Impact of porous microsponges in minimizing myotoxic side effects of simvastatin.

Simvastatin (SV) is a poorly soluble drug; its oral administration is associated with a significant problem: Myopathy. The present study aims to formulate SV microsponges that have the potential to minimize the myotoxicity accompanying the oral administration of the drug. SV microsponges were prepared by exploiting the emulsion solvent evaporation technique. The % entrapment efficiency (%EE) of the drug approached 82.54 ± 1.27%, the mean particle size of SV microsponges ranged from 53.80 ± 6.35 to 86.03 ± 4.79 µm in diameter, and the % cumulative drug release (%CDR) of SV from microsponges was significantly higher than that from free drug dispersion much more, the specific surface area of the optimized microsponges formulation was found to be 16.6 m2/g revealed the porosity of prepared microsponges. Histological and glycogen histochemical studies in the skeletal muscles of male albino rats revealed that microsponges were safer than free SV in minimizing myotoxicity. These findings were proven by Gene expression of Mitochondrial fusion and fission (Mfn1) & (Fis1) and (Peroxisome proliferator-activated receptor gamma co-activator 1α) PGC-1α. Finally, our study ascertained that SV microsponges significantly decreased the myotoxicity of SV.

Detection of Salmonella spp. by Traditional and PCR Assays in Raw Milk, Maysan, Iraq.

Salmonella spp are characterized as rod- shaped, motile, gram- negative bacteria which has the ability to infect animals and human. Salmonella spp occasionally causes sickness while in most cases not lead to severe symptoms. Analyzing milk for Salmonella spp. is not routine but traditional culture methods are used to evaluate the health condition of the dairy products. However, the antibody-based and nucleic-acid- based methods are practical for identifying Salmonella spp. Therefore, this research was designed to evaluate the use of traditional culture methods and PCR in detection of the presence of Salmonella spp. in raw milk samples in, Maysan Iraq. A total number of 130 raw milk samples collected from Maysan Iraq. All the samples were analyzed for the presence of Salmonella spp. using traditional culture method and polymerase chain reaction (PCR). The culture method used in this experiment were done by using pre-enrichment, enrichment, selective plating and biochemical tests. The results of this traditional technique were compared with the results obtained from PCR method. The PCR was performed using a 284bp sequence of the invA gene. The results showed that 8 (7.07%) of samples were identified as salmonella positive using traditional culture technique but 14 (12.3%) samples were detected as salmonella positive by PCR method. The results of the current research revealed that the traditional culture based methods are generally time costuming and labor intensive but the development of new rapid methods including DNA based methods such as PCR are more sensitive and have dramatically decreased the time necessary for the detection of bacteria.

Presence of Escherichia coli O157:H7 in Dairy Farms located in Najaf, Baghdad, Kirkuk, and Erbil, Iraq.

It has been approved that one of the most dangerous foodborne pathogenic bacteria is E. coli O157:H7, which is responsible for several infection and death cases worldwide. It is well documented that in the developing countries E. coli O157:H7 is considered the main causative pathogen of human gastrointestinal infections. Therefore, the current research was aimed to evaluate the prevalence of E. coli O157:H7 in dairy cattle's milk using a rapid method, in Iraq (Najaf, Baghdad, Kirkuk, and Erbil). Over a period of 6 months (During hot months) samples were obtained and investigated by culturing on selective media (CT-SMAC). The multiplex PCR (m-PCR) also used for milk sample direct investigation. Using biochemical tests the recorded data showed that, 2 recognized isolates were E. coli, while the recorded data obtained from m-PCR assay revealed that none of the isolated E. coli was toxigenic E.coli O157:H7. The results of m-PCR on the milk samples revealed that 45 milk samples contained at least one of the following genes: O157, H7, stx1, stx2 genes. Also the results of the m-PCR revealed that 2 samples (raw milk) were toxigenic O157:H7 positive. In conclusion, to the best of authors' knowledge, this investigation was the first report on the prevalence of E. coli O157:H7 in the raw milk samples in Iraq. The results showed that the proportion of contaminated milk samples contaminated with E. coli O157:H7 identified in the current survey were similar to that the results of the previously published research from different dairy products across different countries in the Middle East region.

