The Involvement Of Endoplasmic Reticulum Stress Associated Apoptosis In Detrusor Impairment In Diabetic Cystopathy

Diabetes mellitus(DM) is currently one of the most common chronic metabolic diseases all over the world, which can affect multiple organ systems progressively and seriously. As the most important urologic complication of DM, diabetic cystopathy(DCP) has a prevalence ranging from 25% to 83%, and is characterized by many urological dysfunctions including detrusor overactivity, diminished first sensation, elevated maximal cystometric capacity, impaired bladder compliance, and increased postvoid residual. An accumulating body of evidence has indicated the possible pathogenesis of DCP is a multifactorial and profound process involved with the structural and functional impairments of DSM, the diabetic induced nerve damage, and the secondary alterations of urothelium, in which DSM could be considered the dominant target and the site of pathogenesis effect. Nevertheless, the exact mechanisms of DCP are still far from clear, and the promising prevention and treatments to this refractory disease remain to be explored.Apoptosis, which can result in excessive cell loss, has been reported to play a critical role in the development of DCP by several studies. As an important subcelluar network with the membranous structure, the endoplasmic reticulum(ER) is associated with the intrinsic pathway of apoptosis and has been suggested to be pivotal in the lipid biosynthesis, protein folding, and protein maturation. Many adverse events, such as hypoxia, calcium homeostasis, gene mutation, free radical stimulus, as well as the enhanced protein synthesis, can perturb the functions of ER and lead to the accumulation of unfolded or misfolded proteins consequently. This process is defined as the activation of the ERS. Under the moderate ERS condition, the proteins still can be folded, packaged, and modified accurately in the ER with the help of an effective self-screening mechanism named unfolded protein response(UPR). The process of protein translation will be halted, following the repair to the unfolded or misfolded proteins by upregulating the expression of ER resident molecular chaperons, such as GRP78. In the early stage of ERS, the UPR can attenuate cellular stress and restore the normal functions of the impaired cell, but if the disturbance is prolonged along with the more severe ERS condition, the UPR will activate the cell apoptotic pathway to guarantee quality control of cells. In the process of ERS-related apoptosis, CHOP is a key initiator, and the activation of Caspase12-dependent pathway is considered another evident manifestation.The ERS-related apoptosis has been suggested to play a critical role in the development of many diabetic complications. However, it remains unknown whether ERS is involved in the pathogenesis of DCP. In our study partⅠ, we investigated the ERS-associated apoptosis, as well as the expressions of three ERS-hallmarked proteins in DSM of different STZinduced diabetic rats. Furthermore, we designed a cell experiment as the study part Ⅱ,providing the high-glucose stimulation in vitro with differenrt durations as the simulation of the hyperglycemia environment in vivo, to investigate the relationship between ERSassociated apoptosis and high-glucose stimulation in high-glucose cultured detrusor smooth muscle cells(DSMCs).Chapter 1 The study on the involvement of endoplasmic reticulum stress associated apoptosis in detrusor impairment in STZ-induced diabetic ratsPart 1 The evaluation of time-dependent changes on bladder function in STZ-induced diabetic ratsObjective: To evaluate and characterize the time-dependent changes on bladder function in STZinduced diabetic rats with the progressionMethods:A total of 50 female Sprague–Dawley rats were used in the present study, and they were randomly divided into five groups(n=10 for each group): normal control, DM for 4 weeks(DM4w), DM for 8 weeks(DM8w), DM for 12 weeks(DM12w), and DM for 16 weeks(DM16w). Diabetes was induced by a single intraperitoneal injection of STZ in DM4 w, DM8 w, DM12 w, and DM16 w group. The rats in the normal control group were all treated with vehicle only. After 3 days of the injection, the tail venous blood samplings from all the rats were collected and the blood glucose concentration of each rat was measured. When the blood glucose level was higher than 300 mg/d L, the diabetic rat model was considered to be induced successfully, and was used in the following