Increased expression of dendrin in the dorsal horn of the spinal cord during stress is regulated by sex hormones.

Chronic stress has various effects on organisms and is sex-specific. The aim of the study was to describe the expression of synapse strengthening protein, dendrin, in the spinal cord (SC) and the dependence of its expression on chronic stress and sex hormones. Thirteen-month-old female and male rats were castrated (ovariectomy [F-OVX] or orchidectomy [M-ORX]) or sham-operated (F-SH or M-SH), respectively. At age 15 months, three 10-day-sessions of sham stress (control, C) or chronic stress (S) were conducted. Dendrin expression was present in the thoracic SC segments and the dorsal root ganglia (DRG). In the SC, dendrin expression was prominent in superficial laminae of the dorsal horn and lamina X (central canal). The M-ORX-S group had the highest dendrin expression in the dorsal horn, being significantly higher than the M-ORX-C or M-SH-S groups (P < 0.05). Dendrin expression was significantly higher in the F-SH-S group than the F-SH-C group (P < 0.05), as well as in the F-SH-S than the M-SH-S (P < 0.05). Co-localization with the α-d-galactosyl-specific isolectin B4 (IB4) in central projections of the DRG neurons in the dorsal horn of the SC was 7.43 ± 3.36%, while with the calcitonin gene-related peptide (CGRP) was 8.47 ± 4.45%. Localization of dendrin was observed in soma and nuclei of neurons in the dorsal horn. Dendrin expression in pain-processing areas of the SC, the DRG neurons and their peripheral projections suggest possible roles in pain perception and modulation. Stress-induced increase in dendrin expression and its dependence on sex hormones may partially explain sex-specific pain hypersensitivity induced by stress.

Ultrastructural characterization of vitamin D receptors and metabolizing enzymes in the lipid droplets of the fatty liver in rat.

Vitamin D is a steroid hormone with numerous actions in the organism. There are strong evidences that relate vitamin D deficiency with liver lipid metabolism disturbances, but the mechanism of this action is still unknown. In our previous work we postulated the localization and accumulation of vitamin D receptor (VDR) in membrane of the lipid droplets (LDs) in hepatocytes. In this study, we applied the transmission electron microscopy (TEM) to confirm this hypothesis by using a long-term (6 months) high sucrose intake rat model that was previously found to be appropriate for research of the hepatic lipid accumulation. In addition to the VDR, we also found key vitamin D metabolizing enzymes, 1α-hydroxylase and CYP 24 associated with the membrane of the LDs. A light-microscopy data revealed significant increase in expression of VDR and CYP 24 in liver of high-sucrose treated rats, in comparison to controlones. According to the best of our knowledge, this is a first study confirming the presence of the VDR in the membrane of the LDs in general and also in particular in LDs of the hepatocytes that were accumulated as a consequence of the prolonged high sucrose intake. Moreover, we found association of main vitamin D metabolizing enzymes with LD membrane. These results provide a new insight in the possible relation of vitamin D signalling system with LD morphology and function and with the lipid metabolism in general.

Dietary DHA/EPA supplementation ameliorates diabetic nephropathy by protecting from distal tubular cell damage.

