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.

Prevalence of burnout among health care workers in the Federation of Bosnia and Herzegovina during the coronavirus disease-2019 pandemic: a cross-sectional study.

AIM: To investigate the prevalence of burnout syndrome among health care workers in the Federation of Bosnia and Herzegovina (FBiH) during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: This cross-sectional study was conducted in May and June 2021 using an online survey based on Copenhagen Burnout Inventory. The questionnaire underwent forward and backward translation, preliminary pilot testing, and was assessed for reliability and validity. Personal burnout, work-related burnout, and patient-related burnout were assessed. The survey was sent to the members of the Union of Physicians and Dentists in FBIH, who were asked to forward the link to their medical technicians and nurses. RESULTS: A total of 77% of participants experienced some form of burnout. As many as 32% experienced all three forms of burnout. Those actively involved in tackling the COVID-19 pandemic more often experienced burnout. In personal and work-related burnout domains, higher level of burnout was reported among female respondents. Higher work-related and patient-related burnout was reported by physicians compared with medical technicians/nurses. Higher level of patient-related burnout was reported in health care workers aged 30-39 and 50-59 years, among respondents working in primary care, and among physicians. CONCLUSION: The majority of health care workers showed moderate or high levels of personal and work-related burnout, with a lower level of patient-related burnout. There is a need for further research into the causes of burnout, as well as for the implementation of organizational interventions aimed to minimize workplace burnout.

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.

The Expression Pattern of Bcl-2 and Bax in the Tumor and Stromal Cells in Colorectal Carcinoma.

Background and objectives: The epithelial and stromal tissues both play a role in the progression of colorectal cancer (CRC). The aim of this study was to assess the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax in the epithelium as well as the lamina propria of normal colonic controls, low-grade tumor samples and high-grade tumor samples. Materials and Methods: A total of 60 samples consisting of both normal colonic and carcinoma samples was collected from the Department of Pathology, Cytology and Forensic Medicine, University Hospital Center, Split from January 2020 to December 2021. The expression of Bcl-2 and Bax markers was semi-quantitatively and quantitatively evaluated by recording immunofluorescence stain intensity and by counting stained cells in the lamina propria and epithelium. Analysis of positive cells was performed using the Mann-Whitney test. Results: In all samples, Bcl-2 was significantly more expressed in the lamina propria when compared with the epithelium. Bax was significantly more expressed in the epithelium of normal and low-grade cancer samples when compared with their respective laminae propriae. The percentage of Bcl-2-positive cells in lamina propria is about two times lower in high-grade CRC and about three times lower in low-grade CRC in comparison with healthy controls. Contrary to this, the percentage of Bax-positive cells was greater in the epithelium of low-grade CRC in comparison with healthy control and high-grade CRC. Conclusions: Our study provides a new insight into Bcl-2 and Bax expression pattern in CRC. Evaluation of Bcl-2 expression in the lamina propria and Bax expression in the epithelium could provide important information for colorectal cancer prognosis as well as potential treatment strategies.

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.

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.

Expression pattern of apoptosis-inducing factor in the kidneys of streptozotocin-induced diabetic rats.

BACKGROUND: It is believed that tubulo-interstitial fibrosis and atrophy in diabetic patients are directly associated with the progression of chronic kidney disease, CKD. AIF is one of the crucial factors responsible for mitochondrial apoptosis, however, it can also promote cell survival independently from its role in apoptosis, and therefore can be potentially used as a tool in prevention of the onset of CKD in diabetic patients. Our aim was to investigate the significance of AIF expression in the development of CKD by observing the expression of AIF in 2 weeks' and 2 months' kidneys of diabetic rats compared to their controls. METHODS: Male Sprague-Dawley rats were treated with 55 mg/kg streptozotocin (model of type 1 diabetes mellitus; DM group) or citrate buffer (control). After 2 weeks and 2 months kidney samples were collected and analysed in different renal areas. RESULTS: Characteristic morphologic changes were found between the 2 months' control and 2 months' diabetic groups. Those changes, including fibrosis and possible replacement of podocytes with connective tissue were mainly present in the glomeruli. AIF expression was seen in the both cortex, and in the collecting ducts of the medulla. Strong intensity of AIF expression was seen in proximal and distal convoluted tubules in both diabetic groups. In the control groups the glomeruli showed no AIF staining but moderate staining was seen in both diabetic groups. Overall, the percentage of AIF positive cells in the glomeruli was the lowest. The greatest rise in cell positivity was displayed from the 2 weeks' control group to 2 weeks' diabetes group (38 %) in glomeruli. The cell positivity of the 2 weeks' diabetic group is significantly reduced to 18 % in the 2 months' diabetic group in glomeruli. A similar pattern was seen in the proximal tubular cells (92 % positivity 2 weeks diabetic groups; 89 % positivity 2 months diabetic groups), as well as in the distal tubules. The highest percentage of AIF positive cells was seen in the collecting ducts, more than 80 % in all groups. CONCLUSIONS: Our study provides insight into AIF expression pattern during short term diabetes model, confirming possible dual role of AIF, not only in apoptosis but also in cell function and homeostasis, and proving AIF as potential therapeutic target and marker of advancement of CKD.

