The Effect Of Safingol-Loaded Magnetic Nanoparticles On Glomerular Filtration Rate In Rats With Fulminant Hepatic Failure

Objective:Acute liver failure（ALF）of different reasons is common in China.Hepatorenal syndrome（HRS）is one of the severe complications in ALF.In Patients with severe liver damage the vasoactive substance and TNF-αincreased obviously.TNF-αwas shown to potentiates IP₃R1 expression through increasing the activity of PKC-αin human glomerular smooth musle cells and mesangial cells,which causes their increasing of reactivity to vasoactive substances,increasing of renal perfusion pressure,decreasing of glomerular filtration rate（GFR）,then finally cause progressive,functional renal failure without structural changes in the kidney.GFR could be improved by silenceing PKC-αgene using lentivirus mediated interference in HRS resulting from fulminant hepatic failure.So,whether Safingol,a PKC-αinhibitor,could suppress TNF-α-induced expression of IP₃R1 levels by inhibiting the activity of PKC-α,then improve GFR in fulminant hepatic failure with HRS.It is beset with difficulties for safingol to apply clinically due to its dose dependent vascular stimulation,hemolysis,hematuresis,liver and kidney injury.Magnetic nanoparticles（MNPs）for drug delivery is the use of ferromagnetic material carrying drugs to directional movement and positioning in the body by the effect of the magnetic field in vitro.So we choose MNP as drug delivery system for increasing drug concentration in kidney to reduce the occurrence of side effects.In this study we firstly built a drug delivery system for safingol with MNP by EDC/NHS,and research in vitro whether the Drug-loading nanoparticles could restrain the activation of PKC-αand decrease the expression of IP₃R1 in rat glomerulus mesangial cells（RGMCs）,then study in vivo whether the Drug-loading nanoparticles could improve the GFR of rat with HRS.Materials and methods:1、Materials1)Rat GMCs（HBZY-1）（CRL-2573）was obtained from American Type Culture（ATCC,Manassa,VA）.Cell line was cultured at 37℃in a 5%CO2 humidified incubator with high glucose DMEM medium（containing 10%fetal bovine serum（FBS）,100U/I penicillin,100mg/l streptomycin）.2)Male Sprague-Dawley rats weighting around 90±5g were house in stainless steel mesh cages under controlled conditions of temperature（23±3℃）and light illumination for 12h a day.The animals were allowed access to food and tap water throughout the acclimatization for a week and experimental periods.2、Methods1)Fe₃O₄nanoparticles conjugated with safingol through EDC/NHS catalytic amidation between the carboxylic acid end groups on the surface of the magnetic nanoparticles and the amine groups on safingol.Then electrophoresis in 1%agarose gel was carried out for drug-loading nanoparticles.2)The appearance of the nanoparticles before and after conjugation were mesured by transmission electron microscope（TEM）.3)The conjugate pruduct was proven by Fourier Transform Infrared spectroscopy（FTIR）.4)The drug louding capacity of nanparticles is calculated through testing residual concentration of safingol by High Performance Liquid Chromatography（HPLC）.5)The MTS method was used for evaluate the effects on cell proliferation of magnetic nanoparticles and safingol loaded nanoparticles.6)The localization of the conjugate was proven by iron staining.7)In vitro non-radioactive phosphorylation method was used to test the activity of PKC-α.8)Western blot were used to test the effect of MNP-Sa and safingol on expression of PKC-α,PPKC-α,IP₃R1 protein in RGMCs.9)Real Time PCR were used to test the effect of MNP-Sa and safingol on expression of PKC-α,IP₃R1 mRNA in RGMCs.10)Western blot were used to test the effect of MNP-Sa and safingol with different concentrations on expression of