The Influence of Rumen-Protected Choline and α-tocopherol Supplementation on Early Lactating Dairy Cows Metabolism.

It is well documented that choline is known as one of the essential ingredients of phospholipids. Choline acts as a determinative element for appropriate cell membrane functions. On the other hand α-tocopherol (Vit E) is a fat-soluble vitamin. This vitamin acts as a strong antioxidant in the living body's defense system against oxidative stress. Lipid peroxidation in peripartum and early lactating cows is significantly increased while the level of serum Vit E is decreases dramatically. These concomitant physiological changes demonstrate a higher level of oxidative stress subsequently leads to serious health issues in dairy cows. Therefore, the present research was designed to investigate the following items in dairy cattle: 1) evaluation of the possible changes in serum protein fractions, and 2) comparing the oxidative status of orally RPC and vitamin E supplementation in dairy cows in early lactation period. In the current study 30 early lactating primiparous and multiparous Holstein cows (body condition score (BCS)=2.51 ± 0.10) were used beginning five weeks postpartum. All the animals were randomly divided in to three groups (n=10) (number of lactation=2.61). The animals were randomly assigned to receive one of the following treatments. Group 1 served as control group were not received any supplement. The second group was supplemented with 90 g/d of RPC (Reashre Choline, Balchem, USA). The third group was administrated 4400 IU/d vitamin E (Roche, Vitamins Ltd; Switzerland). In the current study, serum protein electrophoresis showed four main fractions as follows: albumin, α-globulin, ß-globulin, and γ-globulin. The recorded data showed that the percentages of albumin and γ-globulin fractions were higher in treated groups compared to the control group. In the animals supplementing with RPC and vitamin E the percentages of serum albumin increased to the value of 37. 70±1.63 and 38.21±1.28 respectively compare to the control group (34.69±1.21), which were significant (P<0.05).

Knowledge and Attitude of Healthcare Practitioners Toward Therapeutic Drug Monitoring Practices in the Najran Region, Kingdom of Saudi Arabia.

BACKGROUND: Therapeutic drug monitoring (TDM) is one of the useful clinical tools that aim to improve and ensure the best therapeutic effects while avoiding drug-related toxicity. We aimed to evaluate the knowledge and attitude toward TDM practices among healthcare practitioners in the Najran region, Saudi Arabia. METHODOLOGY: A cross-sectional study was conducted between May 2021 and October 2021 for assessing the awareness and attitude of licensed doctors and pharmacists working in Saudi Arabia regarding TDM practices. A 31-item questionnaire was distributed to the healthcare professionals via an electronic link. Descriptive and inferential statistics were used to report the data. RESULTS: A total of 392 participants submitted questionnaires. More than half of the professionals (55.9%) had an overall awareness of the TDM, while only 3.1% did not. Only 16% and 23% of those who were surveyed indicated that TDM is used at the beginning of medication administration and when shifting from one drug to another, respectively. The majority of the professionals responded that TDM is revealed with laboratory changes in liver and kidney function (81%), and TDM is specified with suspected therapeutic failure (93%). Only half of the respondents claimed that they had ever requested or suggested a TDM in their practice. More than 90% of the respondents claimed that they were aware of the indications for vancomycin (n = 381), gentamicin (n = 375), lithium (n = 369), digoxin (n = 380), and theophylline (n = 369). CONCLUSION: This study found that the majority of the healthcare professionals in Najran were aware of the TDM, particularly those with more than 10 years of professional experience. In addition, TDM service is not widely available in smaller hospitals in Najran. There is a need to conduct customized training programs for junior healthcare professionals, thus increasing the levels of awareness toward TDM. Future studies in Saudi Arabia can explore the role of clinical pharmacists in the provision of TDM services by assessing the clinical and economic outcomes of pharmacist interventions in TDM.

Co-Evaporated CuO-Doped In2O3 1D-Nanostructure for Reversible CH4 Detection at Low Temperatures: Structural Phase Change and Properties.

In order to improve the sensitivity and to reduce the working temperature of the CH4 gas sensor, a novel 1D nanostructure of CuO-doped In2O3 was synthesized by the co-evaporation of Cu and In granules. The samples were prepared with changing the weight ratio between Cu and In. Morphology, structure, and gas sensing properties of the prepared films were characterized. The planned operating temperatures for the fabricated sensors are 50-200 °C, where the ability to detect CH4 at low temperatures is rarely reported. For low Cu content, the fabricated sensors based on CuO-doped In2O3 showed very good sensing performance at low operating temperatures. The detection of CH4 at these low temperatures exhibits the potential of the present sensors compared to the reported in the literature. The fabricated sensors showed also good reversibility toward the CH4 gas. However, the sensor fabricated of CuO-mixed In2O3 with a ratio of 1:1 did not show any response toward CH4. In other words, the mixed-phase of p- and n-type of CuO and In2O3 materials with a ratio of 1:1 is not recommended for fabricating sensors for reducing gas, such as CH4. The gas sensing mechanism was described in terms of the incorporation of Cu in the In2O3 matrix and the formation of CuO and In2O3 phases.

Irradiation and prolonged storage of red cells are associated with increased adverse events.

BACKGROUND: Red blood cell (RBC) transfusion is associated with the most transfusion-related adverse events (AE). Recent clinical studies showed no significant difference in transfusion-associated mortality between fresh and older RBCs. However, the impact of storage duration as well as irradiation on nonfatal yet much more common complications has not been fully investigated. MATERIALS/METHODS: In this retrospective study of RBC transfusion-associated AEs, a total of 188,562 units of leucocyte-reduced RBCs were transfused in approximately 5·5 years. After excluding washed, deglycerolized, autologous or directed RBCs and RBCs transfused during a massive transfusion protocol, 149,052 units were analysed. Attributes of RBCs including storage time, collection method, CMV serological status and gamma irradiation, as well as the recipient's gender, were analysed. A total of 358 RBC transfusion AEs were categorized into allergic and non-allergic reactions and analysed. RESULTS: Univariate and multivariate logistic analyses showed that irradiated RBCs were associated with a significantly increased frequency of non-allergic reactions (OR (95% CI): 1·89 (1·52, 2·35); P < 0·001). There was a significant association between the frequency of non-allergic reactions and the storage time of irradiated RBCs (OR (95% CI): 1·024 (1·001, 1·048); P = 0·042). In contrast, there was no association between the frequency of allergic reactions and the storage time of irradiated RBCs or between the age of non-irradiated RBCs and the frequency of non-allergic reactions. CONCLUSIONS: Prolonged storage of irradiated RBCs was associated with a significant increase in non-allergic transfusion reactions. Overall, the irradiated RBCs appeared to cause more non-allergic reactions compared with non-irradiated RBCs.

Prolonged platelet storage associated with increased frequency of transfusion-related adverse events.

BACKGROUND: Platelets (PLTs) have been associated with the highest rate of transfusion-associated adverse events (AEs) among all blood products. Most of PLT-associated AEs are considered to have an inflammatory mechanism. However, it is still unclear whether prolonged storage of platelets is associated with an increased rate of transfusion-related AEs, especially in the era of universal prestorage leucoreduction. METHODS/MATERIALS: In this retrospective study, 52 649 PLT products consisting of about 80% apheresis PLTs and 20% whole blood-derived (WBD) PLTs were transfused to 9415 patients from July 2011 to March 2017. All the PLTs were leucoreduced prior to storage. All but 69 units of the apheresis PLTs were irradiated and none of WBD PLTs were irradiated. During this period, a total of 284 AEs that were reported to the transfusion service were analysed. RESULTS: Univariate and multivariate logistic analyses showed that apheresis/irradiated PLTs and PLT age were associated with a significantly increased frequency of inflammation type AEs (OR (95% CI): 2·24 (1·32, 4·15) and 1·30 (1·12, 1·52), respectively). There was a significant increase in the frequency of inflammation AEs associated with prolonged storage of apheresis/irradiated PLTs [OR (95% CI): 1·26 (1·03, 1·53)]. In contrast, there was no association between allergic symptoms and PLT age. Moreover, the frequency of transfusion AEs associated with apheresis/irradiated PLTs (57·2/10 000) was significantly higher than that of WBD/nonirradiated PLTs (26·0/10 000) (P < 0·01). CONCLUSION: Prolonged storage of apheresis/irradiated PLTs was associated with a higher frequency of inflammation AEs. Apheresis/irradiated PLTs caused more AEs than WBD/nonirradiated PLTs.