The aim was to explore the influence of experimental diabetes mellitus type 1 (DM1) and potential protective/deleterious effects of different dietary n-6/n-3 PUFA ratios on renal phospholipid composition and pathological changes caused by DM1. Male Wistar rats were injected with 55 mg/kg streptozotocin or citrate buffer (control group). Control (C) and diabetic groups (STZ) were fed with n-6/n-3 ratio of ≈ 7, STZ + N6 with n-6/n-3 ratio ≈ 60 and STZ + DHA with n-6/n-3 ratio of ≈ 1 containing 16% EPA and 19% DHA. Tissues were harvested 30 days after DM1 induction. Blood and kidneys were collected and analysed for phospholipid fatty acid composition, pathohystological changes, ectopic lipid accumulation and expression of VEGF, NF-kB and special AT-rich sequence-binding protein-1 (SATB1). Pathological changes were studied also by using transmission electron microscopy, after immunostaining for VEGF. Substantial changes in renal phospholipid fatty acid composition resulted from DM1 and dietary PUFA manipulation. Extensive vacuolization of distal tubular cells (DTCs) was found in DM1, but it was attenuated in the STZ + DHA group, in which the highest renal NF-kB expression was observed. The ectopic lipid accumulation was observed in proximal tubular cells (PTCs) of all diabetic animals, but it was worsened in the STZ + N6 group. In DM1, we found disturbance of VEGF-transporting vesicular PTCs system, which was substantially worsened in STZ + DHA and STZ + N6. Results have shown that the early phase of DN is characterized with extent damage and vacuolization of DTCs, which could be attenuated by DHA/EPA supplementation. We concluded that dietary fatty acid composition can strongly influence the outcomes of DN.

Immunohistochemical expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the normal human kidney development.

AIM: Present study analyses the co-localisation of RIP5 with FGFR1, FGFR2 and HIP2 in the developing kidney, as RIP5 is a major determinant of urinary tract development, downstream of FGF-signaling. METHODS: Paraffin embedded human kidney tissues of 16 conceptuses between the 6th-22th developmental week were analysed using double-immunofluorescence method with RIP5/FGFR1/FGFR2 and HIP2 markers. Quantification of positive cells were performed using Kruskal-Wallis test. RESULTS: In the 6th week of kidney development RIP5 (89.6%) and HIP2 (39.6%) are strongly expressed in the metanephric mesenchyme. FGFR1 shows moderate/strong expression in the developing nephrons (87.3%) and collecting ducts (70.5%) (p < 0.05). RIP5/FGFR1 co-localized at the marginal zone and the ureteric bud with predominant FGFR1 expression. FGFR2 (26.1%) shows similar expression pattern as FGFR1 (70.5%) in the same kidney structures. RIP5/FGFR2 co-localized at the marginal zone and the collecting ducts (predominant expression of FGFR2). HIP2 is strongly expressed in collecting ducts (96.7%), and co-localized with RIP5. In 10th week, RIP5 expression decrease (74.2%), while the pattern of expression of RIP5 and FGFR1 in collecting ducts (33.4% and 91.9%) and developing nephrons (21.9% and 32.4%) (p < 0.05) is similar to that in the 6th developmental week. Ureter is moderately expressing RIP5 while FGFR1 is strongly expressed in the ureteric wall. FGFR2 is strongly expressed in the collecting ducts (84.3%) and ureter. HIP2 have 81.1% positive cells in the collecting duct. RIP5/FGFR1 co-localize in collecting ducts and Henley's loop. CONCLUSIONS: The expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the human kidney development might indicate their important roles in metanephric development and ureteric muscle layer differentiation through FGF signaling pathways.

Time course and expression pattern of the neuronal markers in the developing human spinal cord.

The aim of this study was to examine the spatio-temporal appearance of different neuronal cell subtypes by analyzing expression patterns of several neuronal markers (calretinin, neurofilament 200 (NF200), vanilloid receptor 1(VR1) and calcitonin gene-related peptide (CGRP)) of the embryonic human spinal cord (SC). Developing human SCs from 11 human conceptuses beetwen 5-10 developmental weeks (DW) were examined by light and electron microscopy and immunofluorescence. Light and electron microscopy revealed different embryonic stages of recognizable structure of the SC. NF200, CGRP and VR1 positive cells were observed in SCs during 5th-6th DW. NF200 was predominantly expressed in the ventral part, indicating presence of motoneurons. As development advanced, NF200 was mainly expressed in the marginal zone. Expression of CGRP was intense during all of the investigated periods, predominantly during the 5th-6th DW pointing to neural sensory differentiation, as opposed to the last DW when reduced expression of CGRP in the marginal layer indicated the terminations of the sensory afferents. Expression of VR1 was highest in the intermediate zone, at the beginning and at the end of the investigated periods, pointing to VR1 spatial pattern in the visceral afferents in the grey matter, while the first signs of calretinin were found in the 9th-10th DW ventrally. Delineating the relationships between factors involved in processes of neuronal differentiation as well as spatial and temporal arrangement of SC interrelated neurons can provide a useful information about normal SC development as well as the insight in possible causes of anomalies and disorders during embryonic life.

