Jen Ai Lee

Aristolochic acid-induced accumulation of methylglyoxal and Ne-(carboxymethyl)lysine: An important and novel pathway in the pathogenic mechanism for aristolochic acid nephropathy

Aristolochic acid, found in the Aristolochia species, causes aristolochic acid nephropathy (AAN) and can develop into renal failure. Methylglyoxal (MGO) is a highly cytotoxic compound generated from the metabolic process of glucose or fatty acids. It binds to proteins and forms N(ε)-(carboxymethyl)lysine (CML), which contributes to aging and diabetes mellitus complications. However, no relevant literature explores the relationship of MGO and CML with AAN. By injecting AA (10mg/kg BW) into C3H/He mice for 5 consecutive days, we successfully developed an AAN model and observed tubular atrophy with decreased renal function. Creatinine clearance also decreased from 10.32 ± 0.79 ml/min/kg to 2.19 ± 0.29 ml/min/kg (p<0.01). The concentration of MGO in kidney homogenates increased 12 × compared to the control group (from 18.23 ± 8.05 μg/mg of protein to 231.16 ± 17.57 μg/mg of protein, p<0.01), and CML was observed in the renal tubules of the mice by immunohistochemistry. Furthermore, compared to the control group, GSH levels decreased by 0.32 × (from 2.46 ± 0.41 μM/μg of protein to 0.78 ± 0.15 μM/μg of protein, p<0.01), whereas intra-renal antioxidant capacity decreased by 0.54×(from 6.82 ± 0.97 U to 3.71 ± 0.25 U; unit is equivalent to μM Trolox/mg of protein, p<0.01). In this study, we found that serious kidney damage induced by AA is related to an increase and accumulation of MGO and CML.

Determination of time-dependent accumulation of d-lactate in the streptozotocin-induced diabetic rat kidney by column-switching HPLC with fluorescence detection

For better understanding the complete metabolism and the physiological role of D-lactate, the concentrations of D-lactate in the serum, liver and kidney of normal and diabetic rats were determined by our established column-switching HPLC method with pre-column fluorescence derivatization. Eight-week-old male Sprague-Dawley rats were administered with streptozotocin (STZ) (80 mg/kg) or citrate buffer intraperitoneally. The tissues were then removed and homogenized after 4, 8, 12 and 16 weeks of drug administration, respectively. The homogenates were centrifuged at 1200 × g for 10 min, then the supernatants were derivatized with a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), separated on an ODS column followed by a Chiralpak AD-RH chiral column for enantioseparation. The results showed that the D-lactate content elevated in all the 3 examined tissues under diabetic stages. In addition, D-lactate concentrations in rat kidney were accumulated significantly and time-dependently in diabetic groups after receiving STZ for 4, 8, 12 and 16 weeks (2.99, 13.11, 18.19, 23.23 vs. 0.79 μmol/mg protein as control group). Moreover, the kidney of induced 12-week diabetic rat renal showed some histological changes of progressive diabetic nephropathy. The results suggest that D-lactate may be used as a marker of diabetic nephropathy.

Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity.

AIMS Aristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and N(ε)-(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger. METHODS Mice were orally administered water or metformin for 15 days (12 or 24 mg kg(-1)day(-1)), and injected AA (5mgkg(-1)day(-1)) intraperitoneally for 8 days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed. KEY FINDINGS Compared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p<0.05). Further, compared to control treatment, administration of 12 mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33 ± 9.78 vs. 5.89 ± 2.64 μg/mg of protein, respectively, p<0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity. SIGNIFICANCE This study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further.

Urinary d-lactate levels reflect renal function in aristolochic acid-induced nephropathy in mice.