Ghrelin receptor regulates HFCS-induced adipose inflammation and insulin resistance.

BACKGROUND AND OBJECTIVES: High fructose corn syrup (HFCS) is the most commonly used sweetener in the United States. Some studies show that HFCS consumption correlates with obesity and insulin resistance, while other studies are in disagreement. Owing to conflicting and insufficient scientific evidence, the safety of HFCS consumption remains controversial. SUBJECTS/METHODS: We investigated the metabolic consequences of mice fed a (a) regular diet, (b) 'Western' high-fat diet or (c) regular diet supplemented with 8% HFCS in drinking water (to mimic soft drinks) for 10 months. Adipose tissue macrophages (ATMs) have emerged as a major pathogenic factor for obesity and insulin resistance. ATMs consist of proinflammatory F4/80(+)CD11c(+) macrophages and anti-inflammatory F4/80(+)CD11c(-) macrophages. In this study, we assessed the effects of HFCS on ATMs in intra-abdominal fat. RESULTS: We found that HFCS feeding in mice induced more severe adipose inflammation and insulin resistance than even the higher-calorie-containing 'Western' high-fat diet, and these HFCS-induced deleterious effects were independent of calorie intake or body fat content. We showed that similar to 'Western' high-fat diet, HFCS triggered a robust increase of both proinflammatory ATMs and anti-inflammatory ATMs in intra-abdominal fat. Remarkably, however, the anti-inflammatory ATMs were much less abundant in HFCS-fed mice than in high-fat-fed mice. Furthermore, we showed that deletion of the ghrelin receptor (growth hormone secretagogue receptor, GHS-R) ameliorates HFCS-induced adipose inflammation and insulin resistance. HFCS-fed GHS-R-null mice exhibit decreased proinflammatory ATMs in intra-abdominal fat, reduced adipose inflammation and attenuated liver steatosis. CONCLUSION: Our studies demonstrate that HFCS has detrimental effects on metabolism, suggesting that dietary guidelines on HFCS consumption for Americans may need to be revisited. GHS-R deletion mitigates the effects of HFCS on adipose inflammation and insulin resistance, suggesting that GHS-R antagonists may represent a novel therapy for insulin resistance.

Renal cell apoptosis in chronic obstructive uropathy: the roles of caspases.

BACKGROUND: Apoptosis of tubular and interstitial cells is well documented in kidneys with chronic obstructive uropathy (COU) and probably plays an important role in the pathogenesis of this condition. The molecular control of apoptosis in COU remains poorly understood. Apoptosis in general is known to proceed initially along distinct pathways, which later converge into a common arm characterized by orderly activation of caspases. Caspases are cytosolic enzymes that belong to a 12-member family and serve as effector molecules for apoptosis. The role of individual caspases in mediating renal cell apoptosis in kidneys with COU is studied. METHODS: Kidneys were harvested from sham-operated mice and mice with COU created by left ureter ligation at days 4, 7, 15, 20, and 30. The following studies were performed: (1) determination of dried kidney weight; (2) in situ end labeling of fragmented DNA to detect apoptotic tubular and interstitial cells; (3) ribonuclease protection assay with specific anti-sense RNA probes for caspases 1, 2, 3, 6, 7, 8, 9, 11, and 12 to detect the expression of individual caspases; (4) immunostaining for caspases; and (5) assay for caspase 3. To assess the role of caspases in COU-associated renal cell apoptosis, the frequencies of apoptotic tubular and interstitial cells were separately quantitated for each experimental time point, and their patterns of variation were correlated with those of individual caspases. RESULTS: The obstructed kidneys showed progressive tissue loss (60% of control at day 15). Apoptosis of both tubular and interstitial cells was seen in obstructed kidneys. Tubular cell apoptosis peaked at four days after ureter ligation (13-fold of control), remained high between days 4 to 15, and thereafter decreased rapidly. Apoptotic interstitial cells were scanty initially, but gradually increased throughout the entire experiment. Apoptosis was minimal throughout the experiment in control and contralateral kidneys. In control and contralateral kidneys, caspases 2, 3, 6, 7, 8, and 9 mRNAs were expressed at low levels, whereas those for caspases 1, 11, and 12 were not detected. The obstructed kidneys displayed increased expression of all tested caspases. Caspases 1, 11, and 12 mRNAs were detected in obstructed kidneys in a common pattern characterized by a sharp increase at day 4, followed by a decrease until day 20, and a subsequent sharp increase until the end of the study at day 30. A similar pattern was noted for other caspases (2, 3, 6, 7, 8, and 9), which maximally reached twofold to fourfold that of controls. Immunostaining for caspases 1, 2, 3, 6, 7, 8, and 9 showed the same pattern characterized by focal and weak expression in proximal tubules of control or contralateral kidney, contrasting with increased staining in atrophic or dilated tubules of obstructed kidneys. Interstitial cells also displayed staining for several caspases, which paralleled the increasing density of interstitial cells toward the end of the experiment. Caspase-3 assay showed a marked increased activity in obstructed kidneys that reached fourfold and sevenfold of control at days 4 and 30, respectively. The rise and fall of caspase mRNAs between days 4 and 30 paralleled a similar fluctuation in tubular cell apoptosis. The subsequent increase of mRNAs was correlated with a continuous rise of interstitial cell apoptosis. CONCLUSIONS: Urinary obstruction in mice induces apoptosis of both tubular and interstitial cells in the affected kidney in a distinctive pattern that parallels an increased expression of caspases. This correlation suggests that these caspases mediate COU-associated renal cell apoptosis. Among the evaluated caspases, increased renal caspase 3 activity implies its central role in renal cell apoptosis associated with urinary obstruction.

Mechanism of chronic obstructive uropathy: increased expression of apoptosis-promoting molecules.