Expression and localization of DAB1 and Reelin during normal human kidney development.

AIM: To explore the spatial and temporal expression patterns of DAB1 and Reelin in the developing and postnatal healthy human kidneys as potential determinants of kidney development. METHODS: Paraffin-embedded fetal kidney tissue between the 13/14th and 38th developmental weeks (dw) and postnatal tissue at 1.5 and 7 years were stained with DAB1 and Reelin antibodies by double immunofluorescence. RESULTS: During the fetal kidney development and postnatal period, DAB1 and Reelin showed specific spatial expression pattern and diverse fluorescence intensity. During the fetal period, DAB1 was strongly expressed in the distal convoluted tubules (DCT), with strong reactivity, and diversely in the proximal convoluted tubules (PCT) and glomeruli. In the postnatal period, DAB1 expression decreased. The strongest Reelin expression in early fetal stages was observed in the PCT. In the postnatal period, Reelin expression decreased dramatically in all observed structures. These two markers were colocalized during early developmental stages, mostly in PCT, DCT, and podocytes. CONCLUSION: The appearance of DAB1 and Reelin during fetal kidney development confirms their potential significant role in the formation of kidney structure or function. High DAB1 expression in the DCT implies its regulatory role in tubular formation or function maintenance during development. Reelin was highly expressed in human kidneys at early fetal stages, mostly in the PCT, while at later fetal stages and postnatal period its expression decreased.

Reabsorption in the proximal tubuli-ultrastructural evidence for a novel aspect of renal VEGF trafficking.

Many clinical and experimental studies have revealed VEGF as an important factor in the pathophysiology of renal damage during diabetes mellitus (DM). Anti-VEGF therapy is in clinical use for treatment of DM and other diabetes-related (and unrelated) diseases. Nevertheless, little is known about the metabolism of VEGF in the kidneys. In order to determine the ultrastructural localization of VEGF in the kidney, we study the distribution of VEGF in the kidney of rats by using immunogold immunohistochemistry. Our light-microscopic data showed remarkable re-distribution of VEGF in proximal tubular cells (PTCs) during prolonged hyperglycemia, a DM type 2 model (DM2), which was confirmed by transmission electron microscopy (TEM) findings. TEM findings revealed an initial presence of VEGF in the vesicular transport apparatus of PTCs in healthy rats and its gradual translocation to the apical membrane of PTCs after renal damage caused by high sucrose treatment. The presented data add to our understanding of kidney VEGF trafficking, providing novel insight into the renal metabolism and pharmacodynamics of the cytokine. This could have a high impact on the use of VEGF and anti-VEGF therapy in different renal diseases.

Expression of Epithelial and Mesenchymal Differentiation Markers in the Early Human Gonadal Development.

Expressions of cytokeratin 8 (CK8), vimentin, nestin, and alpha-smooth-muscle-actin (alpha-SMA) were analyzed in the developing gonads of 12, 5-9 week old (W) human conceptuses by immunohistochemistry and immunofluorescence. During the investigated period, the number of CK8 positive cells increased from 56% to 92% in the gonadal surface epithelium, from 50% to 60% in the stroma, and from 23% to 42% in the medulla. In the early fetal period, the cell expression of CK8 increased in all gonadal parts, whereas primordial germ cells (PGC) remained negative. The expression of vimentin increased in the gonad stroma (gs) from 73% to 88%, and in the surface epithelium from 18% to 97% until ninth W. The medulla had the highest expression of vimentin in the seventh to eighth W (93%). Vimentin and CK8 colocalized in the somatic cells, while some PGCs showed vimentin expression only. Initially, nestin was positive in the gonad surface epithelium (8%) and stroma (52%), however during further development it decreased to 1% and 33%, respectively. In the early fetal period, the nestin positive cells decreased from 44% to 31% in the gonad medulla. Alpha-SMA was positive only in the blood vessels and mesonephros. The described pattern of expression of intermediate filaments (IF) in developing human gonads suggests their role in the control of PGC apoptosis, early differentiation of gs cells and cell migration. Both epithelial and mesenchymal origins of follicular cells and possible epithelial-to-mesenchymal transition of somatic cells is proposed. Lastly, IF intensity expression varies depending on the cell type and developmental period analyzed. Anat Rec, 300:1315-1326, 2017. © 2017 Wiley Periodicals, Inc.

