Investigating the Role of Ischemia vs. Elevated Hydrostatic Pressure Associated with Acute Obstructive Uropathy

Obstructive uropathy can cause irreversible renal damage. It has been hypothesized that elevated hydrostatic pressure within renal tubules and/or renal ischemia contributes to cellular injury following obstruction. However, these assaults are essentially impossible to isolate in vivo. Therefore, we developed a novel pressure system to evaluate the isolated and coordinated effects of elevated hydrostatic pressure and ischemic insults on renal cells in vitro. Cells were subjected to: (1) elevated hydrostatic pressure (80 cm H₂O); (2) ischemic insults (hypoxia (0% O₂), hypercapnia (20% CO₂), and 0 mM glucose media); and (3) elevated pressure + ischemic insults. Cellular responses including cell density, lactate dehydrogenase (LDH) release, and intracellular LDH (LDH_i), were recorded after 24 h of insult and following recovery. Data were analyzed to assess the primary effects of ischemic insults and elevated pressure. Unlike pressure, ischemic insults exerted a primary effect on nearly all response measurements. We also evaluated the data for insult interactions and identified significant interactions between ischemic insults and pressure. Altogether, findings indicate that pressure may sub-lethally effect cells and alter cellular metabolism (LDH_i) and membrane properties. Results suggest that renal ischemia may be the primary, but not the sole, cause of cellular injury induced by obstructive uropathy.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

目的 探讨结缔组织生长因子(CTGF)对肾小管上皮细胞向间充质细胞转化的影响.方法 体外培养的人肾小管上皮细胞(HK-2),按是否用CTGF处理将其分为实验组和对照组.直接免疫荧光法、间接免疫化学检测培养72 h后肾小管上皮细胞E-cadherin、波形蛋白、α-平滑肌肌动蛋白((α-SMA)、细胞外信号调节激酶2(ERK2)的表达;Western blot方法 在72 h检测E-cadherin、波形蛋白和ERK2的表达;Boyden小室在1、3、5 d检测细胞迁移能力的变化.结果 (1)实验组上皮细胞表型标志蛋白E-cadherin随着时间的延长而表达递减,波形蛋白表达渐增,ERK2表达上调;(2)在第1天,实验组与对照组细胞迁移能力差异无统计学意义,第3天实验组迁移至滤膜下面细胞多于对照组,第5天实验组细胞迁移数明显高于对照组[(45.0±1.1):(14.0±1.2),P<0.05].结论 在体外,人肾小管上皮细胞由CTGF刺激表现出向间充质细胞转化的特性,并且ERK2信号转导通路可能参与了对这一过程的调控效应.     

糖尿病大鼠AGEs与nephrin表达的研究

目的：探讨终末期糖基化产物（AGEs）、肾病蛋白（nephrin）在糖尿病大鼠肾脏组织中的表达及其在糖尿病肾病发生、发展中的作用。方法：用免疫组化方法检测链脲佐菌素诱发糖尿病及正常对照SD大鼠肾脏中AGEs、nephrin的表达,并计算其阳性指数,同时测定各组大鼠24 h尿蛋白定量及肾功能等一般生化指标,并对大鼠肾脏AGEs、nephrin与蛋白尿进行相关分析。结果：与对照组相比,糖尿病组大鼠肾小球上AGEs表达明显增加（P〈0.01）,nephrin表达明显减少（P〈0.01）,24 h尿蛋白排泄量明显增加（P〈0.01）,大鼠肾小球AGEs的表达和nephrin的表达呈负相关（r=-0.921,P〈0.01）;nephrin蛋白表达与24 h尿蛋白排泄量呈负相关（r=-0.904,P〈0.01）;AGEs表达与24 h尿蛋白排泄量呈正相关（r=0.974,P〈0.01）。结论：AGEs可能介导了nephrin的表达缺失,进而导致足细胞损伤并加速了糖尿病肾病的进展。     

Effect of desensitising toothpastes on dentinal tubule occlusion: A dentine permeability measurement and SEM in vitro study

Objectives

The purpose of this study was to evaluate the in vitro effectiveness of a new bioglass-containing and two commercial desensitising toothpastes on dentinal tubule occlusion after citric acid challenge or artificial saliva (AS) immersion.

Methods

One hundred dentin discs from human third molars were used. Specimens were randomly divided into five groups (n = 20), Group 1: EDTA-treated dentin; Group 2: brushing with distilled water; Group 3: brushing with Novamin; Group 4: brushing with Sensodyne Freshmint; Group 5: brushing with Colgate Sensitive. In each group, samples were then equally split into two subgroups (n = 10) to test two post-treatments: 6% citric acid challenge or 24 h immersion in artificial saliva. Dentine permeability of each specimen was measured before and after each treatment using a hydrostatic device working at 20 cm H₂O pressure. Data were analysed by two-way repeated measures ANOVA to determine if there were any significant differences within or between groups. Dentine morphology and surface deposits were observed by SEM.