BACKGROUND: We have demonstrated that renal tubular and interstitial cells undergo pronounced apoptosis during the course of chronic obstructive uropathy (COU). Apoptosis is a complex cellular process consisting of multiple steps, each of which is mediated by families of related molecules. These families may include receptor/ligand molecules such as Fas, Fas ligand, tumor necrosis factor receptor-1 (TNFR-1), and TNF-related apoptosis inducing ligand (TRAIL); signal transduction adapter molecules such as Fas-associated death domain (FADD), TNFR-1 associated death domain (TRADD), receptor-interacting protein (RIP), Fas-associated factor (FAF), and Fas-associated phosphatase (FAP); or effector molecules such as caspases. However, the mechanism of tubular cell apoptosis, as well as the pathogenetic relevance of these apoptosis-related molecules in COU, remains poorly understood. METHODS: Kidneys were harvested from sham-operated control mice and mice with COU created by left ureter ligation sacrificed in groups of three at days 4, 15, 30, and 45. To detect apoptotic tubular and interstitial cells, in situ end labeling of fragmented DNA was performed. To detect the expression of apoptosis-related molecules, ribonuclease protection assay was used with specific antisense RNA probes for Fas, Fas ligand, TNFR-1, TRAIL, FADD, TRADD, RIP, FAF, FAP, and caspase-8. Immunostaining for Fas, Fas ligand, TRAIL, TRADD, RIP, and caspase-8 was also performed. To assess the role of these molecules in COU-associated renal cell apoptosis, the frequencies of apoptotic tubular and interstitial cells were separately quantitated for each experimental time point, and their patterns of variation were correlated with those of apoptosis-related molecules. RESULTS: The obstructed kidneys displayed increased apoptosis of both tubular and interstitial cells. Tubular cell apoptosis appeared at day 4 after ureter ligation, peaked (fivefold of control) at day 15, and decreased gradually until the end of the experiment. In contrast, interstitial cell apoptosis sustained a progressive increase throughout the experiment. Apoptosis was minimal at all experimental time points for control and contralateral kidneys. Compared with control and contralateral kidneys, the ligated kidneys displayed a dynamic expression of mRNAs for many apoptosis-related molecules, which included an up to threefold increase for Fas, Fas ligand, TNF-R1, TRAIL, TRADD, RIP, and caspase-8, and an up to twofold increase for FADD and FAP, but there was little change for FAF. These mRNAs increased between days 4 and 15, decreased until day 30, but then increased again until day 45. The rise and fall of mRNAs between days 4 and 30 paralleled a similar fluctuation in tubular cell apoptosis in that period. The subsequent increase of mRNAs was correlated with a continuous rise of interstitial cell apoptosis. We demonstrated a positive immunostaining for Fas and Fas ligand in the tubular cells at early time points as well as in interstitial inflammatory cells at later time points. Although increased expression of TRAIL, TRADD, RIP, and caspase-8 was noted in tubular cells, there was no staining for these molecules in interstitial cells. CONCLUSION: The current study documents a dynamic expression of several molecules that are known to mediate the most crucial steps of apoptosis. It implicates these molecules in COU-associated renal cell apoptosis and in the pathogenesis of this condition. It also lays the foundation for interventional studies, including genetic engineering, to evaluate the molecular control of apoptosis associated with COU.

Osmotically relevant membrane signaling complex: association between HB-EGF, beta(1)-integrin, and CD9 in mTAL.

The integral membrane proteins cluster of differentiation-9 (CD9), beta(1)-integrin, and heparin-binding epidermal growth factor-like (HB-EGF) exist in association in many cell lines and are linked to intracellular signaling mechanisms. Two of the proteins (CD9 and beta(1)-integrin) are induced by hypertonicity, suggesting that their related signaling processes may be relevant to osmotic stress. The validity of this hypothesis rests upon coexpression and physical association between these molecules in nephron segments that are normally exposed to high and variable ambient osmolality. In this work, we show that CD9 and beta(1)-integrin are induced in rat kidney medulla after dehydration. Immunohistochemistry and immunoprecipitation studies show that CD9, HB-EGF, and beta(1)-integrin are coexpressed and physically associated in medullary thick ascending limbs (mTAL), nephron segments that are normally exposed to high and variable extracellular osmolality. Our findings are consistent with the existence of a cluster of integral membrane proteins in mTAL that may initiate or modulate osmotically relevant signaling pathways.

Regulation of stanniocalcin in MDCK cells by hypertonicity and extracellular calcium.

Differential display RT-PCR cloning method was applied to poly(A)(+) RNA isolated from Madin-Darby canine kidney (MDCK) cells in isotonic or hypertonic medium. A differentially expressed 360-bp PCR fragment was isolated, subcloned, sequenced, and used to screen an MDCK cDNA library constructed in lambdaZapII. A composite sequence of two overlapping cDNA clones provided 1,053 bp of sequence that was 93% identical to human stanniocalcin and corresponded to the 3'-end of the mRNA. Although the fish homolog of this hormone inhibits calcium uptake by the gill and intestine, the function of mammalian stanniocalcin remains unknown. Stanniocalcin cDNA probe hybridizes to a 4.4-kb mRNA that is induced eightfold by hypertonicity, in a manner that is dependent on medium organic osmolytes. The mRNA induction correlates with increased total cellular content of the protein and its concomitant release to the medium, consistent with secretion for autocrine or paracrine activity. Furthermore, induction of the mRNA by hypertonicity is dependent on extracellular calcium and displays a threshold phenomenon. The data suggest that kidney stanniocalcin may have a role in the adaptation of kidney cells to osmotic stress, in a manner that is extracellular calcium dependent.

