Candesartan upregulates angiotensin-converting enzyme 2 in kidneys of male animals by decreased ubiquitination.

Candesartan is a common angiotensin-II receptor-1 blocker used for patients with cardiovascular and renal diseases. Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of blood pressure (BP), and also a major receptor for coronaviruses. To determine whether and how candesartan upregulates ACE2, we examined BP and ACE2 in multi-organs from male and female C57BL/6J mice treated with candesartan (1 mg/kg, i.p.) for 7 days. Relative to the vehicle, candesartan lowered BP more in males than females; ACE2 protein abundances were increased in kidneys, not lungs, hearts, aorta, liver, spleen, brain, or serum, only from males. Ace2-mRNA was similar in kidneys. Candesartan also decreased BP in normal, hypertensive, and nephrotic male rats. The renal ACE2 was increased by the drug in normal and nephrotic male rats but not spontaneously hypertensive ones. In male mouse kidneys, ACE2 was distributed at sodium-hydrogen-exchanger-3 positive proximal-convoluted-tubules; ACE2-ubiquitination was decreased by candesartan, accompanied with increased ubiquitin-specific-protease-48 (USP48). In candesartan-treated mouse renal proximal-convoluted-tubule cells, ACE2 abundances and activities were increased while ACE2-ubiquitination and colocalization with lysosomal and proteosomal markers were decreased. The silence of USP48 by siRNA caused a reduction of ACE2 in the cells. Thus, the sex-differential ACE2 upregulation by candesartan in kidney from males may be due to the decreased ACE2-ubiquitination, associated with USP48, and consequent degradation in lysosomes and proteosomes. This is a novel mechanism and may shed light on candesartan-like-drug choice in men and women prone to coronavirus infections.

Renal CD81 interacts with sodium potassium 2 chloride cotransporter and sodium chloride cotransporter in rats with lipopolysaccharide-induced preeclampsia.

The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)-induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS-PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS-PE than controls on GD14. On GD18, LPS-PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co-immunoprecipitated with NKCC2 or NCC in kidney homogenates and co-immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS-PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co-immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS-induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS-PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE.

Morroniside ameliorates glucocorticoid-induced osteoporosis and promotes osteoblastogenesis by interacting with sodium-glucose cotransporter 2.

CONTEXT: Morroniside (MOR) possesses antiosteoporosis (OP) effects, but its molecular target and relevant mechanisms remain unknown. OBJECTIVE: We investigated the effects of MOR on glucocorticoid-induced OP and osteoblastogenesis and its underlying mechanisms. MATERIALS AND METHODS: The effects of MOR (10-100 µM) on the proliferation and differentiation of MC3T3-E1 cells were studied in vitro. The glucocorticoid-induced zebrafish OP model was treated with 10, 20 and 40 µM MOR for five days to evaluate its effects on vertebral bone density and related osteogenic markers. In addition, molecular targets prediction and molecular docking analysis were carried out to explore the binding interactions of MOR with the target proteins. RESULTS: In cultured MC3T3-E1 cells, 20 µM MOR significantly increased cell viability (1.64 ± 0.12 vs. 0.95 ± 0.16; p < 0.01) and cell differentiation (1.57 ± 0.01 vs. 1.00 ± 0.04; p < 0.01) compared to the control group. MOR treatment significantly ameliorated vertebral bone loss in the glucocorticoid-induced OP zebrafish model (0.86 ± 0.02 vs. 0.40 ± 0.03; p < 0.01) and restored the expression of osteoblast-specific markers, including ALP, Runx2 and Col-І. Ligand-based target prediction and molecular docking revealed the binding interaction between MOR and the glucose pockets in sodium-glucose cotransporter 2 (SGLT2). DISCUSSION AND CONCLUSIONS: These findings demonstrated that MOR treatment promoted osteoblastogenesis and ameliorated glucocorticoid-induced OP by targeting SGLT2, which may provide therapeutic potential in managing glucocorticoid-induced OP.

Glucose-induced decrease of cystathionine ß-synthase mediates renal injuries.