Results

All three desensitising toothpastes significantly reduced dentine permeability and created precipitates on the treated dentine surfaces. Moreover, the reductions in dentine permeability showed partial recovery after a citric acid and artificial saliva immersion. Sensodyne showed significant resistant to acid attack and Novamin exhibited the lowest permeability after artificial saliva immersion for 24 h.

Conclusions

The application of the three toothpastes resulted in effective dentinal tubule occlusion. However, the new bioglass-containing toothpaste (Novamin) represented excellent occlusion effects after brushing treatment and AS immersion, while Sensodyne demonstrated more reduction in permeability when citric acid challenged.     

Dentine desensitization induced by prophylactic and air-polishing procedures: An in vitro dentine permeability and confocal microscopy study

Objectives

The exposure of dentinal tubules causes fluid movement and dentinal hypersensitivity. This study aimed at evaluating the dentine permeability after prophylactic measures performed on exposed dentine after immersion in artificial saliva and citric acid challenge. Confocal microscopy was performed to evaluate the percentage of occluded tubules (OCT%) and the changes in dentine morphology.

Methods

Prophy-powders and pastes were tested in this study. An oxalic acid liner was used as a positive control. Dentine discs from human third molars were treated with each material and the dentine permeability was evaluated using a fluid filtration system working at 20 cm H₂O. Artificial saliva and citric acid were used for the determination of changes in dentine permeability. The percentage of tubule occlusion capability (OCT%) was evaluated using confocal microscopy.

Results

All the products used in this study were able to significantly reduce the dentine permeability of acid-etched specimens. The use of the bioactive glass and sodium bicarbonate showed the highest values in dentine permeability reduction. However, the air-polishing procedures performed with Sylc bioactive glass powder created a dentine surface resistant to citric acid attack.

Conclusion

Bioactive glass is suitable for treatment of dentinal hypersensitivity by creating a dentine surface resistant to citric acid attack.     

Water permeability,hybrid layer long-term integrity and reaction mechanism of a two-step adhesive system

Objectives

Our aim was to investigate the reaction mechanism of formation of the hybrid layer by a HEMA-containing self-etch adhesive and to study fluid filtration, contact angle and interfacial ultrastructure by SEM following a 1 year ageing period.

Methods

Acidic behaviour and chemical interactions between Silorane System Adhesive and dentine were studied by potentiometric titrations, atomic absorption spectroscopy and infrared spectroscopy. The hydrophilicity of the adhesive was evaluated using the sessile drop method and dentine permeability by hydraulic conductance. The morphological study of the dentine/adhesive system interface was conducted using SEM.

Results

The Silorane System Adhesive behaved as a multi-acid with several different pK_a values. When the adhesive was in contact with dentine, the acid was progressively consumed and calcium ions were released. The acrylate substituted phosphonate bound strongly to apatite crystals. The polyacrylic acid copolymer reacted with calcium ions and formed an interpenetrating polymer network (IPN). Water contact angle measurements showed rapid spreading on primer (angles reached 15° at 30 s) and larger contact angles when the Silorane bonding layer was added (from over 60° to 44°). A thick, homogeneous hybrid layer was observed both initially and after 1 year of ageing, with a corresponding hydraulic conductance of −48.50% initially and −52.07% at 12 months.

Conclusion

The Silorane System Adhesive is capable of both dissolving calcium ions and binding to apatite surfaces. The results showed the hydrophilicity of the adhesive, which formed an IPN-like hybrid layer that conserved adequate impermeability over a 1-year period.     

Influence of the degree of dentine mineralization on pulp chamber temperature increase during resin-based composite (RBC) light-activation

Objectives

To analyse the influence of the degree of dentine mineralization on the pulp chamber temperature increase during composite light-activation.

Methods

Dentine discs (2 mm thick) obtained from recently extracted teeth or those with extensive dentine sclerosis were analysed by FT-IR spectrometry in order to choose the two discs with the greatest difference in the degree of mineralization. A model tooth was set up with the dentine discs between a molar with the pulp chamber exposed and a crown with a standardized class II cavity. A K-type thermocouple was introduced into the molar root until it came into contact with the dentine discs and the cavity was filled with P60 resin composite. The temperature rise was measured for 120 s after light-activation began: Standard (S) 600 mW/cm²/40 s; Ramp (R) 0 → 800 mW/cm²/10 s + 800 mW/cm²/10 s; Boost (B) 850 mW/cm²/10 s and LED (L) 1.300 mW/cm²/40 s (n = 10). The same protocol was repeated after grinding the dentine discs to 1.0 and 0.5 mm thickness.

Results

The temperature increase was significantly higher in dentine with high degree of mineralization (p < 0.05). With respect to the dentine thickness, the following result was found: 2 mm < 1 mm < 0.5 mm (p < 0.05). The light-activation mode also presented significant difference as follows: S > R = L > B (p < 0.05).

