Deficiency of cannabinoid receptor of type 2 worsens renal functional and structural abnormalities in streptozotocin-induced diabetic mice.

A functionally active endocannabinoid system is present within the kidney. The cannabinoid receptor type 2 (CB2) is expressed by both inflammatory cells and podocytes, and its activation has beneficial effects in experimental diabetic nephropathy. To further explore the role of CB2 in diabetic nephropathy, we studied renal functional and structural abnormalities in streptozotocin-induced diabetic CB2 knockout mice. In diabetic mice, deletion of the CB2 receptor albuminuria, the downregulation of podocin and nephrin, mesangial expansion, overexpression of extracellular matrix components, monocyte infiltration, and reduced renal function were all exacerbated. To investigate the relative contributions of podocytes and monocytes to the phenotype of diabetic knockout mice, bone marrow transplantation experiments were performed. The lack of CB2 on bone marrow-derived cells was shown to be important in driving the enhanced glomerular monocyte accrual found in diabetic knockout mice. Absence of CB2 on resident glomerular cells had a major role in worsening diabetic nephropathy, both functional and structural abnormalities, likely by enhanced MCP-1 and CB1 signaling. Studies in cultured podocytes demonstrated that CB2 expression is not altered by a high glucose milieu but is downregulated by mechanical stretch, mimicking glomerular capillary hypertension. Thus, CB2 deletion worsens diabetic nephropathy, independent of bone marrow-derived cells.

The smell of moulting: N-acetylglucosamino-1,5-lactone is a premoult biomarker and candidate component of the courtship pheromone in the urine of the blue crab, Callinectes sapidus.

Female blue crabs (Callinectes sapidus) in their pubertal moult stage release unidentified sex pheromone molecules in their urine, causing males to respond with courtship behaviours including a display called courtship stationary paddling and a form of precopulatory guarding called cradle carry. We hypothesized that pheromones are mixtures of molecules and are more concentrated in urine of pubertal premoult females compared with other moulting stages and thus that these molecules are biomarkers (i.e. metabolites that can be used as an indicator of some biological state or condition) of pubertal premoult females. We tested this hypothesis by combining bioassay-guided fractionation and biomarker targeting. To evaluate the molecular mass of the putative pheromone by bioassay-guided fractionation, we separated urine from pubertal premoult females and intermoult males by ultrafiltration into three molecular mass fractions. The <500 Da fraction and the 500-1000 Da fraction but not the >1000 Da fraction of female urine induced male courtship stationary paddling, but none of the fractions of male urine did. Thus, female urine contains molecules of <1000 Da that stimulate courtship behaviours in males. Biomarker targeting using nuclear magnetic resonance (NMR) spectral analysis of the 500-1000 Da fraction of urine from premoult and postmoult males and females revealed a premoult biomarker. Purification, nuclear magnetic resonance, mass spectrometry and high pressure liquid chromatography analysis of this premoult biomarker identified it as N-acetylglucosamino-1,5-lactone (NAGL) and showed that it is more abundant in urine of premoult females and males than in urine of either postmoult or juvenile females and males. NAGL has not been reported before as a natural product or as a molecule of the chitin metabolic pathway. Physiological and behavioural experiments demonstrated that blue crabs can detect NAGL through their olfactory pathway. Thus, we hypothesize that NAGL is a component of the sex pheromone and that it acts in conjunction with other yet unidentified components.

Garlic supplemented diet attenuates gentamicin nephrotoxicity in rats.

PURPOSE: To demonstrate the effect of a 4% pulverized garlic supplemented diet on the nephrotoxicity induced by gentamicin in rats. MATERIALS AND METHODS: Twenty four healthy male Wistar rats, weighing between 220 - 260 grams, were divided into three groups. The rats were randomly assigned to either the gentamicin injection without garlic supplementation group (Group I, n = 8), gentamicin injection with garlic supplementation group (Group II, n = 8), and control group (Group III, n = 8). Urine from the rats was collected and the volume (mL), microalbumin (mg/L), creatinine (mg/dL), Na (mmol/L), K (mmol/L), Cl (mmol/L), P (mg/dL), N-acetyl glucosamine (NAG) (U/L) and pH values were measured. Then urea (mg/dL), creatinine (mg/dL), total protein (g/dL) and cystatin (mg/L) values were measured for the blood samples obtained from tail veins. RESULTS: The median NAG value for the control group (52.050 U/L) was similar to value for Group II (56.400 U/L), which received gentamicin and the garlic diet. However, the median NAG value for Group I (77.030 U/L), which received gentamicin without garlic supplementation, was determined to be statistically significantly higher (p = 0.010) than the value for the control group. In addition, the mean cystatin value for Group II (1.360 U/L) was found to be statistically significantly lower than the value for the Group I (2.240 U/L) (p = 0.015). CONCLUSIONS: In this study we showed the effect of 4% pulverized garlic supplemented diet for preventing nephrotoxicity induced by gentamicin in rats by using as parameters NAG in urine samples and cystatin C in serum samples.

The protective effect of aminoguanidine on doxorubicin-induced nephropathy in rats.

Reactive oxygen species and cytokines have been implicated in the nephrotoxicity induced by doxorubicin. The goal of the present study was to determine protective effect of aminoguanidine on doxorubicin-induced nephrotoxicity in rats. Different groups of male Wistar rats received doxorubicin (67.75 mg/kg/i.p./2 days), aminoguanidine alone and aminoguanidine (200 and 400 mg/kg/i.p./30 days) prior to doxorubicin, respectively. Doxorubicin significantly increased serum creatinine (505%), blood urea nitrogen (333%), nitric oxide (406%), and plasma tumor necrosis factor-alpha (706%) as well as urinary albumin (452%) and N-acetyl-ß-D-glucosaminidase (415%) compared to control. Moreover, renal glutathione (334%), superoxide dismutase (283%), and catalase (513%) were significantly reduced accompanied with elevation in renal malondialdehyde compared to control. Pretreatment with aminoguanidine mitigated such changes in all mentioned parameters. Histopathological changes showed that doxorubicin-caused significant structural damages to kidneys that were reduced with aminoguanidine. Results indicate that reactive oxygen species and cytokines are involved in doxorubicin-induced nephrotoxicity, which can be reduced by aminoguanidine.

Early initiation of enzyme replacement therapy improves metabolic correction in the brain tissue of aspartylglycosaminuria mice.

Aspartylglycosaminuria (AGU) is a lysosomal storage disease caused by deficient activity of glycosylasparaginase (AGA), and characterized by motor and mental retardation. Enzyme replacement therapy (ERT) in adult AGU mice with AGA removes the accumulating substance aspartylglucosamine from and reverses pathology in many somatic tissues, but has only limited efficacy in the brain tissue of the animals. In the current work, ERT of AGU mice was initiated at the age of 1 week with three different dosage schedules of recombinant glycosylasparaginase. The animals received either 3.4 U of AGA/kg every second day for 2 weeks (Group 1), 1.7 U/kg every second day for 9 days followed by an enzyme injection once a week for 4 weeks (Group 2) or 17 U/kg at the age of 7 and 9 days (Group 3). In the Group 1 and Group 3 mice, ERT reduced the amount of aspartylglucosamine by 34 and 41% in the brain tissue, respectively. No therapeutic effect was observed in the brain tissue of Group 2 mice. As in the case of adult AGU mice, the AGA therapy was much more effective in the somatic tissues than in the brain tissue of the newborn AGU mice. The combined evidence demonstrates that a high dose ERT with AGA in newborn AGU mice is up to twofold more effective in reducing the amount of the accumulated storage material from the brain tissue than ERT in adult AGU animals, indicating the importance of early detection and treatment of the disease.

Crush syndrome after the Wenchuan earthquake: new experience with regional citrate anticoagulation continuous veno-venous hemofiltration.