Exogenous hydrogen sulfide (H2 S) protects kidneys from diabetic injuries in animal models. In order to explore the role of endogenous H2 S in diabetic nephropathy, we determined the renal H2S producing enzymes in vivo and in vitro. In diabetic mice, H2 S levels in blood and kidney were decreased while cystathionine ß-synthase (CBS), mainly located in mouse renal proximal convoluted tubules (PCT), was reduced selectively. In cultured mouse PCT cells treated with high glucose, CBS protein and activity was reduced while ubiquitinated CBS was increased, which was abolished by a proteasome inhibitor MG132 at 1 hour; high glucose drove CBS colocalized with proteasome 26S subunit ATPase6, indicating an involvement of ubiquitination proteasome degradation. At 48 hours, high glucose also selectively decreased CBS protein, concentration-dependently, but increased the ubiquitination of CBS; silence of CBS by siRNA increased nitrotyrosine, a marker for protein oxidative injury. Nitrotyrosine was also increased by high glucose treatments. The increases of nitrotyrosine either by cbs-siRNA or by glucose were restored by GYY4137, indicating that the H2 S donor may protect kidney from oxidative injury induced by CBS deficiency. In diabetic kidneys, ubiquitinated CBS and nitrotyrosine were increased but restored by GYY4137. The treatment also ameliorated albuminuria and renal morphologic changes in diabetic mice. Our findings suggest that high glucose induces reduction of renal CBS protein and activity in vivo and in vitro that is critical to the pathogenesis of diabetic kidney disease.

miR-532-3p inhibits osteogenic differentiation in MC3T3-E1 cells by downregulating ETS1.

BACKGROUND: Many studies had identified that MicroRNAs (miRNAs) could affect bone metabolism by regulating the expression of various proteins. This study explored the effect and mechanism of miR-532-3p on osteogenic differentiation. METHODS: We analyzed the content of miR-532-3p in osteoporosis patients, osteoporosis rats, and osteogenic induced MC3T3-E1 cells. MiR-532-3p mimic or inhibitor utilized to alter intracellular miR-532-3p content. MTT method executed to detect the effect of miR-532-3p on osteoblast proliferation. Real-time qPCR, Western blot, alkaline phosphatase staining, and alizarin red staining utilized to ascertain the influence of miR-532-3p on osteogenic differentiation. Then, databases and a dual-luciferase reporter gene assay used to verify the target of miR-532-3p. Furthermore, the lentiviral vector was utilized to overexpress interesting target gene expression and checked whether the target gene was involved in the regulation of osteogenic differentiation by miR-532-3p. RESULTS: MiR-532-3p expression boosted in low bone mineral density (BMD) patients and rats. In MC3T3-E1 cells, miR-532-3p expression gradually decreased as osteogenic induction matures. MiR-532-3p mimic negatively regulated succinate dehydrogenase (SDH) activity, alkaline phosphatase (ALP) activity, mineralization ability, the osteogenic-associated gene (Col1A1, Runx2, ALP, OPN, and OCN) and E-26 transformation specific-1 (ETS1) expression of MC3T3-E1 cells. Things are the opposite of the miR-532-3p inhibitor. ETS1 identified as the miR-532-3p target gene, and miR-532-3p could inhibit its expression. Besides, improved ETS1 expression could rescue the suppressive effect of miR-532-3p mimic on osteogenic differentiation. CONCLUSION: miR-532-3p can suppress osteogenic differentiation by downregulating ETS1 expression.

General and fast synthesis of graphene frameworks using sugars for high-performance hydrogen peroxide nonenzymatic electrochemical sensor.