Urinary d-lactate is highly correlated to diabetic nephropathy - a progressive kidney disease in renal glomeruli. In this study, we used a C3H/3e mouse model to investigate the relationship between urinary d-lactate and aristolochic acid nephropathy where the glomerular structure is not affected. The nephropathy was induced using intravenous injections of aristolochic acid at a dosage of 10 mg/kg per day for 5 days and was characterized biochemically and histologically. The urinary excretions of proteins, N-acetyl-β-d-glucosaminidase and serum creatinine were determined and connected to histological conventional findings. Urinary d-lactate was analyzed using column-switching high-performance liquid chromatography with fluorescence detection. The results showed a remarkable increase of urinary markers, including of urinary proteins and N-acetyl-β-d-glucosaminidase, and the histological examination confirmed a diagnosis of acute tubule necrosis. The ratio of d-lactate to creatinine in the urine of aristolochic acid-treated mice was approximately 36 times greater than that of the mice in the control group (p < 0.05). The ratios for the two groups of mice were 311.00 ± 71.70 and 8.60 ± 1.80 µmol/mmol creatinine, respectively. These data confirm in vivo that urinary d-lactate reflects renal injury conditions in aristolochic acid-treated mice and may be a marker for the assessment of nephropathy.

Elevated urinary d-lactate levels in patients with diabetes and microalbuminuria

Diabetic nephropathy (DN) has become the major cause of end-stage renal disease. Early detection of disease risk, to enable intervention before advanced renal damage occurs, is an important goal. Microalbuminuria has been used to monitor renal damage in clinical settings for years. In this study, we divided patients with diabetes into different groups based on their microalbumin values to elucidate the relationship between urinary D-lactate and corresponding microalbumin values. Group DM1 comprised of patients with an albumin-to-creatinine ratio (ACR) of less than 30 μg albumin/mg creatinine (normal range); Group DM2 comprised of patients with an ACR of 30-299 μg albumin/mg creatinine (microalbuminuria); and Group DM3 comprised of patients with an ACR of ≥300 μg albumin/mg creatinine (macroalbuminuria). The urinary D-lactate concentration of patients with diabetes was determined by pre-column fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), and the accuracy (recovery) and precision (relative standard deviation; RSD) were validated. The measured values showed an accuracy that was in the acceptable range (91.59-112.96%), with an RSD in the range of 3.13-13.21%. The urinary D-lactate levels of the 3 diabetic groups (groups DM1, DM2, and DM3) were significantly higher than those of healthy subjects (78.31 ± 22.13, 92.47 ± 21.98, and 47.29 ± 17.51 vs. 6.28 ± 2.39 nmol/mg creatinine, respectively; p<0.05), with urinary D-lactate levels in the DM2 group being the highest. This modified fluorescence-based, high-performance liquid chromatography method to quantify D-lactate concentrations in the urine of patients with diabetes was established. Also, measuring the new risk marker identified in this study (D-lactate) in combination with microalbumin may facilitate the prevention of DN.

Determination of lipoprotein lipase activity in post heparin plasma of streptozotocin-induced diabetic rats by high-performance liquid chromatography with fluorescence detection

The activity of lipoprotein lipase (LPL), an enzyme responsible for lipoprotein metabolism, would vary in diseases and metabolic disorders. For determination of LPL activity, a highly sensitive high performance liquid chromatography (HPLC) method using a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was applied to determinate the oleic acid (OA) generated from triolein by LPL activity without multiple solvents extraction step. We studied the optimal conditions of the reaction including the effect of emulsifiers, deproteinizing solvents, and the concentration of bovine serum albumin (BSA). Ten millimolar concentrations of triolein, 5% of BSA, 1% of Gum arabic (GA), and acetonitrile showed the optimum conditions for measuring the LPL activity. The accuracy values for the determination of LPL activity in 10 microL of rat post heparin plasma were 108.73 approximately 114.36%, and the intra- and inter-day precision values were within 1.28% and 2.91%, respectively. The limit of detection was about 4.53 nM (signal-to-noise ratio 3). The proposed method was applied to determination of LPL activity in post heparin plasma of normal and streptozotocininduced diabetic rats associated with 52.3% reduction. The established assay system could be used for determining LPL activity in different physiological and pathological conditions to clarify the relationship between LPL activity and diabetes mellitus.

Proteome analysis of altered proteins in streptozotocin-induced diabetic rat kidney using the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry method.