Renal expression of sigma 1 receptors in diabetic rats.

The purpose of this study was to determine the changes in the expression of sigma 1 receptors (σ1Rs) in the kidney of diabetic rats, which could indicate their possible role in the pathogenesis of diabetic nephropathy (DN). Sprague-Dawley rats were were given intraperitoneal injection of 55 mg/kg streptozotocin (STZ) in order to induce type I of diabetes (DM1). Control and diabetic rats were sacrificed 2 weeks or 2 months after DM1 induction. Expression of σ1Rs was determined in kidneys of the experimental rats, using immunohistochemistry. The most prominent expression of σ1Rs was found in distal tubuli (DT). Results have shown significant increase in renal σ1Rs section percentage area of rats 2 months after DM1 induction, compared to both control group at the same age and diabetic group 2 weeks after induction (P < 0.01 both). Similarly, a number of immunoreactive DT increased in diabetic group 2 months after induction, compared to DM1 group 2 weeks after induction (P < 0.001). We also found a decrease of a number of immunoreactive DT 2 weeks post DM1 induction (P < 0.01). However, the same was found during maturation of the control rats (P < 0.001). In addition, a strong co-expression of σ1R and proinflammatory factor TGFß was seen in vacuolated DT. The results indicate to the potential role of σ1Rs in postnatal maturation of the rat kidneys and in distal tubular damage in the pathogenesis of the diabetic nephropathy. We conclude that σ1Rs could be potential target in treatment of the diabetic nephropathy.

Changes in snail and SRF expression in the kidneys of diabetic rats during ageing.

BACKGROUND: Diabetic nephropathy is a progressive condition which develops for many years. We analyzed expression of Snail and serum response factor (SRF), epithelial-mesenchymal transition (EMT) regulatory transcription factors with a key role in renal fibrosis, in different renal areas of diabetic rats during ageing. METHODS: Male Sprague-Dawley rats were treated with 55 mg/kg streptozotocin (model of type 1 diabetes mellitus; DM group) or citrate buffer (control). DM group received insulin weekly to prevent ketoacidosis. After 2 weeks, 2, 6 and 12 months kidney samples were collected and analysed in different renal areas. RESULTS: Snail expression was located within cortex in proximal convoluted tubules, in control and DM groups, in the cytoplasm. Percentage of Snail-positive cells in control groups was high and decreased with time, whereas in DM groups the highest percentage was after 2 weeks. In all time points, smaller percentage of Snail expression was seen in DM groups compared to controls. SRF expression was mostly located in the proximal convoluted tubules, always in the cytoplasm. In control groups SRF was expressed in all time periods in proximal convoluted tubules, with decrement after 12 months. Percentage of SRF-positive cells was higher in control groups compared to DM in all time points, with the exception of 12 months. To a smaller degree, SRF expression was seen in the glomeruli and distal convoluted tubules, with more SRF positive cells in DM compared to their control groups. CONCLUSIONS: While Snail expression remained lower in diabetic tissues, compared to controls, expression of SRF increased in diabetic tissues in the second part of the year. These changes may need long time to develop, and, in line with earlier reports, it is possible that insulin treatment of DM rats once a week reduces possibility of EMT and development of renal fibrosis even in the long term.

Students' Practices and Knowledge on Antimicrobial Usage and Resistance in Split, Croatia: The Education of Future Prescribers.

Aims: To evaluate the effect of biomedical students' ongoing education, we assessed their knowledge and attitudes toward antimicrobial use. Study Design: A cross-sectional study was carried out among the students of four study programs: Medicine in Croatian, Medicine in English, Dental medicine, and Pharmacy. The anonymous questionnaire was distributed to students who attended classes from April to May 2018. Results: A total of 947 (86%) out of 1,107 students enrolled at the University of Split School of Medicine participated in this study. A third of dental students (51/159) and a quarter of medical (113/458) and pharmacy students (32/130) believed that paracetamol was an antibiotic that reduces pain. However, the percentage significantly decreased from the first to the final years. Only 31% of the final year dental medicine students (5/16) named a correct guideline for the usage of antimicrobial drugs, 23% of medical students (18/78), and none in the English program. Pharmacy students were the most informed, since 76% of the final year students (16/21) named Intersectoral Coordination Mechanism for the Control of Antimicrobial Resistance (ISKRA) guidelines. Conclusion: The students showed poor knowledge on the use of guidelines for antibiotic use, highlighting the need for changes in the existing curricula, including a more effective course on antimicrobial prescribing.