3D graphene frameworks (GFs) are fast and scalably synthesized via a general and facile method from the rich biomass of sugars with the aid of molten salts, using glucose as the prototype, to obtain an effective sensing platform for sensitive nonenzymatic hydrogen peroxide (H2O2) detection. The electroactive area of the GFs/GCE (0.1437 cm2) is obviously higher than that of bare GCE (0.0653 cm2). The GFs are found to exhibit remarkable electrocatalytic activity toward H2O2 reduction while avoiding enzyme loading. The electrochemical sensor for H2O2 based on GFs displays a low detection limit of 0.032 ± 0.005 µM (S/N = 3) at a working potential of - 0.55 V in 0.01 M N2-saturated phosphate-buffered saline (PBS, pH = 7.4) by an amperometric method. The sensor has good selectivity over other compounds such as ascorbic acid, dopamine, uric acid, NaCl, citric acid, and glucose. Moreover, the sensor shows excellent reproducibility with a relative standard deviation of 3.7% and acceptable stability after 30 days of usage. Furthermore, it can detect H2O2 released from living tumorigenic cells in real time. Most importantly, it is demonstrated that such GFs can be obtained from a variety of sugars (sucrose, fructose, lactose, and maltose). This work may offer a new general avenue for the synthesis of 3D GFs and promote the development of electrochemical sensors. Graphical abstract We have reported a general and fast method to synthesize GFs from sugars (glucose, sucrose, fructose, lactose, and maltose) with the addition of molten Na2CO3 salt as a template. The developed GFs can be applied as excellent electrode materials for efficient electrochemical sensing of H2O2.

Characterizations and Assays of α-Glucosidase Inhibition Activity on Gallic Acid Cocrystals: Can the Cocrystals be Defined as a New Chemical Entity During Binding with the α-Glucosidase?

Cocrystallization with co-former (CCF) has proved to be a powerful approach to improve the solubility and even bioavailability of poorly water-soluble active pharmaceutical ingredients (APIs). However, it is still uncertain whether a cocrystal would exert the pharmacological activity in the form of a new chemical entity, an API-CCF supramolecule. In the present study, gallic acid (GA)-glutaric acid and GA-succinimide cocrystals were screened. The solubility, dissolution rate and oral bioavailability of the two cocrystals were evaluated. As expected, AUCs of GA-glutaric acid and GA-succinimide cocrystals were 1.86-fold and 2.60-fold higher than that of single GA, respectively. Moreover, experimental evaluations on α-glucosidase inhibition activity in vitro and theoretical simulations were used to detect whether the two cocrystals would be recognized as a new chemical entity during binding with α-glucosidase, a target protein in hypoglycemic mechanisms. The enzyme activity evaluation results showed that both GA and glutaric acid displayed α-glucosidase inhibition activity, and GA-glutaric acid cocrystals showed strengthened α-glucosidase inhibition activity at a moderate concentration, which is attributed to synergism of the two components. Molecular docking displayed that the GA-glutaric acid complex deeply entered the active cavity of the α-glucosidase in the form of a supramolecule, which made the guest-enzyme binding configuration more stable. For the GA and succinimide system, succinimide showed no enzyme inhibition activity, however, the GA-succinimide complex presented slightly higher α-glucosidase inhibition activity than that of GA. Molecular docking simulation indicated that the guest molecules entering the active cavity of the α-glucosidase were free GA and succinimide, not the GA-succinimide supramolecule.

Reduction of Interferences Using Fe-Containing Metal-Organic Frameworks for Matrix Separation and Enhanced Photochemical Vapor Generation of Trace Bismuth.

Photochemical vapor generation (PVG) has emerged as a promising sample introduction method for atomic spectrometry in recent years. Despite its great success, a major impediment for the wide application of PVG is the interferences from the coexisting ions, especially transition-metal ions. In this work, iron and 1,3,5-benzenetricarboxylic (Fe-BTC), a Fe-containing metal-organic framework (MOF) material, was synthesized and first used as a platform integrating the sample matrix separation, preconcentration, and photocatalysis for the highly selective determination of elements by PVG. Bismuth was selected as a model analyte. Fe-BTC served not only as the photocatalyst for the PVG of Bi3+ but also as an efficient absorbent for the separation of analytes from the sample matrix. Compared with the previous PVG system, the performance of tolerating interferences toward coexisting ions of the proposed method has been greatly improved after using Fe-BTC-based matrix separation. Thus the excess of 10 mg L-1 of Co2+ and 100 mg L-1 of Cu2+, Ni2+, and Fe3+ caused no obvious interferences for 1 µg L-1 of Bi determination. Under the optimal conditions, the limit of detection (LOD, 3σ) of the developed method was 0.3 ng L-1 with the inductively coupled plasma-mass spectrometry (ICPMS) measurement, which could be lowered down to 0.04 ng L-1 after ten times of preconcentration with Fe-BTC prior to analysis. This method was successfully applied for the analysis of Bi in complicated sample matrices of soil (GBW07401), sediment (GBW07310), nickel-iron alloy (GBW01622), and nickel alloys (GBW01641) by the external calibration method.

