxia inducible factor-1α activation on the 7th day after folic acid injection, which ameliorated ultrastructural abnormalities, promoted ATP production, and attenuated excessive reactive oxygen species production both in renal tissue and mitochondria. This was mainly mediated by balancing of mitochondrial dynamics, as indicated by down-regulation of mitochondrial fission 1 and dynamin-related protein 1 as well as up-regulation of mitofusin 1 and optic atrophy 1. Moreover, FG-4592 pretreatment attenuated renal tubular epithelial cell death, kidney inflammation, and subsequent interstitial fibrosis. In vitro, TNF-α-induced HK-2 cells injury could be ameliorated by FG-4592 pretreatment. In summary, our findings support the protective effect of FG-4592 against folic acid-induced mitochondrial dysfunction; therefore, FG-4592 treatment can be used as a useful strategy to facilitate tubular repair and mitigate acute kidney injury progression.

Kallistatin correlates with inflammation in abdominal aortic aneurysm and suppresses its formation in mice.

BACKGROUND: Kallistatin (KS), encoded by SERPINA4, was suggested to play a protective role in many cardiovascular diseases. However, its role in the pathogenesis of abdominal aortic aneurysm (AAA) remains unclear. The aim of this study was to examine the potential association of KS with AAA pathogenesis. METHODS: We examined KS (SERPINA4) expression in human AAA by PCR, immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay (ELISA) and analyzed correlations between kallistain and clinical data. We then analyzed the effect of recombinant KS on AAA formation and the Wingless (Wnt) signaling pathway in a mouse AAA model developed by angiotensin II (AngII) infusion to apolipoprotein E-deficient (ApoE-/-) mice. RESULTS: In AAA tissue samples, KS was significantly increased compared with samples from the control group (P<0.001, P<0.001, respectively). Clinically, decreased SERPINA4 expression in AAA tissue samples represented an increased rate of iliac artery aneurysm [odds ratio (OR): 0.017; P=0.040]. And decreased plasma KS level represented a high risk for rupture (OR: 0.837; P=0.034). KS inhibited AAA formation and blocked the Wnt signaling pathway in AngII-infused ApoE-/- mice. CONCLUSIONS: The present study demonstrates that aberrant changes in KS expression occur in AAA. KS plays an important anti-inflammatory role and showed important clinical correlations in AAA. Decreased KS (SERPINA4) level is a risk factor of AAA rupture. Our pre-clinical animal experiments indicate that treatment with recombination KS suppresses AngII-induced aortic aneurysm formation and might be a new target for the drug therapy of AAA.

Pretreatment with Roxadustat (FG-4592) Attenuates Folic Acid-Induced Kidney Injury through Antiferroptosis via Akt/GSK-3ß/Nrf2 Pathway.

Folic acid- (FA-) induced kidney injury is characterized by the tubule damage due to the disturbance of the antioxidant system and subsequent interstitial fibrosis. FG-4592 is an inhibitor of prolyl hydroxylase of hypoxia-inducible factor (HIF), an antioxidant factor. The present study investigated the protective role of FG-4592 pretreatment at the early stage of the kidney injury and long-term impact on the progression of renal fibrosis. FG-4592 was administrated two days before FA injection in mice. On the second day after FA injection, the mice with FG-4592 pretreatment showed an improved renal function, compared with those without FG-4592 pretreatment, indicated by biochemical and histological parameters; meanwhile, the cellular content of iron, malondialdehyde, and 4-hydroxynonenal histologically decreased, implying the suppression of iron accumulation and lipid peroxidation. Simultaneously, upregulation of HIF-1α was found, along with Nrf2 activation, which was reflected by increased nuclear translocation and high-expression of downstream proteins, including heme-oxygenase1, glutathione peroxidase4, and cystine/glutamate transporter, as well as ferroportin. Correspondingly, the elevated levels of antioxidative enzymes and glutathione, as well as reduced iron accumulation, were observed, suggesting a lower risk of occurrence of ferroptosis with FG-4592 pretreatment. This was confirmed by reversed pathological parameters and improved renal function in FA-treated mice with the administration of ferrostatin-1, a specific ferroptosis inhibitor. Furthermore, a signal pathway study indicated that Nrf2 activation was associated with increased phosphorylation of Akt and GSK-3ß, verified by the use of an inhibitor of the PI3K that phosphorylates Akt. Moreover, FG-4592 pretreatment also decreased macrophage infiltration and expression of inflammatory factors TNF-α and IL-1ß. On the 14th day after FA injection, FG-4592 pretreatment decreased collagen deposition and expression of fibrosis biomarkers. These findings suggest that the protective role of FG-4592 pretreatment is achieved mainly by decreasing ferroptosis at the early stage of FA-induced kidney injury via Akt/GSK-3ß-mediated Nrf2 activation, which retards the fibrosis progression.

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.

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.

A microwave antigen retrieval method using two heating steps for enhanced immunostaining on aldehyde-fixed paraffin-embedded tissue sections.

Antigen retrieval is an immunohistochemical procedure that results in better exposure of target antigens in aldehyde-fixed, paraffin-embedded tissue sections to antibodies. However, the commercially recommended or conventional protocols for antigen retrieval do not always succeed in expressing the target antigen. Here, an improved method was developed for antigen retrieval from aldehyde-fixed, paraffin-embedded histological sections. Proliferating cell nuclear antigen (PCNA), tight junction proteins Claudin-2 and Claudin-7, and water channel aquaporins in kidney tissue were selected as test antigens. Typically, PCNA and Claudin-2 and Claudin-7 show negative, weak, or nonspecific immunoreactions with conventional antigen retrieval methods using microwave heating. In the present study, microwave heating was performed twice with an interval of 30 min between the two steps to allow the buffer solution to cool. Sodium citrate buffer (10 mM sodium citrate, pH 6.0) was used for PCNA, and Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 9.0) was used for the Claudins. Compared with conventionally prepared tissues, the tissues exhibited both enhanced and specific immunostaining, and well-preserved morphology. In conclusion, the conventional protocol could be supplemented with a second microwave heating step to improve the expression of antigens that do not respond well to the conventional method.

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.

Down-regulation of integrin ß1 and focal adhesion kinase in renal glomeruli under various hemodynamic conditions.

Given that integrin ß1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK) is the most essential intracellular integrator in the integrin ß1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possible interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT) and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin ß1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin ß1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin ß1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin ß1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases.