Syndecans, Exostosins and Sulfotransferases as Potential Synovial Inflammation Moderators in Patients with Hip Osteoarthritis.

The gradual deterioration of articular cartilage was thought to be the central event in osteoarthritis (OA), but recent studies demonstrated the importance of low-grade synovitis in the progression of OA. The Syndecan (SDC) family of membrane proteoglycans is known to be involved in the regulation of inflammation, but there is limited evidence considering the role of syndecans in OA synovitis. Our study aimed to investigate the hip OA synovial membrane expression patterns of SDC1, SDC2 and SDC4, as well as exostosins and sulfotransferases (enzymes involved in the polymerisation and modification of syndecans' heparan sulphate chains). Synovial membrane samples of patients with OA (24) were divided into two groups according to their Krenn synovitis score severity. The immunohistochemical expressions of SDC1, SDC2, SDC4, EXT1, EXT2, NDST1 and NDST2 in synovial intima and subintima were then analysed and compared with the control group (patients with femoral neck fracture). According to our study, the immunoexpression of SDC1, NDST1 and EXT2 is significantly increased in the intimal cells of OA synovial membrane in patients with lower histological synovitis scores and SDC4 in patients with higher synovitis scores, in comparison with non-OA controls. The difference in the expression of SDC2 among the OA and non-OA groups was insignificant. SDC1, SDC4, NDST1 and EXT2 seem to be involved as inflammation moderators in low-grade OA synovitis and, therefore, should be further investigated as potential markers of disease progression and therapeutic goals.

Immunohistochemical Expression Pattern of FGFR1, FGFR2, RIP5, and HIP2 in Developing and Postnatal Kidneys of Dab1-/- (yotari) Mice.

This study aimed to explore how Dab1 gene functional silencing influences the spatial and temporal expression patterns of fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), receptor-interacting protein kinase 5 (RIP5), and huntingtin-interacting protein 2 (HIP2) in the developing and postnatal kidneys of the yotari mice as potential determinants of normal kidney formation and function. Dab1-/- animal kidneys exhibit diminished FGFR1/FGFR2 expression in all examined developmental stages, whereas RIP5 cell immunoreactivity demonstrated negligible variation. The HIP2 expression revealed a discernible difference during the postnatal period, where we noted a significant decrease in almost all the observed kidney structures of yotari animals. An extracellular signal-regulated kinase (Erk1/2) and mammalian target of rapamycin (mTOR) expression in yotari kidneys decreased in embryonic and postnatal developmental phases for which we can hypothesize that the Erk1/2 signaling pathway in the yotari mice kidneys is dependent on Reelin with Dab1 only partially implicated in Reelin-mediated MEK/Erk1/2 activation. The impairment of FGFR1 and FGFR2 expression suggests the involvement of the observed markers in generating the CAKUT phenotype resulting in renal hypoplasia. Our study demonstrates the critical role of HIP2 in reducing cell death throughout nephrogenesis and maturation in wild-type mice and indicates a possible connection between decreased HIP2 expression in postnatal kidney structures and observed podocyte injury in yotari. Our results emphasize the crucial function of the examined markers throughout normal kidney development and their potential participation in kidney pathology and diagnostics, where they might serve as biomarkers and therapeutic targets.

A Lack of GD3 Synthase Leads to Impaired Renal Expression of Connexins and Pannexin1 in St8sia1 Knockout Mice.

The aim of this study was to determine the effects of altered ganglioside composition on the expression of Cx37, Cx40, Cx43, Cx45, and Panx1 in different kidney regions of St8sia1 gene knockout mice (St8sia1 KO) lacking the GD3 synthase enzyme. Experiments were performed in twelve male 6-month-old mice: four wild-type (C57BL/6-type, WT) and eight St8sia1 KO mice. After euthanasia, kidney tissue was harvested, embedded in paraffin wax, and processed for immunohistochemistry. The expression of connexins and Panx1 was determined in different regions of the kidney: cortex (CTX.), outer stripe of outer medulla (O.S.), inner stripe of outer medulla (IN.S.), and inner medulla (IN.MED.). We determined significantly lower expression of Cx37, Cx40, Cx45, and Panx1 in different parts of the kidneys of St8sia1 KO mice compared with WT. The most consistent decrease was found in the O.S. where all markers (Cx 37, 40, 45 and Panx1) were disrupted in St8si1 KO mice. In the CTX. region, we observed decrease in the expression of Cx37, Cx45, and Panx1, while reduced expression of Cx37 and Panx1 was more specific to IN.S. The results of the present study suggest that deficiency of GD3 synthase in St8sia1 KO mice leads to disruption of renal Cx expression, which is probably related to alteration of ganglioside composition.

Aberrations in FGFR1, FGFR2, and RIP5 Expression in Human Congenital Anomalies of the Kidney and Urinary Tract (CAKUT).

This study aimed to explore the spatio-temporal expression patterns of congenital anomalies of kidney and urinary tract (CAKUT) candidate genes, Fibroblast Growth Factor Receptor 1 (FGFR1), Fibroblast Growth Factor Receptor 2 (FGFR2) and Receptor-Interacting Protein Kinase 5 (RIP5), in human fetal kidney development (CTRL) and kidneys affected with CAKUT. Human fetal kidneys from the 22nd to 41st developmental week (duplex, hypoplastic, dysplastic, and controls) were stained with antibodies and analyzed by epifluorescence microscopy and RT-qPCR. The effect of CAKUT candidate genes on kidney nephrogenesis and function is confirmed by statistically significant variations in the spatio-temporal expression patterns of the investigated markers. The nuclear localization of FGFR1, elevated expression score of FGFR1 mRNA, the increased area percentage of FGFR1-positive cells in the kidney cortex, and the overall decrease in the expression after the peak at the 27th developmental week in dysplastic kidneys (DYS), suggest an altered expression pattern and protein function in response to CAKUT pathophysiology. The RT-qPCR analysis revealed a significantly higher FGFR2 mRNA expression score in the CAKUT kidneys compared to the CTRL. This increase could be due to the repair mechanism involving the downstream mediator, Extracellular Signal-Regulated Kinase 1/2 (ERK1/2). The expression of RIP5 during normal human kidney development was reduced temporarily, due to urine production and increased later since it undertakes additional functions in the maturation of the postnatal kidney and homeostasis, while the expression dynamics in CAKUT-affected kidneys exhibited a decrease in the percentage of RIP5-positive cells during the investigated developmental period. Our findings highlight the importance of FGFR1, FGFR2, and RIP5 as markers in normal and pathological kidney development.

CRKL, AIFM3, AIF, BCL2, and UBASH3A during Human Kidney Development.

We aimed to investigate the spatio-temporal expression of possible CAKUT candidate genes CRKL, AIFM3, and UBASH3A, as well as AIF and BCL2 during human kidney development. Human fetal kidney tissue was stained with antibodies and analyzed by fluorescence microscopy and RT-PCR. Quantification of positive cells was assessed by calculation of area percentage and counting cells in nephron structures. Results showed statistically significant differences in the temporal expression patterns of the examined markers, depending on the investigated developmental stage. Limited but strong expression of CRKL was seen in developing kidneys, with increasing expression up to the period where the majority of nephrons are formed. Results also lead us to conclude that AIFM3 and AIF are important for promoting cell survival, but only AIFM3 is considered a CAKUT candidate gene due to the lack of AIF in nephron developmental structures. Our findings imply great importance of AIFM3 in energy production in nephrogenesis and tubular maturation. UBASH3A raw scores showed greater immunoreactivity in developing structures than mature ones which would point to a meaningful role in nephrogenesis. The fact that mRNA and proteins of CRKL, UBASH3A, and AIFM3 were detected in all phases of kidney development implies their role as renal development control genes.

Expression Pattern of α-Tubulin, Inversin and Its Target Dishevelled-1 and Morphology of Primary Cilia in Normal Human Kidney Development and Diseases.