Enhanced Photochemical Vapor Generation for the Determination of Bismuth by Inductively Coupled Plasma Mass Spectrometry.

An enhanced photochemical vapor generation (PVG) sample introduction procedure is developed for the determination of trace Bi with inductively coupled plasma mass spectrometry (ICP MS) by the addition of iron. Gas chromatography mass spectrometry (GC-MS) reveals that (CH3)3Bi is the major component of the volatile Bi species formed in the presence of 20% (v/v) acetic acid, 5% (v/v) formic acid, and 60 µg mL-1 Fe3+ under UV irradiation. The addition of Fe3+ not only largely increases the PVG efficiency of Bi3+ but also accelerates the reaction kinetics of photochemical reduction of Bi3+. The analytical sensitivity was enhanced 30-fold using PVG for sample introduction compared to that for direct solution nebulization detection by ICP MS detection. Furthermore, the proposed method shows much better tolerance of interference from Cu2+ and Ni2+ than that from conventional hydride generation (HG). Under the optimized conditions, a detection limit of 0.3 ng L-1 was obtained for Bi by ICP MS determination. The relative standard deviation (RSD) was 2.5% for seven replicate measurements of 0.5 ng mL-1 Bi3+ standard solution. The proposed method has been successfully applied for the determination of Bi in environmental samples, including water samples, and certified reference material of soil (GSS-1) and sediments (GSD-5a and GSD-10) with satisfying results.

Treatment of cerebrospinal fluid leak after spine surgery.

Owing to the complexity of spinal surgery, there is a great prevalence of dural tear causing cerebrospinal fluid (CSF) leakage. Many studies focused on suture repair for dural tear to stop CSF leak. Now some new treatment strategies have shown a promising effect that is listed as follows: 1) creating watertight dural closure to stop CSF leak with the help of dural substitute material; and 2) retarding CSF leak by changing pressure difference, including reducing the subarachnoid fluid pressure, increasing the epidural space pressure and both. In fact several methods mentioned above are usually combined to treat CSF leak. However, no update review summarized the relevant studies implemented in recent years. In this review, the authors would compare the effects of different dural closure techniques, and introduce the latest treatment methods and mechanisms.

Subfascial drainage for management of cerebrospinal fluid leakage after posterior spine surgeryd---A prospective study based on Poiseuille's law.

PURPOSE: Up to date, some approaches retarding the flow of cerebrospinal fluid (CSF) could be regarded as direct applications of the fluid mechanics (Poiseuille's law). However, there is a lack of the research on the efficacy of subfascial drainage for management of CSF leak after spine surgery based on the law. This is a prospective and comparative study on subfascial drainage for CSF. METHODS: Every four months in the three years from January 2010 to December 2012, the patients were enrolled respectively in Group A, Group B and Group C, in which, the drainage tube was discontinued within postoperative 3-4 days, 5-6 days, 7-10 days. Results and complications of postoperative CSF leak were investigated, and mean wound healing time (MWHT) of the three groups was compared. RESULTS: A total of 108 cases (Group A/B/C:35/32/41) of CSF leak following posterior spine surgery were admitted to Tianjin Union Medicine Center, and 92 cases have been followed up for more than 1 year (follow-up rate of 85.2%). Preoperative demographics were similar among the 3 groups. In Group A, 7 patients developed CSF leak through the wound (CSFLW), of which 5 cases had to undergo reoperation. One case in Group A was confirmed to have pseudomeningocele at the 1st month after surgery. The MWHT was (16.6±3.6) days. In Group B, 3 patients developed CSFLWand cured by reoperation, in which 1 case of superficial infection recovered well after reoperation. MWHT was (11.4±2.2) days. In Group C, CSFLWwas not found and MWHT was (10.1±2.9) days. The differences of MWHT among Groups A, B and C were statistically significant. CONCLUSION: Postoperative subfascial drainage, which is used to decrease the subfascial space pressure (P2), would help wound healing. When it is placed for more than 7 days, the wound resistance (Rw) would be strong enough to withstand the subarachnoid pressure (P1). Meanwhile, the power trans- duction in a sequence of Rw > P2 > P1 will indirectly retard CSF leak at the durotomy site and accordingly facilitate the healing of damaged spinal dura mater.

Surgical treatment of traumatic lower limb pseudoaneurysm.

OBJECTIVE: To summarize our experience in surgical treatment of traumatic lower limb pseudoaneurysm. METHODS: Twenty patients with traumatic lower limb pseudoaneurysm were surgically treated in our department from January 2007 to January 2012. The treatment protocols included interventional covered-stent placement (10 cases), spring coil embolization (2 cases), and surgical operation (8 cases). Surgical operations included pseudoaneurysm repair (2 cases), autologous-vein transplantation (1 case), and artificial-vessel bypass graft (5 cases). RESULTS: All the patients were successfully treated without aggravating lower limb ischemia. Pseudoaneurysm disappeared after treatment. A surgical operation is suitable to most pseudoaneurysms, but its damage is relatively obvious and usually leads to more bleeding. It also requires a longer operating time. Compared to a surgical operation, interventional therapy is less traumatic and patients usually have a quicker recovery (P<0.05). All patients were followed up once per month for 12-36 months by color Doppler ultrasound examination. There were no cases of pseudoaneurysm recurrence. CONCLUSION: Both surgical operation and interventional therapy are safe and effective in the treatment of pseudoaneurysm.

[The significance of lumbar MRI in the diagnosis of recurrented lumbar disc herniation after surgery].

OBJECTIVE: To retrospectively evaluate the clinical evaluation of preoperative lumbar T2 sagittal MRI image in predicting the recurrent lumbar disc herniation (RLDH). METHODS: Between January 2009 and April 2011, 28 patients were diagnosed as recurrent L4-5 disc herniation within 2.5 years after surgery, and 25 of them were included in the study as relapse group. At the same time, selected 25 patients implemented the same surgical methods in the same level as a control group randomly, they were all with good to excellent result and the follow-up time was at least 2.5 years. There was no statistical significance between the two groups in gender, age and body mass index(BMI) (P > 0.05). The lumbar MRI image of two groups of patients before surgery were collected and analyzed, with the disc degeneration grade classified. The χ(2) test was used to analyzed the difference of degeneration between the two groups of patients before surgery. Rank correlation analysis evaluated the correlation between disc degeneration and the period of time from the first operation to the recurrence. RESULTS: In terms of preoperative lumbar disc degeneration, there were 22 cases of low-grade disc degeneration and 3 cases of advanced disc degeneration in the relapse group and 5 cases and 20 cases respectively in the control group. there was significant difference between two groups (χ(2) = 23.27, P < 0.05), low-grade disc degeneration (gradesIand III) was significantly more frequent in the relapse group than in the control group. The patients with low-grade disc degeneration had a higher risk of recurrence, that was the risk of recurrent disc herniation increased by a factor of 4.4 from advanced disc degeneration to low-grade disc degeneration(OR = 4.4, 95%CI:1.983-9.765, P < 0.05). In cases of recurrence, the time interval between primary surgery and the recurrence of the patient with advanced disc degeneration was longer compared with low-grade disc degeneration (r = 0.733, P < 0.05). CONCLUSIONS: Preoperative lumbar MRI image may suggest the possibility of the recurrence lumbar disc herniation.Light disc degeneration is an important risk factor for recurrent disc herniation, and the time interval between primary surgery and the recurrence is positively correlated with severity of disc degeneration.

DOPAMINE D4 RECEPTOR DOWN-REGULATES RENAL SODIUM CHLORIDE COTRANSPORTER VIA UBIQUITINATION-ASSOCIATED LYSOSOME DEGRADATION.