PPKC-αprotein in RGMCs.11)Cell intracellular calcium concentration([Ca²⁺]i)was measured to test the effect of MNP-Sa and safingol on[Ca²⁺]i release in RGMCs.12)60 rats were randomly divided into 6 groups:the blank control group（NS group）,D-Gal N/LPS group,D-Gal N/LPS+MNP group,D-Gal N/LPS+MNP-Sa0.25μM group,D-Gal N/LPS+MNP-Sa0.125μM group,D-Gal N/LPS+safingol 10μM group.The microosmotic pumps with FITC-inulin were implanted into rat’s abdominal cavity and permanent magnets were implanted subcutaneous near renals.On day 7 post-pump implantation saline,MNP,MNP-Sa and safingol were injected into tail vain 30min before the injection of Gal N/LPS.13)Liver and kidney specimens were fixed in 10%formalin and 2.5%glutaraldehyde,the embedded in paraffin for histopathological analysis.Paraffin-embedded liver and kidney tissue was cut into 4μm thich sections.The sections were stained with hematoxylin and eoin and analyzed under a light microscope.14)Serum levels of alanine aminotransferase（ALT）,Urea nitrogen（BUN）,creatinine（Cr）,potassium ino（K⁺）,sodium ion（Na⁺）,chloride ion（Cl^-）were determined using commercial kits.15)Serum were collected to determine FITC-inulin after 12 hours.Fluorescence of FITC was determined using a Thermo Scientific Varioskan Flash with 485-nm excitation and read at 538-nm emission.GFR was evaluated using the equation inulin clearance=inulin infusion rate/steady-state blood inulin concentration.GFR was expressd in microliters per minute（GFR1）,microliters per minute per kilogram body weight（GFR2）,microliters per minuter per gram kidney weight（GFR3）.The model of HRS was considered to be successful,according to GFR and other results determined above.16)The effect of MNP-Sa on glomerular inulin space（GIS）and[Ca²⁺]i in glomeruli were determined.17)Western blot and Real Time PCR methods were used to test PKC-α,PPKC-α,IP₃R1protein and mRNA in rat’s kidney.Results:1.Fe₃O₄nanoparticles conjugated with safingol through EDC/NHS catalytic reaction:there was no significant difference in electrophoresis between MNP-Sa and MNP in 1%agarose gel;2.Through TEM observation of drug-loading nanoparticles morphology:the diameter of MNP is about 15 nm in scattered distribution,and MNP-Sa is about 15 to 20 nm in diameter,scattered distribution,with no obvious condensation Through TEM observation.3.the FTIR spectra of MNP-Sa showed the wave crest around 3219 cm^-1cm and1194 cm^-1/1105^-1which represent the stretching vibration of N-H bonds and C-N bonds seperately,that the FTIR spectra of MNP didn’t have;4.the molar ratio of MNP and safingol in MNP-Sa is about1:8.44;5.The MTS method MNP-Sa and safingol effect on cell proliferation rate of study:compared with saline control group,there was no significant difference in cell proliferation activity（P>0.05）when incubate RGMCs with MNP-Sa（0.125μM and0.25μM）,MNP（0.125μM and 0.25μM）,and safingol（5μM and 10μM）for 12h;6.the Prussian blue staining of RGMCs incubate with MNP-Sa for 12h showed a large number of dyed blue grain in cytoplasm,but there was no blue dye in NS control group;7.there was no significant difference in levels of PKC-αmRNA in RGMCs treated with MNP-Sa and safingol compared with NS control group（P>0.05）,but the IP₃R1 mRNA decreased significantly in MNP-Saand Sa group compared with NS group（P<0.05）;8.A significant induction of PKC-αactivity occurred in MNP-Sa and Sa group compared with NS group（P<0.05）.there was no significant difference in levels of PKC-αactivity in RGMCs of MNP-Sa and Sa group（P>0.05）;9.There was no significantly difference in PKC-αprotein expression in these four groups（P>0.05）.the levels of PPKC-αprotein and IP₃R1 protein in MNP-Sa and Sa group were significantly decreased compared with the NS group（P<0.05）;10.the levels of PPKC-αprotein were significantly reduced in safingol groups with different concentration（5μM,7.5μM,10μM）and MNP-Sa group compared with NS group（P<0.05）,and that effect was almost equal in these four groups（P>0.05）;11.We found a markedly increase of[Ca²⁺]i in RGMCs when treated with ET-1 in NS group,but that was obviously decreased by MNP-Sa0.125mM,Sa5mM and Sa7.5mM（P<0.05）,and that effect was almost equal in these three groups（P>0.05）,but not in Sa2.5mM group（P<0.05）;12.Histological evaluation:Dramatic damages occurred in liver 12 hours after treatment with D-Gal N/LPS.The damaged was characterized by massive hepatocellular necrosis without liver cell regeneration.There was no obvious injury in glomerulus,proximal tubule and distal tubule in kidney pathology.13.There was a markedly increase in serum ALT,TBIL,Cr,BUN,K+,Na+levels in rats exposed to Gal N/LPS（Table）.The Cr in G/L+MNP-Sa and G/L+Sa group had no significant difference compared with NS control group.14.The inulin clearance was measured 7 days after micro-osmotic pumps implantation and 12 h after Gal N/LPS exposure,when the FITC-inulin arrived a steady-state in blood and weight loss of rats recover.Then the GFR was calculated（table）.The GFR1,GFR2and GFR3 decreased obviously in rats exposed to Gal N/LPS.But MNP-Sa may improve the GFR compared with G/L group.GFR1 and GFR3 in G/L+MNP-Sa group decreased markedly compared with NS group（P<0.05）,but increased obviously compared with G/L group（P<0.05）.GFR3 decreased than NS group and increased than G/L group although with no statistical significance.15.The ratio of GIS（GIS2/GIS1）reflect the changes in glomerular size,that indicate the vascular dilation/constriction degree indirectly.MNP-Sa could decrease the GIS（GIS2/GIS1）in rats with FHF,that represent MNP-Sa could decrease the blood vessel contraction in glomeruli.The GIS in G/L（3.83±0.23）and G/L+MNP（3.80±0.33）group increased significantly（P<0.05）compared with NS control group（1.76±0.29）.Compared with G/L group the GIS in G/L+Sa（2.79±0.17）and G/L+MNP-Sa（1.84±0.33）group decreased significantly（P<0.05）.16.MNP-Sa could decrease the increasing times of[Ca²⁺]i in glomeruli after ET-1stimulation in rats with FHF,which represent that MNP-Sa could decrease the[Ca²⁺]i release in glomeruli when exposed to ET-1,a vasoactive substance.The increasing times of[Ca²⁺]i in glomeruli after ET-1 stimulation in G/L（2.42±0.10）and G/L+MNP（2.12±0.08）group increased significantly（P<0.05）compared with NS control group（1.33±0.03）.Compared with G/L group the increasing times of[Ca²⁺]i in glomeruli when exposed to ET-1 in G/L+Sa（1.45±0.03）and G/L+MNP-Sa（1.31±0.02）group decreased significantly（P<0.05）,but had no statistical difference compared with NS group（p>0.05）.17.There was no significant difference in levels of PKC-αmRNA and PKC-αprotein between each group.But in rats with FHF,safingol and MNP-Sa could suppress the expression of P-PKCαprotein,IP3R1 mRNA and IP3R1 protein.The P-PKCαprotein,IP3R1 mRNA and IP3R1 protein increased significantly in G/L group compared with NS group（p<0.05）,but in G/L+Sa and G/L+MNP-Sa group they were suppressed significantly（p<0.05）Conclusion:1.Safingol can be loaded to water-soluble carboxylic iron oxide magnetic nanoparticles through the EDC/NHS catalytic reaction.The drug-loading nanoparticles have no obvious cytotoxicity and can be uptaken by RGMCs;2.MNP-Sa（0.125μM,drug-loading of safingol as 1.1μM）and safingol（5μM）can significantly inhibit the activation of PKC-α,the expression of P-PKCαprotein,reduce the level of IP₃R1mRNA and protein expression,reduce the[Ca²⁺]i in RGMCs,without affecting the expression of PKC-αprotein and mRNA level;3.The glomerular filtration rate decreased significantly in rats withacute liver failure,while MNP-Sa and Safingol can improve the GFR obviously.