Attitudes of dental practitioners towards antimicrobial therapy in Croatia and Bosnia and Herzegovina.

INTRODUCTION: The objective of this study was to assess perceptions and attitudes amongst dental practitioners in relation to antibiotic usage and antibiotic resistance. METHODS: Self-administered questionnaire was given to dental practitioners employed in south Croatia, west Herzegovina and Sarajevo, Bosnia and Herzegovina (N = 115). RESULTS: 81.7% of respondents agreed the usage of antimicrobials is frequently uncritical and unnecessary. 83.5% of dental practitioners reported that they have used guidelines in their practice; however, only 9 out of 115 stated valid guidelines. One-third of the respondents agreed or were undecided that the usage of antimicrobials in every oral inflammatory process treatment is justified. Furthermore, 13% was undecided and 26% agreed that pregnant women and breastfeeding women should not use any antimicrobials. However, three quarters of respondents considered they had satisfactory knowledge on antimicrobials. DISCUSSION: The respondents considered they had satisfactory knowledge on antimicrobials, which was in contrast to the knowledge shown, but also expressed the need for additional education. Therefore, adequate measures include the creation of the local guidelines, their implementation, and updating the practitioners' knowledge on antibiotic use and resistance through continuous educational courses.

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.

Sigma-1 Receptor Expression in DRG Neurons During a Carrageenan-Provoked Inflammation.

Sigma 1 receptor (σ1R) is a non-opioid receptor that modulates pain perception and is strongly expressed in dorsal root ganglion (DRG) neurons. We studied the changes in the expression of σ1R in different sub-populations of DRG neurons during the first 48 hr in a carrageenan-induced inflammation rat model, with σ1R being a possible base for the development of neuropathic pain after inflammation. Twenty Sprague Dawley rats were divided into five groups (N = 4 in each group): the control (C) group was sacrificed immediately; all other animals received an intraplantar injection of 0.1 mL 2% carrageenan and were sacrificed in 6, 12, 24 or 48 hr after the injection and DRGs were collected and processed for immunohistochemistry. σ1R fluorescence intensity decreased slightly but significantly in up to 24 hr post-carrageenan injection in all sub-populations of DRG neurons (ib4+; ib4- medium, ib4- large and ib4- in total; P < 0.05 - P < 0.001), with the exception of the ib4- small neurons (<25 µm; P > 0.05). This decrement was followed by a subsequent increase in σ1R fluorescence intensity 48 hr after the plantar carrageenan injection (P < 0.05 - P < 0.0001). The same trend was also observed in the CGRP+ population of the DRG neurons, in the total population as well as in the CGRP+ small (<25 µm) and larger CGRP (>25 µm) sub-populations (P < 0.05 - P < 0.001). The presented results may contribute to further understanding of role of σ1R in the development of peripheral sensitization during inflammation. They may also be valuable for the therapeutic application of σ1R antagonists, particularly in the adjustment of the antagonist's dosage in a particular time window. Anat Rec, 302:1620-1627, 2019. © 2019 American Association for Anatomy.

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.

Changes in expression of special AT-rich sequence binding protein 1 and phosphatase and tensin homologue in kidneys of diabetic rats during ageing.

Background: Diabetic nephropathy (DN) is a common complication of diabetes mellitus (DM). We studied the expression of special AT-rich sequence binding protein 1 (SATB1) and phosphatase and tensin homologue (PTEN) in the kidneys of diabetic rats during ageing. Methods: Male Sprague Dawley rats were injected with 55 mg/kg streptozotocin (STZ) (DM group) or with citrate buffer (control group). Kidneys were collected after 2 weeks, 6 months and 12 months, and were analysed in three different kidney structures: glomeruli, proximal (PCT) and distal convoluted tubules (DCT). Sections were stained immunohistochemically, using SATB1 and PTEN. Results: Significant differences in marker expression were observed after 2 weeks, with higher SATB1 expression and lower PTEN expression in diabetic rats. PTEN was more highly expressed in controls after 6 and 12 months. After 12 months, there was higher SATB1 expression in diabetic rats. In the glomeruli, control rats had higher PTEN expression, whereas diabetic rats had higher SATB1 expression, after 12 months. PTEN expression increased from 2 weeks to 12 months in both the PCT and DCT of control rats. SATB1 was expressed exclusively in the PCT of diabetic rats after 2 weeks, and its expression in the DCT was higher in controls. After 6 months, both the PCT and DCT showed higher SATB1 expression in diabetic rats. Conclusions: The major changes in expression of SATB1 and PTEN occur after 2 weeks of DM onset, particularly in the PCT, implying an early onset of pathophysiological changes in diabetic kidneys, which would normally occur with ageing. These findings help to contribute to our understanding of changes associated with DN and guide towards possible appropriate treatment modalities.