BACKGROUND: The thiazide-sensitive sodium chloride cotransporter (NCC) is the major apical sodium transporter located in the mammalian renal distal convoluted tubule (DCT). The amount of sodium reabsorbed in the DCT through NCC plays an important role in the regulation of extracellular fluid volume and blood pressure. Dopamine and its receptors constitute a renal antihypertensive system in mammals. The disruption of Drd4 in mice causes kidney-related hypertension. However, the pathogenesis of D4R-deficiency associated hypertension is not well documented. METHOD: We assessed the effects of D4R on NCC protein abundances and activities of DCT in mice with renal or global Drd4-deficiencies and expressing human D4.7 variant and in cultured mouse DCT cells, and explored the molecular mechanism. RESULTS: NCC inhibitor hydrochlorothiazide enhanced the natriuresis in Drd4-/- mice. Renal NCC protein was greater while ubiquitination of NCC was less in Drd4-/- than Drd4+/+ mice. Silencing of D4R in cultured mouse DCT cells increased NCC protein but decreased NCC ubiquitination. D4R agonist had opposite effects that were blocked by the antagonist. In mouse kidneys and DCT cells D4R and NCC colocalized and co-immunoprecipitated. Moreover, D4R-agonist promoted the binding between the two proteins demonstrated by fluorescence resonance energy transfer. D4R agonism internalized NCC, decreased NCC in the plasma membrane, increased NCC in lysosomes and reduced NCC-dependent-intracellular-sodium transport. The lysosomal inhibitor chloroquine prevented the D4R-induced NCC-reduction. A shortened NCC half-life was suggested by its decay under cycloheximide-chase. Ubiquitin-specific-protease 48 (USP48, a deubiquitinating enzyme) was increased in the kidneys and cells with Drd4-deficiency while D4R stimulation decreased it in vitro and reduction of USP48 with siRNA decreased NCC expression. The mice carrying human D4.7 variant or with renal supcapsular-Drd4-siRNA-delivery developed hypertension with increased NCC. CONCLUSION: Our data demonstrates that D4R downregulates NCC by promoting USP48-associated deubiquitination and subsequent internalization, lysosome relocation and degradation.

A low-salt diet with candesartan administration is associated with acute kidney injury in nephritis by increasing nitric oxide.

A low-salt diet may activate the renin-angiotensin-aldosterone system (RAAS) and is often applied simultaneously with RAAS inhibitors, especially for treatment of proteinuric nephritis. To explore the effect of a low-salt diet combined with angiotensin receptor blockers (ARB) on kidney function, the proteinuric nephritis model was induced by single intravenous injection of doxorubicin, and then the SD rats were administrated with candesartan intraperitoneal injection and fed with different salt diets. Rats with low-salt plus candesartan, not either alone, experienced acute kidney injury (AKI) at day 7 and could not self-restore when extending the experiment time from 7 days to 21 days, unless switching low-salt to normal-salt. Among three nitric oxide synthetases (NOS), endothelial NOS (eNOS) was obviously elevated and PI3K-Akt-eNOS signal pathway was activated. NG-Nitro-L-Arginine Methyl Ester (L-NAME), an eNOS inhibitor, reversed the decreased blood pressure and recovered the kidney dysfunction induced by low-salt with candesartan. The increased TUNEL-positive cells, Bax/Bcl-2 and cleaved-caspase3 protein abundance was ameliorated by L-NAME in vivo. In vitro, sodium nitroprusside, a nitric oxide donor, can also increase Bax/Bcl-2 and cleaved-caspase3 protein level in HK-2 cell. Thus, low-salt diet combined with candesartan in nephritis rats led to AKI, and the mechanism involved the increase of eNOS/NO, which linked to the decrease of blood pressure and the increase of apoptosis. This study provides practical guidance for salt intake in cases of RAS inhibitor usage clinically.

GYY4137, a H2S donor, ameliorates kidney injuries in diabetic mice by modifying renal ROS-associated enzymes.