PURPOSE: Acute renal failure (ARF) related to crush syndrome is usually treated with hemodialysis. Continuous veno-venous hemofiltration (CVVH) has seldom been adopted in this situation due to the main drawback of continuous anticoagulation. The purpose of this study was to evaluate the effectiveness and safety of regional citrate anticoagulation (RCA)-CVVH in two crush syndrome patients following the Wenchaun earthquake. METHODS: Two victims from the Wenchuan earthquake in Southwest China were admitted to our hospital on May 23, 2008, 11 days after their injury. The total entrapment time under the rubble was 5.5 and 22.5 hrs respectively. They remained oliguric on admission, in spite of vigorous treatment in the local hospital including aggressive fluid infusion, fasciotomy and intermittent hemodialysis. On admission, their serum myoglobin levels were 765 and 829 ng/mL, respectively. Further debridement and drainage were performed. RCA-CVVH was conducted; the citrate containing substitution fluid was infused in a pre-dilution manner at a rate of 4 l/h; calcium was infused through a separate access to the venous inlet of the double lumen catheter. The infusion rate was adjusted according to the serum ionized calcium and whole blood activated clotting time (WBACT). A low dose of low molecular weight heparin (LMWH) was infused at the rate of 150 approximately 300 U/h simultaneously for anticoagulation after anemia had been corrected and their wounds were stable. RCA-CVVH was substituted by conventional CVVH and LMWH anticoagulation when case 2 complicated with hypoxia. RESULTS: RCA-CVVH was well tolerated, hemodynamic status was stable, and no complications related with RCA-CVVH were noted. The body temperature and WBC decreased to normal range, while anemia and hypoalbuminia were corrected. The levels of serum myoglobin and creatine phosphokinase were also decreased to normal range. Their urine volume increased after 20 and 22 days of oliguria and the tubular function of the patients recovered well. Although the second case encountered acute cholecystitis and acute lung injury in the hospital, both the patients recuperated and neither of them underwent amputation. CONCLUSIONS: The present two crush patients have been successfully treated, but due to the limits of the small sample, it is difficult to generalize whether RCA-CVVH is safe enough for crush syndrome with a high risk of bleeding diathesis. Additional investigation with a larger number of patients is required. Fluid equilibrium, nutritional support, prevention of bleeding and infection are fundamental in this situation.

Comparison of kidney injury molecule-1 and other nephrotoxicity biomarkers in urine and kidney following acute exposure to gentamicin, mercury, and chromium.

Sensitive biomarkers are needed to detect kidney injury at the earliest stages. The objective of this study was to determine whether the appearance of kidney injury molecule-1 (Kim-1) protein ectodomain in urine and kidney injury molecule-1/hepatitis A viral cellular receptor-1 (Kim-1/Havcr1) gene expression in kidney tissue may be more predictive of renal injury after exposure to nephrotoxicants when compared to traditionally used biomarkers. Male Sprague-Dawley rats were injected with a range of doses of gentamicin, mercury (Hg; HgCl2), or chromium (Cr; K2Cr2O7). The results showed that increases in urinary Kim-1 and kidney Kim-1/Havcr1 gene expression paralleled the degree of severity of renal histopathology and were detected at lower doses of nephrotoxicants when compared to blood urea nitrogen (BUN), serum creatinine, and urinary N-acetyl-beta-D-glucosaminidase (NAG). In a time course study, urinary Kim-1 was elevated within 24 h after exposure to gentamicin (100 mg/kg), Hg (0.25 mg/kg), or Cr (5 mg/kg) and remained elevated through 72 h. NAG responses were nephrotoxicant dependent with elevations occurring early (gentamicin), late (Cr), or no change (Hg). At 72 h, after treatment with any of the three nephrotoxicants, there was increased Kim-1 immunoreactivity and necrosis involving approximately 50% of the proximal tubules; however, only urinary Kim-1 was significantly increased, while BUN, serum creatinine, and NAG were not different from controls. In rats treated with the hepatotoxicant galactosamine (1.1 mg/kg), serum alanine aminotransferase was increased, but no increase in urinary Kim-1 was observed. Urinary Kim-1 and kidney Kim-1/Havcr1 expression appear to be sensitive and tissue-specific biomarkers that will improve detection of early acute kidney injury following exposure to nephrotoxic chemicals and drugs.

Bilateral pulvinar signal intensity decrease on T2-weighted images in patients with aspartylglucosaminuria.