experiments. Each group was maintained under the identical conditions. The glucose levels and body weight of the rats in each group were detected and recorded before the diabetic induction as well as their euthanization. The bladder weight and bladder weight/body weight were also measured and calculated before the euthanization. Nonstop transvesical CMG was performed in each group and the dynamic parameters including bladder capacity, detrusor leak point pressure(DLPP) and complicance were measured; then full-thickness longitudinal strips approximately 7 to 12 mg and 10×2 mm were prepared from the dorsal part of the bladder body, isometric contraction was recorded.Results: Among the 40 STZ-injected rats, 35 rats reached the diabetic rat model standard of a fasting blood glucose level higher than 300 mg/dl(nine in DM4 w group, eight in DM8 w group, nine in DM12 w group, and nine in DM16 w group). For each group, the numbers of rats that survived at the timepoints of euthanization are as follow: nine in control group, seven in DM4 w group, eight in DM8 w group, eight in DM12 w group, and eight in DM16 w group. Compared with the control group, the mean blood glucose levels in all four diabetic groups elevated significantly, whereas the mean body weight in each diabetic group showed a significant decrease. CMG indicated the mean bladder capacity, DLPP and bladder compliance in 4 diabetic rat groups were all higher than the control group; the mean contractions of strips from diabetic rat groups were also higher than those in controls rats.Conclusion: Diabetes can induce time-dependent impairment to bladder function including the compliance, excitability and autorhythmicity. The compensated alteration of muscle contraction can been seen. The 8th-12 th week after the diabetic induction might be viewed as the the transitional period from the compensated stage to the decompensated stage.Part 2 The observation of time-dependent changes on detrusor morphology in STZ-induced diabetic ratsObjective: To investigate the light-microscopic and ultrastructural changes of detrusor in STZinduced diabetic rats with the diabetes progressionMethods: The STZ-treated rats were euthanized at 4, 8, 12, and 16 weeks(correspondence to DM4 w, DM8 w, DM12 w, and DM16 w group, respectively) after their induction into type 1 diabetic models, and the normal control rats were sacrificed after 3 days of the vehicle injection when the other four STZ-treated groups were confirmed with their establishment of diabetic model. The bladders were harvested soon after the euthanization and the DSM tissues were dissected with the removal of mucosa by using the cotton swabs under a dissecting microscope. The prepared DSM tissue of each rat was sliced into four blocks equally and each block received specific preprocessing immediately depending on the different further biochemical and morphological studies. The DSM tissue samples were fixed in 4% neutral formalin for over 24 h, and then were embedded into paraffin. After cutting into 5-mm thick sections, some of them were further stainned with H&E and the DSM specimen slices of each group were observed under a light microscope; some of them were stainned with Van Gieson solution, the collagen fibers in the DSM specimen slices of each group were observed and their proportions were calculated. The images were captured and saved at 100× magnification by a digital pathology biosystem. Meanwhile, another tissue block of DSM was sliced into 1-mm3 samples and then was placed into 2.5% glutaraldehyde at 4 ℃ for 2 h. After three-time rinsing in sodium cocodylate buffer, the samples were postfixed in 1% osmium tetroxide for 2 h and were dehydrated through graded ethanol. Then, the specimens were embedded in epoxy resin 618 following, the ultra-thin sections were fabricated and stained with 1% uranyl acetate. A transmission electron microscope(TEM) was used to observe and investigate the ultrastructural changes of DSM cells. The digital images were photographed at 25000–30000× magnification.Results: The progressive alterations in histology and ultrastructure along with the prolonged diabetic duration can be seen. The light-microscopic observation and analysis on the H&E stained slices revealed the diabetic DSM fibers in DM4 w group still organized neatly. With the progression of diabetic course, the muscle fiber bundles became disordered and some of them loosely packed and disrupted, indicating the loss of integrity. The muscle bundles became atrophic, and the loose fibrous