Diabetic nephropathy (DN) is a common microvascular complication of both type 1 and type 2 diabetes mellitus and often advances to end-stage renal disease. Oxidative stress plays an important role in the pathogenesis and progress of DN. Hydrogen sulfide (H2S) is considered as a promising candidate for the management of DN. But the antioxidant effects of H2S in DN have not been fully studied. In mouse model induced by high-fat diet and streptozotocin, GYY4137, a H2S donor, ameliorated albuminuria at weeks 6 & 8 and decreased serum creatinine at week 8, but not hyperglycemia. Renal nitrotyrosine and urinary 8-isoprostane were reduced along with the suppressed levels of renal laminin and kidney-injury-molecule 1. Renal NADPH oxidase (NOX) 2 was lower but heme oxygenase (HO) 2, paraoxonase (PON) 1, PON2 were higher in DN+GYY than DN group. NOX1, NOX4, HO1, superoxide dismutases 1-3 were similar between groups. Except for a rise at HO2, all the affected enzymes were unchanged in mRNA levels. The affected reactive-oxygen-species (ROS) enzymes were mainly located in the renal sodium-hydrogen-exchanger positive proximal tubules with similar distribution but changed immunofluorence in GYY4137 treated DN mice. Kidney morphological alterations in DN mice under light and electrical-microscopes were also improved by GYY4137. Thus, exogenous H2S administration may improve the renal oxidative damage in DN by reducing ROS production and enhancing ROS cleavage in kidney via the affected enzymes. This study may shed a light on therapeutic applications in diabetic nephropathy with H2S donors in the future.

Does mismatch of the femoral component aspect ratio influence the range of knee flexion after posterior-stabilized total knee arthroplasty?

OBJECTIVE: To study whether the range of knee flexion (ROF) is affected by geometrical mismatch of the femoral component and the resultant change in the posterior condylar offset (PCO) after high-flexion posterior-stabilized total knee arthroplasty (TKA). METHODS: One hundred osteoarthritic patients (50 males and 50 females) underwent femoral osteotomy by the anterior referencing technique. The PCO for each patient was measured from lateral radiographs before, during and 2 years after TKA. The thickness of the joint cartilage was measured by magnetic resonance imaging before TKA and added onto the radiographic measurement. The relationship between changes in the PCO and improvements in the ROF before, during and 2 years after TKA were statistically analyzed. RESULTS: Compared with the preoperative value, the PCO was reduced by (3.45+/-3.28) mm after TKA, with a significantly larger reduction observed in female patients than male patients (P less than 0.05). When examining the subject population as a whole, there was a significant positive correlation between PCO and ROF improvement during TKA (P less than 0.05), but this improvement was not maintained 2 years after TKA (P larger than 0.05). However, when male and female patients were analyzed separately, there was a significant positive correlation between PCO change and ROF improvement for both sexes at both time points (all P less than 0.05). CONCLUSIONS: Restoration of PCO plays an important role in the optimization of knee flexion even after posterior-stabilized TKA. Femoral components based on Caucasian anatomic characteristics could not match the native anatomy of distal femurs in Chinese population especially female Chinese. Rotated resection of distal femur with anterior re-ferencing technique usually leads to a decreased PCO and therefore reduces maximal obtainable flexion.

Antibiotic-loaded articulating cement spacers in two-stage revision for infected total knee arthroplasty: individual antibiotic treatment and early results of 21 cases.

OBJECTIVE: To detail our early experience and technique of a modified two-stage reimplantation protocol using antibiotic-loaded articulating cement spacers (ALACSs) for treatment of late periprosthetic infection after total knee arthroplasty (TKA). METHODS: From January 2006 to February 2009, a series of 21 patients (21 knees) with late infected TKAs were treated by radical debridement and removal of all components and cement, and then articulating spacers were implanted using antibiotic-impregnated bone cement. For this purpose, 4 g vancomycin powder was mixed with per 40 g cement. Graduated knee motion and partial weight bearing activity were encouraged in the interval period. Each patient received an individual systemic organism-sensitive antimicrobial therapy for 4.9 (range, 2-8) weeks followed by a second-stage TKA revision. All the patients were regularly followed up using the American Knee Society Scoring System. RESULTS: Each case underwent a successful two-stage exchange and had infection eradicated, none had recurrent infection after an average of 32.2 (range, 17-54) months of follow-up. Preoperatively, the mean knee score was 53.5 points, function score was 27.3 points, pain score was 25.7 points, range of motion (ROM) was 82.0 degree extensor lag was 2 degree Between stages, the mean knee score was increased to 61.3 points, function score to 45 points, pain score to 35 points, ROM to 88.2 degree and extensor lag to 3.4 degree At final follow-up, the mean knee score was further increased to 82.1 points, function score to 74.5 points, pain score to 42.1 points, ROM to 94.3 degree and knee extension lag to 1.9 degree The interval period was 11.5 (range, 6-32) weeks. The amount of bone loss was unchanged between stages. No patient developed noticeable dysfunction of the liver or kidney or other complications such as impaired wound healing, deep venous thrombosis, pulmonary embolism, cerebrovascular accidents, etc. CONCLUSIONS: Treating infected TKA with ALACS avoids spacer-related bone loss, preserves knee function between stages, and eradicates infection effectively without significant complications. The early clinical results are inspiring. The authors believe that radical and repeated (if needed) debridement, individual application of systemic antibiotics, and reasonable timing judgement upon the secondary revision are all key factors related to a successful outcome with two-stage reimplantation procedure for infected TKA.