BACKGROUND: Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal disease caused by deficiency of aspartylglucosaminidase. A thalamic T2 signal intensity decrease is associated with lysosomal diseases. PURPOSE: To investigate thalamic signal intensity in AGU by performing a retrospective review of brain magnetic resonance (MR) imaging studies of AGU patients. MATERIAL AND METHODS: A total of 25 MR examinations were available for 11 patients aged between 3 and 32 years (four patients underwent bone marrow transplantation). Of these, 13 examinations were performed after bone marrow transplantation. Five patients had from two to six examinations, and six patients had one examination each. In every patient, the diagnosis of AGU was confirmed by blood and urine tests. Eighteen examinations were performed with a 1.0T imager including dual spin-echo T2 and proton density (PD) axial and coronal images, and 10 examinations also included T1-weighted images. Seven examinations were performed with a 1.5T imager including turbo spin-echo axial and coronal T2-weighted images and axial fluid-attenuated inversion recovery (FLAIR) images; three examinations included T1-weighted three-dimensional magnetization-prepared rapid acquisition gradient-echo (3D MPRAGE) images. The signal intensity of the thalamus and pulvinar in every sequence was compared to that of the putamina. RESULTS: In AGU, thalamic alterations were first detectable on T2-weighted images (25 examinations in 11 patients) from the age of 3 years 6 months, showing decreased signal intensity in 21 of 24 examinations. T1-weighted images (13 examinations) showed slightly increased thalamic signal intensity in five out of seven examinations from the age of 7 years, and PD images (19 examinations) showed decreased signal intensity from the age of 16 years (three examinations). The pulvinar showed decreased signal intensity on spin-echo T2-weighted images for 14 of 18 examinations or on FLAIR sequences for seven of seven examinations from the age of 6 years and 6 months, both in patients with and without bone marrow transplantation, but no pulvinar alterations were observable on T1 and PD images. CONCLUSION: In AGU, the thalamus is affected. Pulvinar changes are visible only on T2-weighted images, and these may be the first changes reported in the group of lysosomal diseases.

Renal safety and extrahepatic defluorination of sevoflurane in hepatic transplantations.

BACKGROUND: The main metabolic pathway for defluorination of sevoflurane in the liver produces inorganic fluoride (Fl). The metabolism and effect of sevoflurane on the kidney is not clear during anhepatic phase in liver transplantation. The goal of the present study was to investigate the metabolism and renal effect of sevoflurane by measuring plasma and urine inorganic fluoride, urinary N-acetyl-glucosaminidase (NAG), and plasma creatinine levels in patients undergoing liver transplantations. METHODS: After institutional approval and informed consent, we studied nine cases of orthotopic liver transplantation after anesthesia was induced with 5 mg . kg(-1) thiopental, 1 mug . kg(-1) fentanyl intravenously, the trachea was intubated after vecuronium bromide 0.1 mg . kg(-1). Anesthesia was maintained with sevoflurane (2%), O(2), and N(2)O at a total gas flow of 6 L . min(-1) using a semiclosed circle system with a sodalime canister. Blood and urine samples were obtained to measure plasma and urine fluoride concentrations and urinary NAG excretions before induction (P0), hourly during resection (P1, P2, P3), every 15 minutes during anhepatic phase (A1, A2, A3), hourly after reperfusion (neohepatic phase) (N1, N2, N3), and postoperative first hour (Po1). Preoperative (T0) and postoperative day 1 (T1), 3 (T3), 7 (T7) plasma blood urea nitrogen (BUN) and creatinine (Cr) levels were also recorded. RESULTS: Mean duration of surgery was 9:06 +/- 0:09 hours. Mean inorganic fluoride concentrations in plasma were in the range of 0.71 +/- 0.30 to 28.73 +/- 3.31 mumole . L(-1). In P3, N1, N2, N3, increases in plasma inorganic fluoride concentrations were significant (P < .05) and reached a peak value at Po1. The mean urine inorganic fluoride concentrations were 12.49 +/- 2.04 to 256.7 +/- 49.62 mumole . L(-1). In A2, A3, N1, N2, and N3, mean urine inorganic fluoride concentrations were significantly increased (P < .05) and the peak value was observed at Po1. Mean NAG concentrations in urine varied (5.6 +/- 1.6 IU . L(-1) to 12.5 +/- 1.14 IU . L(-1)) and peak level was observed at 30 minutes of the anhepatic phase (A2), which did not exceed the normal values for urine NAG levels (1.5 to 6.1 U . L(-1)). No impairment was observed in serum BUN and creatinine levels at any time. While there was only a slight increase in NAG during anhepatic phase, there was no change in plasma F1. CONCLUSIONS: Sevoflurane seemed to have minimal effect on kidney functions of BUN and Cr levels during liver transplantation. Although urine F1 and NAG levels increased during the anhepatic phase plasma F1, BUN, and Cr levels did not, suggesting that renal F1 production may occur in the absence of hepatic function. The renal effect of sevoflurane in chronic liver disease is controversial and must be investigated in further studies.

Extrahepatic metabolism and renal effects of sevoflurane in a case of liver transplantation.