tissue was seen in between muscle bundles. With the diabetic progression, the proportion of collagen fibers showed a significant climbing trend. As for the ultrastructural changes, the TEM images revealed more detailed pathological alterations at the cellular organelle level. The DSM ultrastructure in the control samples was normal in general. There was no significant swelling of the organelles, and the structure in ER zone was tidy without fractures. Compared with the control group, some swelled cisternaes started to show in the DM4 w group mostly in a scattered distribution style. The mild particle fusion and degranulation also could be seen in the zone of ER. The nucleus and chromatin did not show significant changes as compared with the control ones. As the duration progressed, the severity of dilation in cisternaes worsened obviously in the DM8 w group. Moreover, from the initial dispersed style, the swollen cisternaes in ER zone fused into bigger and irregular-shaped vesicles, adjacent to and in which the enhanced particle fusion and degranulation presented. In the DM12 w rats, the destruction to the structure in ER zone aggravated. The fused cisternaes enlarged and the normal structure of mitochondria was damaged significantly and difficult to be identified. Meanwhile, the distorted or pyknotic nucleus contained clusters of unidentified dark-stained granules that could be observed in some of the DSM cells. The more severe subcelluar pathologic manifestations, including highlybubbled ER zone, twisted, and collapsed nucleus, could be seen in the TEM images of the DM16 w rats.Conclusion: Morphological damages to the DSM became augmented with longer duration of diabetes: the time-dependent alterations were characterized with the progressive collapse of muscle bundles under light-microscopy, corresponding to the electron-microscopic organelle destruction. The elevated proportion of collagen fibers indicates the aggrevated bladder fibrosis and impaired compliance in DCP.Part 3 The assessment of detrusor cell proliferation and apoptosis in different phases of STZ-induced diabetic ratsObjective: To evaluate the proliferative and apoptotic levels of DSMCs in the development of diabetic cystopathy.Methods:A commercially available PCNA and TUNEL assay kit were used for the evaluation of DSM cell proliferation and apoptosis respectively. The SABC staining was used in the detection of PCNA expression. Three fields of vision(400×) were randomly selected and the number of PCNA or TUNEL positive cells was counted in a digital pathology biosystem. The percentage of PCNA-positive cells and the apoptotic index(AI)(the TUNEL-positve cells relative to the number of total cells) were calculated eventually.Results: Four time-pointed calculation of the PCNA-positive cell percentage, which indicates the level of cell proliferation, reached its peak at DM8 w point and then showed a relative declined trend. Meanwhile, a significantly higher percentage of TUNEL-positive cell was observed in each STZ-treated group compared with the normal rats, indicating a significantly elevated apoptosis in DSM due to diabetes.Conclusion: The proliferation of DSMCs experiences a rise-and-fall alteration with the progression of DCP in STZ-induced diabetic rats. The 8th to12 th week after the diabetic induction was the vital period for the trend reversal. A uniform elevation of DSMCs apoptosis can be seen during the diabetic progression in STZ-treated rats.Part 4 The study on the involvement of ERS-associated apoptosis in the progression of diabetic cystopathyObjective:To explore the involvement of ERS-associated detrusor muscle apoptosis in STZinduced diabetic rats.Methods: The expressions of GRP78, CHOP, and Caspase12 protein and m RNA in the DSM tissue from controls and diabetic rats at different time points were analyzed using Western blotting and q RT-PCR respectively. We set β-actin as the loading control in Western blotting. The band optical intensity quantification was normalized to β-actin. The m RNA expressions were also normalized to the internal abundance of m RNA for β-actin. Pearson’s correlation analysis was used to investigate the relationship between the expression of GRP78 and PCNA-positive cell percentage and the relationship between the expressions of CHOP, Caspase12 and apoptotic index.Results: The results of Western blotting showed GRP78, CHOP, and Caspase12 proteins in the DSM as single bands migrating at 78, 31, and 45 k Da respectively. The densitometric analysis of the