The spatiotemporal expression of α-tubulin, inversin and dishevelled-1 (DVL-1) proteins associated with the Wnt-signaling pathway, and primary cilia morphology were analyzed in developing kidneys (14th-38th developmental weeks), healthy postnatal (1.5- and 7-years old) and pathologically changed human kidneys, including multicystic dysplastic kidneys (MCDK), focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome of the Finnish type (CNF). The analysis was performed by double immunofluorescence, electron microscopy, semiquantitative and statistical methods. Cytoplasmic co-expression of α-tubulin, inversin and DVL-1 was observed in the proximal convoluted tubules (pct), distal convoluted tubules (dct) and glomeruli (g) of analyzed tissues. During kidney development, the overall expression of α-tubulin, inversin and DVL-1 decreased, while in the postnatal period slightly increased. The highest expressions of α-tubulin and inversin characterized dct and g, while high DVL-1 characterized pct. α-tubulin, inversin and DVL-1 expression pattern in MCDK, FSGS and CNF kidneys significantly differed from the healthy control. Compared to healthy kidneys, pathologically changed kidneys had dysmorphic primary cilia. Different expression dynamics of α-tubulin, inversin and DVL-1 during kidney development could indicate that switch between the canonical and noncanonical Wnt-signaling is essential for normal kidney morphogenesis. In contrast, their disturbed expression in pathological kidneys might be associated with abnormal primary cilia, leading to chronic kidney diseases.

Alteration of Cx37, Cx40, Cx43, Cx45, Panx1, and Renin Expression Patterns in Postnatal Kidneys of Dab1-/- (yotari) Mice.

Numerous evidence corroborates roles of gap junctions/hemichannels in proper kidney development. We analyzed how Dab1 gene functional silencing influences expression and localization of Cx37, Cx40, Cx43, Cx45, Panx1 and renin in postnatal kidneys of yotari mice, by using immunohistochemistry and electron microscopy. Dab1 Δ102/221 might lead to the activation of c-Src tyrosine kinase, causing the upregulation of Cx43 in the medulla of yotari mice. The expression of renin was more prominent in yotari mice (p < 0.001). Renin granules were unusually present inside the vascular walls of glomeruli capillaries, in proximal and distal convoluted tubules and in the medulla. Disfunction of Cx40 is likely responsible for increased atypically positioned renin cells which release renin in an uncontrolled fashion, but this doesn't rule out simultaneous involvement of other Cxs, such as Cx45 which was significantly increased in the yotari cortex. The decreased Cx37 expression in yotari medulla might contribute to hypertension reduction provoked by high renin expression. These findings imply the relevance of Cxs/Panx1 as markers of impaired kidney function (high renin) in yotari mice and that they have a role in the preservation of intercellular signaling and implicate connexopathies as the cause of premature death of yotari mice.

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.

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.

Expression of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) Candidate Genes EDA2R, PCDH9, and TRAF7 in Normal Human Kidney Development and CAKUT.

Approximately half of the cases of chronic kidney disease (CKD) in childhood are caused by congenital anomalies of the kidney and urinary tract (CAKUT). Specific genes were identified as having significant importance in regard to the underlying genetic factors responsible for the CAKUT phenotype, and in our research, we focused on analyzing and comparing the expression levels of ectodysplasin A2 receptor (EDA2R), protocadherin9 (PCDH9), and TNF receptor-associated factor 7 (TRAF7) proteins in the cortex and medulla of healthy control kidneys during developmental phases 2, 3, and 4. We also performed an analysis of the area percentages of the mentioned proteins in the cortical and medullary sections of healthy embryonic and fetal kidneys compared to those affected by CAKUT, including duplex kidneys (DK), horseshoe kidneys (HK), hypoplastic kidneys (HYP), and dysplastic kidneys (DYS). We found that the CAKUT candidate gene proteins EDA2R, PCDH9, and TRAF7 are all expressed during normal human kidney development stages. In DYS, the expression of EDA2R was higher than in normal kidneys, likely due to EDA2R's role in apoptosis, which was upregulated in specific cases and could possibly contribute to the formation of DYS. The expression of PCDH9 was lower in HK, which can be attributed to the possible role of PCDH9 in cell migration suppression. Decreased PCDH9 expression is linked to increased cell migration, potentially contributing to the development of HK. The level of TRAF7 expression was reduced in all examined kidney disorders compared to normal kidneys, suggesting that this reduction might be attributed to the crucial role of TRAF7 in the formation of endothelium and ciliogenesis, both of which are essential for normal kidney development. Further research is required to ascertain the function of these proteins in both the typical development of the kidney and in CAKUT.