In a case of liver transplantation, sevoflurane metabolism was studied to investigate if sevoflurane has an extrahepatic metabolism or possible nephrotoxicity in the presence of chronic liver disease. Plasma blood urea nitrogen (BUN) and creatinine and urine levels of N-acetyl glycosaminidase (NAG) and beta2 microglobulin were assessed intraoperatively and for 11 days postoperatively. We observed a close relation between urine NAG excretion and urine inorganic fluoride levels in the intraoperative period and early postoperative days. The NAG levels were greater than normal despite the peak serum inorganic fluoride concentration of 18.94 micromol/L. No impairment was observed in serum BUN or creatinine levels in these periods.

2-Acetamidoglucal, a new metabolite isolated from the urine of a patient with sialuria.

A new metabolite, namely 2-acetamidoglucal, has been found in the urine of a patient with sialuria in addition to the metabolites N-acetylneuraminic acid, N-acetylmannosamine, N-acetylglucosamine and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reported earlier. the structure has been identified by mass spectrometry and 360 MHz proton nuclear magnetic resonance spectroscopy and verified by synthesis. All accumulated compounds fit into the metabolic pathway for the biosynthesis of CMP-N-acetylneuraminic acid. Sialuria is discussed in terms of a failure of regulation of UDP-N-acetylglucosamine 2-epimerase.

Fibroblast expression of collagens and proteoglycans is altered in aspartylglucosaminuria, a lysosomal storage disease.

Aspartylglucosaminuria (AGU), a lysosomal storage disease caused by deficient activity of aspartylglucosaminidase (E.C. 3.5.1.26), is characterised by progressive mental retardation and variable connective tissue signs. The ultrastructure of collagen fibrils in skin of AGU patients is abnormal and their fibroblasts synthesise reduced amounts of collagens [Näntö-Salonen et al. (1984) Lab invest. 51: 464-468]. In this work we measured the steady-state messenger RNA levels of several extracellular matrix components in skin fibroblast cultures of two patients homozygous for the most prevalent mutation (AGUFin) causing the disease in Finland. In confluent cultures the steady-state mRNA concentrations of type I and III collagens were reduced to 0.5-20% of control values. Almost as marked reduction was observed in the mRNA level of biglycan, a small interstitial proteoglycan whereas that of decorin, a closely related, collagen fibril-associated proteoglycan, was increased several-fold. Elevated decorin and decreased biglycan mRNA levels reflected the amounts of the produced corresponding proteoglycans. The differences in the mRNA levels become more pronounced with the time the cells were in culture. Fibronectin mRNA concentrations were similar in AGU and control fibroblasts. Changes in the expression and synthesis of extracellular matrix components might be related to the connective tissue symptoms of the patients.

Aspartylglycosaminuria: biochemistry and molecular biology.

Aspartylglucosaminuria (AGU, McKusick 208400) is an autosomal recessive lysosomal storage disease caused by defective degradation of Asn-linked glycoproteins. AGU mutations occur in the gene (AGA) for glycosylasparaginase, the enzyme necessary for hydrolysis of the protein oligosaccharide linkage in Asn-linked glycoprotein substrates undergoing metabolic turnover. Loss of glycosylasparaginase activity leads to accumulation of the linkage unit Asn-GlcNAc in tissue lysosomes. Storage of this fragment affects the pathophysiology of neuronal cells most severely. The patients notably suffer from decreased cognitive abilities, skeletal abnormalities and facial grotesqueness. The progress of the disease is slower than in many other lysosomal storage diseases. The patients appear normal during infancy and generally live from 25 to 45 years. A specific AGU mutation is concentrated in the Finnish population with over 200 patients. The carrier frequency in Finland has been estimated to be in the range of 2.5-3% of the population. So far there are 20 other rare family AGU alleles that have been characterized at the molecular level in the world's population. Recently, two knockout mouse models for AGU have been developed. In addition, the crystal structure of human leukocyte glycosylasparaginase has been determined and the protein has a unique alphabetabetaalpha sandwich fold shared by a newly recognized family of important enzymes called N-terminal nucleophile (Ntn) hydrolases. The nascent single-chain precursor of glycosylase araginase self-cleaves into its mature alpha- and beta-subunits, a reaction required to activate the enzyme. This interesting biochemical feature is also shared by most of the Ntn-hydrolase family of proteins. Many of the disease-causing mutations prevent proper folding and subsequent activation of the glycosylasparaginase.