Osmotic response element enhancer activity. Regulation through p38 kinase and mitogen-activated extracellular signal-regulated kinase kinase.

Hypertonicity induces a group of genes that are responsible for the intracellular accumulation of protective organic osmolytes such as sorbitol and betaine. Two representative genes are the aldose reductase enzyme (AR, EC 1.1.1.21), which is responsible for the conversion of glucose to sorbitol, and the betaine transporter (BGT1), which mediates Na+-coupled betaine uptake in response to osmotic stress. We recently reported that the induction of BGT1 mRNA in the renal epithelial Madin-Darby canine kidney cell line is inhibited by SB203580, a specific p38 kinase inhibitor. In these studies we report that the hypertonic induction of aldose reductase mRNA in HepG2 cells as well as the osmotic response element (ORE)-driven reporter gene expression in transfected HepG2 cells are both inhibited by SB203580, suggesting that p38 kinase mediates the activation and/or binding of the transcription factor(s) to the ORE. Electrophoretic gel mobility shift assays with cell extracts prepared from SB203580-treated, hypertonically stressed HepG2 cells further show that the binding of trans-acting factors to the ORE is prevented and is thus also dependent on the activity of p38 kinase. Similarly, treatment of hypertonically stressed cells with PD098059, a mitogen-activated extracellular regulated kinase kinase (MEK1) inhibitor, results in inhibition of the hypertonic induction of aldose reductase mRNA, ORE-driven reporter gene expression, and the binding of trans-acting factors to the ORE. ORE-driven reporter gene expression was not affected by p38 kinase inhibition or MEK1 inhibition in cells incubated in iso-osmotic media. These data indicate that p38 kinase and MEK1 are involved in the regulation of the hyperosmotic stress response.

Cell apoptosis and proliferation in obstructive uropathy.

Distinct patterns of cell proliferation and apoptosis have been recognized for tubular, interstitial, and glomerular cells in chronic obstructive uropathy (OU). In many experimental models of OU, tubular cell apoptosis develops quickly after ureter ligation, peaks between 7 and 24 days postobtruction (about 30-fold of control), and tapers thereafter. Apoptosis initially involves the dilated collecting ducts, but subsequently spreads to other tubules. Tubular cell apoptosis probably accounts for renal tissue loss in OU because a direct correlation between its degree and the decline in dry kidney weight is well-documented. Pronounced tubular cell proliferation occurs shortly after ureter ligation, peaks at about day 6 (60-fold above control), and quickly subsides to baseline. Because the peak of tubular cell proliferation immediately precedes the onset of tubular cell apoptosis, a pathogenetic link may exist between these two processes. Interstitial cell apoptosis occurs with an increasing frequency throughout the course of OU (up to 35-fold above control). Interstitial cell proliferation appears in a bimodal pattern with the early peak coinciding with that of tubular cell proliferation and consisting mostly of fibroblasts, whereas the later peak consists mostly of inflammatory cells. Glomerular cell apoptosis and proliferation are not different from control, which explains, in part, the structural integrity of the glomeruli throughout the disease course. Although the general pathways of cell apoptosis and proliferation are well known, the molecular control of these processes in OU is poorly understood. In addition, whether apoptosis or proliferation of tubular and interstitial cells is differentially regulated remains to be studied. However, several molecules known to be activated or overexpressed in kidney with OU may modulate cell apoptosis and proliferation. The relevant functions of these molecules include induction of apoptosis (angiotensin II, reactive oxygen species, jun-N-terminal kinase, p53), inhibition of the cell cycle (transforming growth factor-beta, p21), inhibition of apoptosis (clusterin, epidermal growth factor, insulin-like growth factor, bcl-2, osteopontin), or promotion of interstitial fibroblast proliferation (platelet-derived growth factor).

The patient with a clotted PTFE graft developing fever.