Unraveling the Impact of Dab1 Gene Silencing on the Expression of Autophagy Markers in Lung Development.

The purpose of this study was to evaluate the effects of Dab1 gene silencing on the immunoexpression of light chain 3 beta (Lc3b), glucose regulating protein 78 (Grp78), heat shock cognate 71 (Hsc70), mammalian target of rapamycin (mTOR) and lysosomal-associated membrane protein 2A (Lamp2a) in the lung tissue of developing yotari (Dab1-/-) and wild-type (wt) mice. The lung epithelium and mesenchyme of the embryos at gestational days E13.5 and E15.5 were examined using immunofluorescence and semi-quantitative methods. In the pulmonary mesenchyme and epithelium, Grp78 and Lc3b of moderate fluorescence reactivity was demonstrated in wt mice for both evaluated time points, while yotari mice exhibited only epithelial reactivity for the same markers. Mild punctate expression of Hsc70 was observed for both genotypes. A significant difference was present when analyzing mTOR expression, where wt mice showed strong perinuclear staining in the epithelium. According to our data, Dab1 gene silencing may result in autophagy abnormalities, which could then cause respiratory system pathologies via defective lung cell degradation by lysosome-dependent cell elimination.

Immunoexpression Pattern of Autophagy Markers in Developing and Postnatal Kidneys of Dab1-/-(yotari) Mice.

The purpose of this study was to compare the immunofluorescence patterns of autophagic markers: Light chain 3 beta (LC3B), Glucose regulating protein 78 (GRP78), Heat shock cognate 71 (HSC70) and Lysosomal-associated membrane protein 2A (LAMP2A) in the developing and postnatal kidneys of Dab1-/- (yotari) mice to those of wild-type samples. Embryos were obtained on gestation days 13.5 and 15.5 (E13.5 and E15.5), and adult animals were sacrificed at postnatal days 4, 11 and 14 (P4, P11, and P14). After fixation and dehydration, paraffin-embedded kidney tissues were sectioned and incubated with specific antibodies. Using an immunofluorescence microscope, sections were analyzed. For statistical analysis, a two-way ANOVA test and a Tukey's multiple comparison test were performed with a probability level of p < 0.05. A significant increase in GRP78 and LAMP2A expression was observed in the renal vesicles and convoluted tubules of yotari in embryonic stages. In postnatal kidneys, all observed proteins showed higher signal intensities in proximal and distal convoluted tubules of yotari, while a higher percentage of LC3B-positive cells was also observed in glomeruli. Our findings suggest that all of the examined autophagic markers play an important role in normal kidney development, as well as the potential importance of these proteins in renal pathology, where they primarily serve a protective function and thus may be used as diagnostic and therapeutic targets.

Expression of Connexins 37, 40 and 45, Pannexin 1 and Vimentin in Laryngeal Squamous Cell Carcinomas.

Approximately 60% of patients with squamous cell carcinoma (LSCC) have regional occult metastatic disease/distant metastases at the time of diagnosis, putting them at higher risk for disease progression. Therefore, biomarkers are needed for early prognostic purpose. The aim of this study was to analyze the expression pattern of connexins (Cx) 37, 40 and 45, pannexin1 (Panx1) and vimentin in LSCC and correlate with tumor grade (G) and outcome. METHODS: Thirty-four patients who underwent (hemi-)laryngectomy and regional lymphadenectomy due to LSCC from 2017 to 2018 in University Hospital Split, Croatia, were studied. Samples of tumor tissue and adjacent normal mucosa embedded in paraffin blocks were stained using the immunofluorescence method and were semi-quantitatively analyzed. RESULTS: The expression of Cx37, Cx40, and Panx1 differed between cancer and adjacent normal mucosa and between histological grades, being the highest in well-differentiated (G1) cancer and low/absent in poorly differentiated (G3) cancer (all p < 0.05). The expression of vimentin was the highest in G3 cancer. Expression of Cx45 was generally weak/absent, with no significant difference between cancer and the controls or between grades. Lower Panx1 and higher vimentin expression were found to be prognostic factors for regional metastatic disease. Lower Cx37 and 40 expressions were present in patients with disease recurrence after the three-year follow-up period. CONCLUSION: Cx37 and Cx40, Panx1, and vimentin have the potential to be used as prognostic biomarkers for LSCC.