bands for all three observed proteins revealed the significant increases in relative abundance at all four time points in comparison with the normal control group. For all four diabetic groups, in spite of the overall elevated expression compared with the control, the detailed patterns varied regarding to the different proteins: the upregulated expression of GRP78 protein reached its peak at the eighth week(DM8w point) after the diabetic confirmation and then showed a relative declined trend, while both CHOP and Caspase12 expressions showed a uniform increase over time. The m RNA expression of GRP78, CHOP, and Caspase12 was significantly increased in all four diabetic groups at all four time points compared with the normal control group. The expression of GRP78 peaked at the 12 th week(DM12w), while CHOP and Caspase12 still maintained the same climbing trend, in parallel with their enhanced protein expressions. Pearson’s correlation analysis revealed thatCaspase12 and CHOP positively correlated with the apoptotic index, and showed GRP78 positively correlated with PCNA-positive cell percentage.Conclusion: ERS-associated apoptosis and UPR exist simultaneously in the course of DCP. The increasingly enhanced ERS-associated apoptosis is an important pathophysiological event with significant negative effects during the progression of DCP. For UPR, the 8th-12 th week after the diabetic induction might be viewed as the the transitional period from the compensated elevating stage to the decompensated declining stage. The occurrence of ERSassociated apoptosis may be involved in the development of DCP and may contribute to the diabetic detrusor impairment.Chapter 2 The study on the effect of endoplasmic reticulum stress in apoptosis of rat detrusor smooth muscle cells stimulated by high glucose in vitroPart 1 The isolating, primary culturing, passaging and identifying of rat detrusor smooth muscle cells in vitroObjective: To provide well-identified DSMCs with good activity for the following experiments via suitable isolating, primary culturing, passaging and identifying techniquesMethods:The detrusor tissue from healthy SD rats were isolated and purified using the method of collagenase Ⅱ(4mg/m L) digestion, and the isolated cells were primary cultured and passaged. The expression of α-smooth muscle actin was detected by immunofluorescent staining and was observed under laser confocal microscope to identify the DSMCs.Results: The primary cultured rat DSMCs with spindle shape were observed under the light microscope. The cell spreading and attachment can be seen after 8 hours of primary culturing, indicating the well activity. Specific green fluorescence of ɑ-SMA in the cytoplasm and blue fluorescence of nucleus were presented under laser confocal microscope. Combined with the specific spindle shape and the results of immunofluorescent staining above, the DSMCs were identified perfectly.Conclusion: The method of collagenase Ⅱ(4mg/m L) digestion is rapid, convenient and highly efficient, which is able to isolated and purified rat DSMCs with well activity. The DSMCs isolated in 24-48 h have enormous proliferative capacity and can meet the following research requirements.Part 2 The observation and evaluation of time-dependent changes on ultrastructural morphology in high-glucose stimulated DSMCs in vitroObjective:To investigate the ultrastructural changes of DSMCs with the time of high-glucose stimulation extended.Methods: The DSMCs were synchronized and were stimulated with normal glucose(NG group)(5 m M) or high glucose(HG group)(25 m M). After the indicated culturing for 12, 24, 48 and 72 h, the cells in each group were collected. The DSMCs was centrifuged and sedimented into 1-mm3 samples and then was placed into 2.5% glutaraldehyde at 4 ℃ for 2 h. After three-time rinsing in sodium cocodylate buffer, the samples were postfixed in 1% osmium tetroxide for 2 h and were dehydrated through graded ethanol. Then, the cell specimens were embedded in epoxy resin 618 following, the ultra-thin sections were fabricated and stained with 1% uranyl acetate. The ultrastructure of DSMCs in each group was observed under TEM.Results: The progressive alterations in ultrastructure along with the prolonged high-glucose stimulation was insignificant. The TEM images of DSMCs in the samples of NG group was normal in general and there was no significant progression with the time extended. No swelling of the organelles, and the structure in ER zone was tidy without fractures. Compared with the NG