Olmesartan medoxomil, a newly developed angiotensin II type 1 receptor antagonist, protects against renal damage in advanced glycation end product (age)-injected rats.

Diabetic nephropathy is a leading cause of end-stage renal disease in industrialized countries. Although the molecular mechanisms for the development and progression of diabetic nephropathy are not fully understood, the formation of advanced glycation end products (AGEs) and activation of the renin-angiotensin system (RAS) have been considered to be the main factors participating in the pathogenesis of diabetic nephropathy. However, functional cross-talk between AGEs and the RAS remains to be elucidated. In this study, we examined the effects of oral administration of olmesartan medoxomil, a newly developed angiotensin II type 1 receptor antagonist, on renal damage in AGE-treated rats. Administration of olmesartan medoxomil significantly inhibited the increase of systolic and diastolic blood pressure levels and urinary N-acetyl-beta-D-glucosaminidase activity in exogenously AGE-injected rats. Furthermore, olmesartan medoxomil treatment also prevented glomerulosclerosis in AGE-treated rats. These results indicate that exogenous AGE treatment could induce renal damage via the activation of the RAS. Our study suggests that olmesartan medoxomil could be a valuable drug for the treatment of diabetic nephropathy by blocking the deleterious effects of AGEs.

Renoprotective effects of azelnidipine, a dihydropyridine-based calcium antagonist in advanced glycation end product (AGE)-injected rats.

Diabetic nephropathy is a leading cause of end-stage renal disease in industrialized countries. Although the molecular mechanisms for the development and progression of diabetic nephropathy are not fully understood, the formation and accumulation of advanced glycation end products (AGEs) have been considered to play a major role in the pathogenesis of diabetic nephropathy. Hypertension is also an independent risk factor for the progression of diabetic nephropathy. However, functional cross-talk between AGEs and blood pressure and their involvement in diabetic nephropathy remain to be elucidated. In this study, we examined the effects of oral administration of azelnidipine, a commercially available dihydropyridine-based calcium antagonist, on renal injury in AGE-treated rats. Administration of azelnidipine inhibited the increase of systolic and diastolic blood pressure levels and urinary N-acetyl-beta-D-glucosaminidase activity in exogenously AGE-injected rats. Furthermore, azelnidipine treatment also prevented glomerulosclerosis in AGE-treated rats. These results indicate that renal damage in AGE-injected rats could be mediated, at least in part, by the elevation of blood pressure. Our present study suggests that azelnidipine would represent a valuable drug for the treatment of diabetic nephropathy by blocking the deleterious effects of AGEs.

Correction of peripheral lysosomal accumulation in mice with aspartylglucosaminuria by bone marrow transplantation.

OBJECTIVE: Bone marrow transplantation has been shown to alleviate symptoms outside the CNS in many lysosomal storage diseases depending on the type and stage of the disease, but the effect on neurological symptoms is variable or still unclear. Aspartylglucosaminuria (AGU) is a lysosomal storage disease characterized by mental retardation, recurrent infections in childhood, hepatosplenomegaly and coarse facial features. Vacuolized storage lysosomes are found in all tissues of patients and uncleaved enzyme substrate is excreted in the urine. The recently generated AGU mouse model closely mimicks the human disease and serves as a good model to study the efficiency of bone marrow transplantation in this disease. METHODS: Eight-week-old AGU mice were lethally irradiated and transplanted with bone marrow from normal donors. The AGA enzyme activity was measured in the liver and the brain and the degree of correction of tissue pathology was analyzed by light and electron microscopy. Reverse bone marrow transplantation (AGU bone marrow to wild-type mice) was also performed. RESULTS: Six months after transplantation the AGA enzyme activity was 13% of normal in the liver, but only 3% in the brain. Tissue pathology was reversed in the liver and the spleen, but not in the brain and the kidney. The urinary excretion of enzyme substrate was diminished but still detectable. No storage vacuoles were found in the tissues after reverse transplantation, but subtle excretion of uncleaved substrate was detected in the urine. CONCLUSION: Liver and spleen pathology of AGU was corrected by bone marrow transplantation, but there was no effect on lysosomal accumulation in the CNS and in the kidneys.

A nitric oxide-donor furoxan moiety improves the efficacy of edaravone against early renal dysfunction and injury evoked by ischemia/reperfusion.