Expression Pattern of DAB Adaptor Protein 2 in Left- and Right-Side Colorectal Carcinoma.

Left-sided and right-sided colorectal cancer (L-CRC and R-CRC) have relatively different clinical pictures and pathophysiological backgrounds. The aim of this study was to investigate the presence of DAB adapter protein 2 (DAB2) as a potential molecular mechanism that contributes to this diversity in terms of malignancy and responses to therapy. The expression of the suppressor gene DAB2 in colon cancer has already been analyzed, but its significance has not been fully elucidated. Archived samples from 34 patients who underwent colon cancer surgery were included in this study, with 13 patients with low-grade CRC and 21 with high-grade CRC. Twenty of the tumors were R-CRC, while 14 were L-CRC. DAB2 expression was analyzed immunohistochemically in the tumor tissue and the colon resection margin was used as a control. Tumors were divided into L-CRC and R-CRC, with splenic flexure as the cutoff point for each side. The results showed that R-CRC had lower DAB2 protein expression compared to L-CRC (p = 0.01). High-grade tumors had reduced DAB2 expression compared to low-grade tumors (p = 0.02). These results are consistent with the analysis of DAB2 gene expression data that we exported from the TCGA Colon and Rectal Cancer Study (COADREAD). In 736 samples of colon cancer, lower DAB2 gene expression was found in R-CRC compared to L-CRC (p < 0.0001). DAB2 gene expression was significantly higher in the sigmoid colon than in the cecum and ascending colon (p < 0.01). The analysis confirmed a lower expression of the DAB2 in tumors with positive microsatellite instability (p < 0.001). In conclusion, DAB2 has a role in the biological differences between R-CRC and L-CRC and its therapeutic and diagnostic potential needs to be further examined.

Disturbances in Switching between Canonical and Non-Canonical Wnt Signaling Characterize Developing and Postnatal Kidneys of Dab1-/- (yotari) Mice.

This study aims to determine the protein expression patterns of acetylated α-tubulin, inversin, dishevelled-1, Wnt5a/b, and ß-catenin in developing (E13.5 and E15.5) and early postnatal (P4 and P14) kidneys of Dab1-/- (yotari) mice, their role in regulating the Wnt signaling pathway, and the possible relation to congenital anomalies of kidney and urinary tract (CAKUT). The analysis of target protein co-expression, observed in the renal vesicles/immature glomeruli, ampullae/collecting ducts, convoluted tubules, metanephric mesenchyme of developing kidneys, but proximal convoluted tubules, distal convoluted tubules and glomeruli of postnatal kidneys, was performed using double immunofluorescence and semi-quantitative methods. The overall expression of acetylated α-tubulin and inversin during normal kidney development increases with higher expression in yotari mice as the kidney acquires mature morphology. An increase in ß-catenin and cytosolic DVL-1 levels, indicating a switch from non-canonical to canonical Wnt signaling, is found in the postnatal kidney of yotari mice. In contrast, healthy mouse kidney expresses inversin and Wnt5a/b in the postnatal period, thus activating non-canonical Wnt signaling. Target protein expression patterns in kidney development and the early postnatal period observed in this study could indicate that switching between canonical and non-canonical Wnt signaling is crucial for normal nephrogenesis, while the defective Dab1 gene product in yotari mice may promote CAKUT due to interfering with this process.

Expression of Pannexin 1 in the Human Kidney during Embryonal, Early Fetal and Postnatal Development and Its Prognostic Significance in Diabetic Nephropathy.