group, DSMCs in HG group at 12 h and 24 h timepoints did not show significant difference in ultrastructural morphology. When the time of stimulation reached the point of 48 h in HG group, some swelled cisternaes started to show. The mild degranulation could be seen in the zone of ER. Moreover, the karyothec of some DSMCs became deshaped and irregular with some folded zone. As the duration progressed, in spite of the distorted or pyknotic nucleus contained clusters of unidentified dark-stained granules that could be observed in some of the DSMCs in HG group at 72 h timepoint, the severity of deformation in the zone of ER did not worsen obviously. Besides, the apoptotic body, which is characteristic symbol of apoptosis, could be seen in every timepoint of both groups.Conclusion: With longer duration of high-glucose stimulation, the morphological damages to the DSMCs did not show a typical mode of time-dependent organelle destruction.The apoptotic bodies could be seen in every timepoint of both groups.Part 3 The assessment of cell proliferation and apoptosis in different phases of high-glucose stimulated DSMCsObjective: To evaluate the proliferative and apoptotic levels of DSMCs at different timepoint of high-glucose stimulation.Methods: MTT colorimetry was used for the evaluation of DSM cell proliferation. The optical densities(OD) in each group at four different timepoints were detected. The Annexin VFITC/PI double-staining and flow cytometry were used for the evaluation of DSM cell apoptosis. The early, late and total apoptotic rates were calculated.Results: Four time-pointed detections of the ODs revealed the significant lower levels of DSM cell proliferation in HG group than in NG group at 24 h, 48 h and 72 h timepoints(P<0.05). Within single group, the NG group showed a significant elevated curve of cell proliferation level. In contrast, the HG group presented a significant declined trend. Meanwhile, the significantly higher early, late and total apoptotic rates were observed in HG group at timepoints of 24 h, 48 h and 72 h compared with the NG group(P<0.05), indicating a significantly elevated apoptosis in DSMCs due to the stimulation of high-glucose.Conclusion: The high-glucose stimulation in vitro has the significant effect of proliferation suppression and apoptosis induction, and this effect became augmented with the time of stimulation extended.Part 4 The assessment of the involvement of ERS-associated apoptosis in the high-glucose stimulated DSMCs in vitroObjective: To explore the involvement of ERS-associated detrusor muscle apoptosis in the highglucose stimulated DSMCs in vitro.Methods: The expressions of GRP78, CHOP, and Caspase12 protein and m RNA in the DSMCs from HG groups at four different timepoints as well as NG group, were analyzed using Western blotting and q RT-PCR respectively. We set β-actin as the loading control in Western blotting. The band optical intensity quantification was normalized to β-actin. The m RNA expressions were also normalized to the internal abundance of m RNA for β-actin. Pearson’s correlation analysis was used to investigate the relationship between the expressions of CHOP, Caspase12 and apoptotic rate measured by flow cytometry.Results: The densitometric analysis of the Western blotting bands for all three observed proteins revealed the significant increases in relative abundance in HG group at all four time points in comparison with the NG group. In spite of the overall elevated expression compared with the NG group, the detailed patterns varied regarding to the different proteins in HG group: the upregulated expression of GRP78 protein reached its peak at 24 h and then showed a relative declined trend, while both CHOP and Caspase12 expressions showed a uniform increase over time. Compared with the NG group, the m RNA expressions of GRP78, CHOP, and Caspase12 in HG group were significantly increased at all four time points. The m RNA expression of GRP78 peaked at 24 h, while CHOP and Caspase12 still maintained the same climbing trend, in parallel with their enhanced protein expressions. Pearson’s correlation analysis revealed that Caspase12 and CHOP positively correlated with the apoptotic rate.Conclusion: Both of the increasingly enhanced ERS-associated apoptosis and the compensateddecompensated transition of UPR are two distinctive pathophysiological characters within the duration of high-glucose stimulation to DSMCs in vitro. Hyperglycemia can induce ERSassociated apoptosis in the progression of DCP directly.