Edaravone (5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one, EDV) is a free-radical scavenger reduces organ ischemic injury. Here we investigated whether the protective effects of EDV in renal ischemia/reperfusion (I/R) injury may be enhanced by an EDV derivative bearing a nitric oxide- (NO-) donor furoxan moiety (NO-EDV). Male Wistar rats were subjected to renal ischemia (45 minutes), followed by reperfusion (6 hours). Administration of either EDV (1.2-6-30 µmol/kg, i.v.) or NO-EDV (0.3-1.2-6 µmol/kg, i.v.) dose-dependently attenuated markers of renal dysfunction (serum urea and creatinine, creatinine clearance, urine flow, urinary N-acetyl-ß-D-glucosaminidase, and neutrophil gelatinase-associated lipocalin/lipocalin-2). NO-EDV exerted protective effects in the dose-range 1.2-6 µmol/kg, while a higher dose (30 µmol/kg) was needed to obtain protection by EDV. Both EDV and NO-EDV modulated tissue markers of oxidative stress and lipid peroxidation. NO-EDV, but not EDV, activated endothelial NO synthase (NOS) and blunted I/R-induced upregulation of inducible NOS, secondary to modulation of Akt and NF-κB activation, respectively. Besides NO-EDV administration inhibited I/R-induced IL-1ß, IL-18, IL-6, and TNF-α overproduction. Overall, these findings demonstrate that the NO-donor moiety contributes to the protection against early renal I/R injury and suggest that NO-donor EDV codrugs are worthy of additional study as innovative pharmacological tools.

Correction of deficient enzyme activity in a lysosomal storage disease, aspartylglucosaminuria, by enzyme replacement and retroviral gene transfer.

The ability of lysosomal enzymes to be secreted and subsequently captured by adjacent cells provides an excellent basis for investigating different therapy strategies in lysosomal storage disorders. Aspartylglucosaminuria (AGU) is caused by deficiency of aspartylglucosaminidase (AGA) leading to interruption of the ordered breakdown of glycoproteins in lysosomes. As a consequence of the disturbed glycoprotein catabolism, patients with AGU exhibit severe cell dysfunction especially in the central nervous system (CNS). The uniform phenotype observed in these patients will make effective evaluation of treatment trials feasible in future. Here we have used fibroblasts and lymphoblasts from AGU patients and murine neural cell lines as targets to evaluate in vitro the feasibility of enzyme replacement and gene therapy in the treatment of this disorder. Complete correction of the enzyme deficiency was obtained both with recombinant AGA enzyme purified from CHO-K1 cells and with retrovirus-mediated transfer of the AGA gene. Furthermore, we were able to demonstrate enzyme correction by cell-to-cell interaction of transduced and nontransduced cells.

Deletion of exon 8 causes glycosylasparaginase deficiency in an African American aspartylglucosaminuria (AGU) patient.

We have indentified a GT-to-TT transversion at the splice donor site of intron 8 in the glycosylasparaginase gene from an African American aspartylglucosaminuria (AGU) patient. This mutation causes abnormal splicing of glycosylasparaginase pre-mRNA by joining exon 7 to 9 and excluding 134 bp exon 8. The effect of the mutation is compounded by a frame shift that occurs after the deletion site resulting in premature translational termination. The truncated AGU protein was neither catalytically active nor processed into mature alpha and beta subunits. Both this and a previously characterized Finnish AGU mutation appear to affect folding of the single-chain precursor of glycosylasparaginase and thereby prevent transport of the enzyme to lysosomes.

Genomic structure of human lysosomal glycosylasparaginase.

The gene structure of the human lysosomal enzyme glycosylasparaginase was determined. The gene spans 13 kb and consists of 9 exons. Both 5' and 3' untranslated regions of the gene are uninterrupted by introns. A number of transcriptional elements were identified in the 5' upstream sequence that includes two putative CAAT boxes followed by TATA-like sequences together with two AP-2 binding sites and one for Spl. A 100 bp CpG island and several ETF binding sites were also found. Additional AP-2 and Sp1 binding sites are present in the first intron. Two polyadenylation sites are present and appear to be functional. The major known glycosylasparaginase gene defect G488----C, which causes the lysosomal storage disease aspartylglycosaminuria (AGU) in Finland, is located in exon 4. Exon 5 encodes the post-translational cleavage site for the formation of the mature alpha/beta subunits of the enzyme as well as a recently proposed active site threonine, Thr206.