Conclusions

The higher the degree of dentine mineralization the greater the increase in pulp chamber temperature. The temperature increase was influenced by the light-polymerization mode and dentine thickness.     

Monitoring bacterial-demineralization of human dentine by electrochemical impedance spectroscopy

Objective

The purpose of this study was two-fold: (1) to monitor bacterial biofilm formation and bacteria-induced demineralization of dentine in situ by using electrochemical impedance spectrum (EIS); (2) to examine the relationship between EIS findings and changes in the chemical composition and ultrastructure of dentine during bacteria-induced demineralization.

Methods

In this study, dentine demineralization was induced by Streptococcusmutans (ATCC 25175) in the presence of sucrose in culture medium and was monitored using two EIS measurement systems (Type A with a working electrode and Type B without a working electrode). Scanning electron microscopy (SEM), field emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX) and X-ray diffraction (XRD) were employed to examine the morphology, element contents and crystallinity of hydroxyapatite (HAP) on the dentine surface. Transverse microradiography (TMR) was used to characterize the lesion depth and degree of mineral loss during demineralization.

Results

The resistance of the bulk dentine (R_d) and the apparent resistance of dentine (R_a) measured from the Type A and Type B EIS systems, respectively, decreased gradually with demineralization. The resistance of the biofilm formed on dentine surface was determined by fitting the EIS data with equivalent circuits. The presence of biofilm slightly increased R_a of dentine before demineralization. However, the electrochemical behavior of biofilm did not affect the decreasing impedance of dentine with demineralization. The SEM, EDX, XRD and TMR results demonstrated that the surface and bulk dentine gradually became more porous due to the loss of minerals during demineralization, which in turn resulted in the decrease in R_d and R_a values obtained from EIS systems.

Conclusions

This investigation highlighted EIS as a potential technique to monitor biofilm formation and bacterial-induced demineralization in situ.     

Evaluation of cystine transport in cultured human kidney cells and establishment of cystinuria type I phenotype by antisense technology

Cystinuria is a rare hereditary disease resulting in recurrent stone formation and the need for repeated invasive interventions. So far, two responsible genes have been identified which encode the two transporters, rBAT and b^0,+AT forming a heterodimer to transport cystine in proximal tubular cells (PTC) and whose defect results in increased excretion of cystine. A human cell line mimicing the phenotype of cystinuria in vitro is yet to be developed. Human kidney (HK)-2 is a PTC line derived from normal HK. After determining the presence of rBAT gene by RT-PCR and Western blot analysis, radioactively labeled cystine (S³⁵) was used to evaluate the functional presence of the amino acid transport in HK-2 cells when cultured in vitro. To achieve a cystinuria type I phenotype in HK-2 cells, the rBAT gene was silenced using antisense oligonucleotides complimentary to human rBAT mRNA. The reduced transport activity of cystine was then determined by radiolabeled cystine uptake measurements. RT-PCR and Western blot confirmed the expression of the rBAT gene in HK-2 cells. Considerable transport of the radio labeled cystine was observed in HK-2 cells and was linearly dependent on the incubation time with the amino acid. The cystine transport in rBAT knockdown cells after incubation with antisense oligonucleotides was significantly lower compared to control (0.76 vs. 0.98%; P = 0.0008), proving a transient knock-down of the rBAT gene. This study demonstrates the presence of the b^0,+ amino acid transport system in human proximal tubular HK-2 cells when cultured in vitro. Inhibition of this transport system is possible by using antisense technology. A permanent inhibition of the cystine transport, based on our model, would be useful for the development and evaluation gene therapeutic approaches. Gunnar Wendt-Nordahl, Sreedhar Sagi contributed equally to this work.     

Regulatory mechanism of “K<Superscript>+</Superscript>-recycling” for Na<Superscript>+</Superscript>-reabsorption in renal tubules

Hypertension is one of the predominant risk factors for the progression of renal impairment, and the most common disorder in industrialized societies. Because reduction of the systemic blood pressure in hypertension can halt the progression of renal impairment, it is imperative to appropriately control the systemic blood pressure. Recent genetic analysis has reconfirmed that renal maladjustment of Na⁺-homeostasis, which determines the extracellular fluid volume, is a key element in the pathogenesis of hypertension. The distal tubules adjust the net Na⁺-excretion according to Na⁺-ingestion and maintain the Na⁺-homeostasis. The distal convoluted tubules and the connecting tubules are the predominant sites for the adjustment in individuals with a modern lifestyle. In these tubules, Na⁺-reabsorption depends on “K⁺-recycling”, which is conducted through K⁺ channels. Because the functional expression of K⁺ channels in these tubules is regulated by signal motifs for intracellular localization, the adjustment of “K⁺-recycling” through the modification of signal motifs could be a new target for the treatment of hypertension.