Changes in neurofilament 200 and tyrosine hydroxylase expression in the cardiac innervation of diabetic rats during aging.

Changes in sensory and sympathetic innervation during diabetes mellitus (DM) can be a predictor of arrhythmias, silent myocardial ischemia, and chronic heart failure, but knowledge about these changes is still unsatisfactory. We analyzed whether prolonged DM induces changes in density of sensory and sympathetic nerve terminals of rat's heart and whether it contributes to cardiomyopathy during aging. DM was induced by i/p injecting 55 mg/kg streptozotocin to male Sprague-Dawley rats, while a control group received a citrate buffer. DM in the rats was validated by measuring blood glucose level. Animals were sacrificed after 2 weeks, 2 months, 6 months, and 12 months. Five areas of cardiac sections were analyzed. Antibodies raised against tyrosine hydroxylase (TH) and neurofilament 200 kDa (NF 200) were used to detect sympathetic and sensory fibers. TH immunoreactive fiber density increased in DM groups 2 weeks after induction, reaching a peek after 2 months, while in the later stages of DM (6 and 12 months), there was no significant difference compared to control. NF 200 immunoreactive fiber density increased 2 weeks after induction compared to control. There was no consistent pattern of change during the given period in both the DM or control groups. In the DM group, we found thickening of the left ventricle wall (P<.05) as the sign of cardiomyopathy. Our findings suggest that hyperglycemia as a hallmark of DM in early stages can lead to proliferation of sympathetic and sensory nerve terminals. This finding can contribute to a better understanding of the occurrence of arrhythmias and silent myocardial ischemia in DM.

Regulation of voltage-gated Ca(2+) currents by Ca(2+)/calmodulin-dependent protein kinase II in resting sensory neurons.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0µM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent.

Expression of calcium/calmodulin-dependent protein kinase II and pain-related behavior in rat models of type 1 and type 2 diabetes.

BACKGROUND: Abnormalities in peripheral nerves and dorsal root ganglia are noticed in the early stage of experimentally provoked diabetic neuropathy. Enzyme calcium/calmodulin-dependent protein kinase II (CaMKII) may have a modulating role in diabetic neuropathy because of its role in calcium homeostasis. METHODS: A model of type 1 diabetes mellitus (DM1) was induced with 55 mg/kg of the streptozotocin and for DM2 induction a combination of high-fat diet and low-dose streptozotocin (35 mg/kg) was used. Pain-related behavior was analyzed using thermal and mechanical stimuli. Two weeks and 2 months after induction of diabetes rats were euthanized, and the expression of CaMKII and its isoforms in the dorsal root ganglia were analyzed using immunofluorescence. RESULTS: Both types of diabetes were successfully induced, as confirmed by hyperglycemia. Increased pain-related behavior became evident in DM1 rats in 2 weeks after diabetes induction, but not in DM2 rats. The expression of total CaMKII and the phosphorylated α isoform of CaMKII increased in DM1 animals concurrently with pain-related behavior. Expression of α, ß, γ, and δ isoforms in DM1 animals and expression of total CaMKII and all of its analyzed isoforms in DM2 animals remained unchanged. CONCLUSIONS: Our findings may indicate involvement of CaMKII in transmission of nociceptive input early in DM1, but not in DM2. CaMKII may be a suitable pharmacological target for diabetic neuropathy.

Sex differences in pain-related behavior and expression of calcium/calmodulin-dependent protein kinase II in dorsal root ganglia of rats with diabetes type 1 and type 2.

Sex differences in pain-related behavior and expression of calcium/calmodulin dependent protein kinase II (CaMKII) in dorsal root ganglia were studied in rat models of Diabetes mellitus type 1 (DM1) and type 2 (DM2). DM1 was induced with 55mg/kg streptozotocin, and DM2 with a combination of high-fat diet and 35mg/kg of streptozotocin. Pain-related behavior was analyzed using thermal and mechanical stimuli. The expression of CaMKII was analyzed with immunofluorescence. Sexual dimorphism in glycemia, and expression of CaMKII was observed in the rat model of DM1, but not in DM2 animals. Increased expression of total CaMKII (tCaMKII) in small-diameter dorsal root ganglia neurons, which are associated with nociception, was found only in male DM1 rats. None of the animals showed increased expression of the phosphorylated alpha CaMKII isoform in small-diameter neurons. The expression of gamma and delta isoforms of CaMKII remained unchanged in all analyzed animal groups. Different patterns of glycemia and tCaMKII expression in male and female model of DM1 were not associated with sexual dimorphism in pain-related behavior. The present findings do not suggest sex-related differences in diabetic painful peripheral neuropathy in male and female diabetic rats.