Pannexins are transmembrane glycoproteins that constitute channels involved in purinergic signaling through ATP release from cells in various physiological and pathological processes. In this study, the distribution of Panx1 expression in different cell populations of healthy postnatal human kidneys and during human embryonic and early fetal development was investigated by double immunohistochemistry. In addition, the glomerular and tubular expression of Panx1 was examined in patients with type 2 diabetes mellitus (DM2) and the control group, and renal Panx1 expression was correlated with serum creatinine. In the 6th week of embryonic development (DW), Panx1 expression was found in mesonephric glomeruli and mesonephric tubules. At the transition from 6th to 7th DW, Panx1 immunoreactivity was found in the mesonephric tubules and mesonephric duct, as well as in the metanephric ureteric bud and ampullae. In the 7th DW, strong Panx1 immunoreactivity was observed in the developing ureteric bud in the metanephros, whereas no Panx1 immunoreactivity was found in the metanephric cup. In the 8th DW, Panx1 expression was also found in the ureteric bud of the metanephros, the renal vesicle and comma-shaped nephron, and the epithelial cells of Bowman's capsule. Expression of Panx1 was found at an early stage in both the paramesonephric duct and the mesonephric duct and diminished toward the 8th DW. During the 6th-10th DW, colocalization of Panx1 with alpha smooth actin (aSMA) was found in developing blood vessels. In the postnatal kidney, strong Panx1 immunoreactivity was present in medullary and cortical collecting duct cells, renin-producing cells, and proximal tubules. Very weak Panx1 immunoreactivity was found in certain distal tubule cells and the thin descending limbs of the loop of Henle. Panx1 immunoreactivity was also found in nephrin-immunoreactive podocytes. Panx1 was not colocalized with aSMA immunoreactivity in the vessels of the postnatal human kidney, but it was present in the endothelium. A significant positive correlation was found between Panx1 expression in glomeruli and serum creatinine only in diabetic patients and was not found in the nondiabetic group. The spatiotemporal expression of Panx1 during the early stages of human kidney development supports its possible role in cellular differentiation, migration, and positioning in the developing human kidney. In addition, our data suggest that glomerular Panx1 expression is a potential indicator of worsening renal function in patients with type 2 diabetes.

Altered Expression of EMT-Related Factors Snail, Wnt4, and Notch2 in the Short-Term Streptozotocin-Induced Diabetic Rat Kidneys.

Background: The aim of this study was to determine the expression of epithelial to mesenchymal transition (EMT)-related transcription factors Snail, Wnt4, and Notch2 with key roles in renal fibrosis, in different renal areas of diabetic rats: glomeruli (G), proximal and distal convoluted tubules (PCT; DCT). Methods: Male Sprague Dawley rats were instilled with 55 mg/kg streptozotocin (diabetes mellitus type I model, DM group) or citrate buffer (control group). Kidney samples were collected 2 weeks and 2 months after DM induction and processed for immunohistochemistry. Results: Diabetic animals showed higher Wnt4 kidney expression both 2 weeks and 2 months post-DM induction, while Snail expression significantly increased only 2 weeks after DM initiation (p < 0.0001). We determined significantly higher expression of examined EMT-related genes in different kidney regions in diabetic animals compared with controls. The most substantial differences were observed in tubular epithelial cells in the period of 2 weeks after induction, with higher Snail and Wnt4 expression in PCT and increased Snail and Notch2 expression in DCT of diabetic animals (p < 0.0001; p < 0.001). Conclusion: The obtained results point to the EMT-related factors Snail, Wnt4, and Notch2 as a potential contributor to diabetic nephropathy development and progression. Changes in their expression, especially in PCT and DCT, could serve as diagnostic biomarkers for the early stages of DM and might be a promising novel therapeutic target in this condition.

Immunohistochemical Expression Pattern of Mismatch Repair Genes in the Short-term Streptozotocin-induced Diabetic Rat Kidneys.