Haemodialysis access graft infection is easily recognizable when local symptoms (warmth, swelling, pain, or drainage) predominate, and endocarditis is a well established complication of infected grafts. We report a case of bacterial endocarditis complicating silent infection in clotted haemodialysis access graft. It is suggested that, clotted non-functioning grafts may be the harbingers of silent infection, and should be suspected as the source of infection in every haemodialysis patient that presents with fever, even in the absence of clinical signs of graft site infection.

Acute effects of vasopressin V2-receptor antagonist on kidney AQP2 expression and subcellular distribution.

The acute effect of treatment with the vasopressin V2-receptor antagonist OPC-31260 (OPC) on aquaporin-2 (AQP2) distribution and expression in rat kidney was examined. Immunofluorescence and semi-quantitative immunoelectron microscopy revealed that 15 and 30 min of OPC treatment resulted in significant reduction in apical plasma membrane labeling of AQP2, with a concomitant increase in labeling of vesicles and multivesicular bodies. In parallel, OPC treatment induced a large increase in urine output [0.6 +/- 0.2 vs. 8.3 +/- 1.0 ml/h (n = 4)]. Northern blotting using a 32P-labeled AQP2 cDNA probe and a digoxigenin-labeled AQP2 RNA probe revealed a band of approximately 1.6 kb corresponding to the predicted size of AQP2 mRNA. In control experiments, thirsting increased, whereas water loading decreased AQP2 mRNA levels. Treatment of rats with OPC caused a significant reduction in AQP2 mRNA within 30 min (52 +/- 21%, n = 8, P < 0.025) and 60 min (56 +/- 7%, n = 4, P < 0.001) of treatment compared with intravenous saline-injected controls. Thus a very rapid reduction in AQP2 mRNA was observed in response to vasopressin-receptor antagonist treatment. The reduction in AQP2 mRNA persisted after 24 h (40 +/- 17%, n = 5, P < 0.05) of OPC treatment. There was a parallel increase in diuresis and reduction in urine osmolality. In conclusion, V2-receptor blockade produced a rapid internalization of AQP2 parallel with a rapid increase in urine output. Furthermore, OPC treatment caused a rapid and significant reduction in AQP2 mRNA expression, demonstrating that for rapid regulation of AQP2 expression, modulation of AQP2 mRNA levels is regulated via vasopressin-receptor signaling pathways.

Silent infection in clotted hemodialysis access grafts.

Thrombotic and infectious complications are frequent causes of hemodialysis vascular access failure and contribute considerably to the cost of care for chronic hemodialysis patients. Although there is clear indication for removal of patent grafts in unresolved bacteremia, there are no guidelines for the management of clotted nonfunctioning grafts. To evaluate for the existence and clinical relevance of silent infection in clotted nonfunctioning hemodialysis grafts, a study was conducted with a series of 20 hemodialysis patients who presented with fever (15 patients), or fever and clinical signs of sepsis (five patients), in whom the source of infection was not immediately localized to any organ system. Comparison was made with 21 asymptomatic patients with clotted grafts who served as control subjects. All patients and control subjects came from a pool of 115 chronic hemodialysis patients in an outpatient hemodialysis unit in the Houston metropolitan area, who were on hemodialysis for a period of time ranging from 3 to 15 yr. Indium scans were performed, followed by removal of the clotted grafts in all patients and control subjects. Bacterial cultures of the recovered surgical material and blood were done concomitantly in all study participants. Indium scans showed positive uptake in or around the clotted grafts in all of the patients and in 15 of the control subjects. Purulent material was found in the grafts in all patients and in 13 of 15 indium scan-positive control subjects. When positive, blood culture pathogens were identical to those cultured from the graft material in all instances. The predominant pathogens were Staphylococcus aureus, followed by Staphylococcus epidermidis. There was no evidence of graft infection in the control subjects if indium scan was negative. Chart review dating back to the start of dialysis revealed five past infectious episodes in the patient group, compared with four in the control group. These findings suggest that clotted nonfunctioning grafts are frequent harbingers of infection. They should be suspected as the source of infection in every hemodialysis patient that presents with fever, even in the absence of clinical signs of graft site infection.