Neuronal differentiation in the developing human spinal ganglia.

The spatiotemporal developmental pattern of the neural crest cells differentiation toward the first appearance of the neuronal subtypes was investigated in developing human spinal ganglia (SG) between the fifth and tenth developmental week using immunohistochemistry and immunofluorescence methods. First neurofilament-200- (NF200, likely myelinated mechanoreceptors) and isolectin-B4-positive neurons (likely unmyelinated nociceptors) appeared already in the 5/6th developmental week and their number subsequently increased during the progression of development. Proportion of NF200-positive cells was higher in the ventral parts of the SG than in the dorsal parts, particularly during the 5/6th and 9/10th developmental weeks (Mann-Whitney, P = 0.040 and P = 0.003). NF200 and IB4 colocalized during the whole investigated period. calcitonin gene-related peptide (CGRP; nociceptive responses), vanilloid receptor-1 (VR1; polymodal nociceptors), and calretinin (calcium signaling) cell immunoreactivity first appeared in the sixth week and eighth week, respectively, especially in the dorsal parts of the SG. VR1 and CGRP colocalized with NF00 during the whole investigated period. Our results indicate the high potential of early differentiated neuronal cells, which slightly decreased with the progression of SG differentiation. On the contrary, the number of neuronal subtypes displayed increasing differentiation at later developmental stage. The great diversity of phenotypic expression found in the SG neurons is the result of a wide variety of influences, occurring at different stages of development in a large potential repertory of these neurons. Understanding the pathway of neural differentiation in the human, SG could be important for the studies dealing with the process of regeneration of damaged spinal nerves or during the repair of pathological changes within the affected ganglia. Anat Rec, 299:1060-1072, 2016. © 2016 Wiley Periodicals, Inc.

Immunohistochemical Studies of Cytoskeletal and Extracellular Matrix Components in Dogfish Scyliorhinus canicula L. Notochordal Cells.

Immunofluorescence and immunohistochemical techniques were used to define the distribution of cytoskeletal (cytokeratin 8, vimentin) and extracellular matrix components (collagen type I, collagen type II, hyaluronic acid, and aggrecan) and bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) in the notochord of the lesser spotted dogfish Scyliorhinus canicula L. Immunolocalization of hyaluronic acid was observed in the notochord, vertebral centrum, and neural and hemal arches, while positive labeling to aggrecan was observed in the ossified centrum, notochord, and the perichondrium of the hyaline cartilage. Type I collagen was observed in the mineralized cartilage of the vertebral bodies, the notochord, the fibrocartilage of intervertebral disc, and the perichondrium. A positive labeling to type II collagen was observed in the inner part of the cartilaginous vertebral centrum and the notochord, as well as in the neural arch and muscle tissue, but there was no appreciable labeling of the hyaline cartilage. The presence of both BMP4 and BMP7 was seen in the mineralized vertebral centrum, notochordal cells, and neural arch. The notochordal cells expressed both cytokeratin 8 and vimentin, but predominantly vimentin. Hyaluronic acid, collagen type I, and collagen type II expression confirmed the presence of a mixture of notochordal and fibrocartilaginous tissue in the intervertebral disc, while BMPs confirmed the presence of an ossification in the cartilaginous skeleton of the spotted dogfish.