We studied the expression of mismatch repair genes (MMRs)-mutS protein homolog 2 (MSH2), PMS2, MutL homolog 1 (MLH1), and yH2AFX in diabetic rat kidneys. Streptozotocin-induced diabetes mellitus type 1 rat model (DM1) was used. Renal samples were collected 2 weeks and 2 months after DM1 induction and immunohistochemical expression of MMR genes in the renal cortex was analyzed. Diabetic animals showed lower MSH2 and higher yH2AFX kidney expression both 2 weeks and 2 months after DM1 induction. MLH1 expression significantly increased 2 weeks after DM1 induction (P<0.0001). The most substantial differences were observed in the period 2 weeks after induction, with lower MSH2 and higher MLH1 expression in the proximal convoluted tubules and distal convoluted tubules (DCT) of diabetic animals (P<0.001). yH2AFX expression significantly increased in the DCT of diabetic animals at both time points (P<0.001; P<0.01). PMS2 expression changed only in the glomeruli, where it significantly decreased 2 months after DM1 induction (P<0.05). We concluded that the most substantial changes in renal expression of MMRs are happening already 2 weeks after diabetes induction, predominantly in the proximal convoluted tubules and DCT. Moreover, DCT could have a critical role in the pathophysiology of diabetic nephropathy (DN) and might be a future therapeutic target in this condition. The obtained results point to the MMRs as a potential factor in the development and progression of DN, as well as the possible link between DN and renal carcinogenesis.

GREB1L, CRELD2 and ITGA10 expression in the human developmental and postnatal kidneys: an immunohistochemical study.

BACKGROUND: Aim of our study is to provide an insight into the genetic expression landscape of GREB1L, ITGA10 and CRELD2 which are important in human genitourinary tract development which might help elucidate the critical stages for the onset of kidney anomalies. METHODS: Morphological parameters were analyzed using immunohistochemistry on human foetal (13-38 w) and postnatal (1.5 and 7.5y) human kidney samples. RESULTS: GREB1L marker had a strong intensity and the highest rate in proximal tubules (PTC) of 1.5 years' kidney (90.25%). In the distal tubules (DCT) there were statistically significant differences in 13 w, 15 w, 16 w, 21 w, 38 w and 7.5y regarding 1.5y (Kruskal-Wallis test, p < 0.001). There was significantly more GREB1L in the glomeruli at 21 w and 38 w in regard to all other stages (Kruskal-Wallis test, p < 0.01). ITGA10 staining intensity was strongest in PCT with the highest rate in 13 w (92.75%), while the lowest rate was found in glomeruli and DCT (Kruskal-Wallis test, p < 0.001). CRELD2 had the strongest staining intensity in PCT with the highest rate in 13 w and 1.5y (92.25%) and lowest in the glomeruli of 7.5 years (24.3 %). In DCT there were statistically significant differences in CRELD2 positive cells in 13 w, 15 w, 16 w, 21 w, 38 w and 7.5y regarding 1.5y (Kruskal-Wallis test, p < 0.01). ITGA10 and CRELD2 co-localised in the postnatal period in DCT. CONCLUSION: High kidney expressions of GREB1L, ITGA10 and CRELD2 even in the postnatal period implicate their importance not only for the onset of CAKUT in the case of their mutation but also for maintenance of kidney homeostasis.

Chronic Stress and Gonadectomy Affect the Expression of Cx37, Cx40 and Cx43 in the Spinal Cord.

The study aimed to determine whether the exposure to chronic stress and/or performance of gonadectomy might lead to disturbance in the expression of connexin (Cx) 37, 40 and 43 in the spinal cord (SC), as a potential explanation for sex differences in stress-related chronic pain conditions. After the rats were sham-operated or gonadectomized, three 10-day sessions of sham or chronic stress were applied. Immunohistochemistry and transmission electron microscopy (TEM) were used to examine Cx localization and expression in the SC. The gonadectomy resulted in an increase of Cx37 expression in the dorsal horn (DH) of the female rats, but chronic stress suppressed the effects of castration. In male rats, only the combined effects of castration and chronic stress increased Cx37 expression. The influence of chronic stress on the DH Cx40 expression was inversely evident after the castration: increased in the ovariectomized female rats, while decreased in the orchidectomized male rats. We did not find any effect of chronic stress and castration, alone or together, on Cx43 expression in the DH, but the percentage of Cx43 overlapping the astrocyte marker glial fibrillary acidic protein (gfap) increased in the male stressed group after the castration. In conclusion, the association of the chronic stress with sex hormone depletion results in disturbances of the SC Cx expression and might be a possible mechanism of disturbed pain perception after chronic stress exposure.