To find new molecular markers for early diagnosis of diabetic nephropathy, we applied fluorogenic derivatization-liquid chromatography-tandem mass spectrometry to identify the differentially expressed proteins in the kidney of control and streptozotocin-induced diabetic rats. The Sprague-Dawley rats were injected with the sodium citrate buffer or streptozotocin and then killed after 1, 4, 12 and 24 weeks. The results showed that seven proteins were significantly changed after 1 week of injection. Only one protein had significantly changed after 4 weeks of injection. However, after 12 weeks of injection, the number of altered proteins rose to 10. After 24 weeks of injection, 18 proteins had altered significantly. Five common proteins were significantly altered at week 12 and 24 after injection, respectively. Importantly, these proteins appeared prior to microalbuminuria and may serve as new biomarkers that are able to improve early detection of and new drug development for diabetic-related nephropathy.

Improvement in Resolution of Laser Capture Microdissection Using Near-Field Probe to Capture Nanoparticles

Purpose: A modified laser capture micro-dissection (LCM) system is developed to improve resolution to 400 nm, using a laser light (808 nm) transmitted by a near-field tip probe. Materials and methods: Using a 150-nm aperture to heat an ethylene vinyl acetate (EVA) film, melted spots on the average of 400 nm in diameter are generated on the underlying target composed of a 20-nm gold-particle monolayer. The near-field tip probe composed of fiber is set on a 2-D nanometer piezoactuator (PZT) for precise capturing of the monolayer of gold particles. The monolayer of gold particles under the target is bound to the EVA film using a laser, while the remaining EVA film stays on the monolayer. Results: The diameter of the melted spots as small as 400 nm are produced and details are provided that demonstrate the feasibility of the nano-operation of this new LCM system. Conclusion: The new LCM system successfully captures nanoparticles and improves resolution of micro-dissection to 400 nm. With this LCM system, the isolation of a single organelle or bacterium is possible.

Construction of a light‐emitting diode fluorescence detector for high‐performance liquid chromatography and its application to fluorometric determination of l‐3‐hydroxybutyrate

This study employed a new light source, a light-emitting diode (LED), for fluorescence detection of high-performance liquid chromatography to measure the concentration of trace constituents in biological fluids. Using l-3-hydroxybutyrate ( l-3HB) as a tested trace compound, the function of the new system was compared with that of the current commercially available model. A detailed schematic diagram of the path of the detection rays in the LED detector is given. A voltage-stabilizer for the drive circuit was designed with an input of 10 V and an output of 8 V, and another voltage regulator was used to maintain a constant 8 V. Then the regulator was used to set the output voltage for the LED at 2.8 V by two external resistors. Replacing the xenon lamp with LED, this system provided higher photon density and a narrow spectrum at a wavelength of 491 nm. At room temperature (22.1°C), the average temperature of six places in the chamber of LED detector was 22.1°C compared with 51.1°C in the xenon detector. The spectra of the excitation light sources were measured. Compared with the xenon lamp, approximately 1.32 times higher excitation intensity was obtained by the LED source. The accuracy of detection of l-3HB in 50 μL of rat serum was 99.85-100.85%, and the intra-day and inter-day precision values were within 8.99 and 13.90%, respectively. The limit of detection of l-3HB was approximately 0.73 µM (signal-to-noise ratio 3). The sensitivity of the proposed LED detector was comparable to that of traditional fluorescence detectors using xenon arc lamps; however, the cost and operating temperature of LED lamps were far lower. This assay system could be further used to detect trace constituents in various samples.

Measurement of blood velocity using Laser Doppler method for the designing module

We built the Dual Beam Mode of the LDA (Laser Doppler Anemometry) frame, set the photodetector at the same side with light source which collect the scattering light of blood cell. Its proper to reduce LDA optical path and convenient for our designing module. The concentration of chicken blood in this study is about 1% and we measured the relations actually between flood velocity and the angle of beams cross on particles, temperature, and the diameter of aqueduct. We found better results while the cross angle was less than 38.8 degree, diameter of aqueduct was 6 mm, and temperature of blood was set to 36 . These parameters can also provide important basis for the LDA module kit that we are designing.