Covalent organic framework-based fluorescent nanoprobe for intracellular pH sensing and imaging.

Lysosomes are the acidic organelles in the cells that play an important role in intracellular degradation and other various cellular functions. The pH disturbance of lysosomes will result in the lysosomal dysfunction and many lysosomal related diseases. In this work, we reported a methoxy-based covalent organic framework (TAPB-DMTP-COF) that a novel pH-responsive fluorescent probe for lysosomal pH imaging in cells. The prepared TAPB-DMTP-COF presented regular crystal structure, low toxicity and good pH responsive property. The rich imine structure in the material enabled pH-responsive properties of the TAPB-DMTP-COF and made it exhibited pH-dependent fluorescence response. Good detection linearity for pH measurements in aqueous solution was achieved by this probe. Moreover, the TAPB-DMTP-COF can be used for the selective lysosomal pH imaging. Confocal fluorescence imaging results demonstrated that the pH fluctuations (from 4.0 to 7.4) and the pH changes in lysosomes can be effectively monitored in situ by the developed probe. This study may provide a new avenue for the intracellular pH sensing, deep study and understanding about the mechanism of diseases related to abnormal lysosomal pH.

Assessment of Urinary Exosomal NHE3 as a Biomarker of Acute Kidney Injury.

The diagnosis of acute kidney injury (AKI) traditionally depends on the serum creatinine (Scr) and urine output, which lack sufficient sensitivity and specificity. Using urinary exosomes as a biomarker has unique advantages. To assess whether urinary exosomal Na+/H+ exchanger isoform 3 (NHE3) protein could serve as a biomarker of AKI, we constructed four AKI rat models: cisplatin (7.5 mg/kg) injected intraperitoneally (IP), furosemide (20 mg/kg, IP) with a low-NaCl (0.03%) diet, a low-NaCl (0.03%) diet with candesartan (1 mg/kg, IP) and bilateral ischemia and reperfusion (I/R) injury for 40 min. Additionally, we assessed six sepsis-associated AKI patients and six healthy volunteers. Urinary exosomes were extracted by ultracentrifugation, and the NHE3 protein abundance was tested by immunoblotting for all the AKI rats and human subjects. The isolated cup-shaped particles with an average diameter of 70 nm and enrichment in CD63 were identified as exosomes. NHE3 abundance was six times higher in exosomes than in the whole urine. In cisplatin-induced AKI rats, urinary exosomal NHE3 was increased on day 2, one day earlier than the increases in Scr and blood urea nitrogen (BUN). In additional rats, urinary exosomal NHE3 decreased along with the decline in Scr after EPO pretreatment. In volume-depletion AKI induced by furosemide injection with a low-NaCl diet, the urinary exosomal NHE3 expression was higher than that in the control. Under a low-NaCl diet with candesartan-related AKI, the urinary exosomal NHE3 was elevated on day 5, earlier than Scr. In I/R-injury AKI, the urinary exosomal NHE3 was also raised compared with that in the control. In humans, the urinary exosomal NHE3 level was also elevated in sepsis-associated AKI patients in comparison with that in the healthy volunteers. The urinary exosomal NHE3 was increased in multiple AKI; it may be used as a diagnostic biomarker of AKI.