You can get to know our methods here.
Mouse models used by us:
• breast cancer isograft (4T1 cell/Balb/C mouse)
• ischemia-reperfusion induced renal fibrosis (NMRI outbread mouse)
Surgical and chemical models:
Endotoxin-ischemia cross-tolerance mouse model: Resistance triggered with increasing doses of endotoxin.
Endotoxin shock mouse model: Introduction of the lipopolysaccharides of gram negative bacteria in order to trigger endotoxin shock.
Diabetes model: Type one diabetes mellitus model: Streptozotocin induced inzulin-deficiency model in mice and rats.
Organ transplant: Ortotropic kidney transplant in rats.
Subtotal nephrectomy: Chronic kidney fibrosis model: we resect the total right kidney and the 2/3 of the left kidney, resulting a 5/6 nephrectomy.
Ischemia-reperfusion: Left kidney is isolated from the circulation by compressing the renal artery and vein. The right kidney is resected as control. (mice and rats)
Renal vein and portal injections: High volume venous infusion of the given organ for renal tubular and hepatocyte transfection.
Hydrodynamic treatment: Rapid (less than 10 ms) venous injection of a high liquid volumen (at least 50% of the circulating volumen) in order to transform abdominal organs with high blood flow (kidneys, liver).
Functional measurements:
Diuretic cage: 24 hour collection of urine for proteinuria and albuminuria tests.
Glomerular damage tests:
Albuminuria: Albumine ELISA essay.
Total urine protein: Total urine protein test on collected urine samples. Mass separation with SDS-PAGE and densitometry.
Inulin, PAH, mannitol clearance: Glomerular filtration rate and renal plasma flow fuctional tests.
CREA/UREA: Blood and urine creatine and urea tests with Reflotron system (Roche).
Tubular damage tests:
NGAL: Urine NGAL (neutrophil gelatinase associated lipocalin) evaluation with ELISA
gamma-GT/CREA: Urine creatinine and gamma-GT with Reflotron system (Roche).
Microproteinuria: SDS-Page and densitometry in order to evaluate low molecular weight proteins in the urine.
Functional tests of the vessels:-
Blood pressure: Telemetry and IITC (piezoelectric) tail blood pressure measurements.
Functional monitoring of the glucose system:
Serum glucose: Serum glucose measurement with Reflotron system (Roche).
OGTT: Oral Glucose Tolerance Test.
Fructosamine: Serum glycated protein measurement.
Liver function: Serum GGT/ALAT (GOT)/ASAT (GPT)/ALP/TG/CHOL with Reflotron system (Roche).
Superoxide-anion tests: Dihydroetidium staining of tissue samples and 3D evaluation of the layers. Detection of superoxide production with confocal microscopy.
Aorta function measurement: Endothel function test on mouse aorta, measurements of contraction and relaxation triggered by farmacons in organ chamber.
Microvessel function measurements: Measurements of reactions to farmacons on isolated resistance vessels in organ chamber.
Molecular biology:
Standard procedures of molecular biology: PCR, Northern blot, enzymatic digestion, DNA/RNA isolation from tissue or cells, primer construction.
Genotyping: Genotyping of the bred laboratory animals.
RT-PCR: Quantitative measurement of the mRNA levels in isolated RNA samples in order to evaluate the level of gene expression.
siRNA:
Electroporation: Evaluation of the effect of siRNA on gene expression.
Hydrodinamic treatment: Venous injection of siRNA in mice in order to examine the effect of siRNA in vivo.
Complement system:
Complement activation and inhibiton: Functional and quantitative tests of the serum complement system with ELISA.
Protein expression:
Examination of the expression level changes of proteins on cell or tissue lysate using Western Blot or ELISA.
Cell cultures:
Primer cell culture: HUVEC (Human Umbilical Vein Endothelial Cells) primer cells isolated from the umbilical chord.
Flow cytometry: Flow cytometry procedures for cell typing using surface markers.
Isolation of blood cells: Isolation of red blood cells, granulocytes and lymphocytes from whole blood.
Lymphocyte activation: Activation of the isolated lymphocytes with PMA or ionomycin, measurement of the produced cytokines with ELISA.
OGD on endothel cells: Evaluation of the effect of oxygen and glucose withdrawal on the superoxide production in HUVEC cells. (dihydroethidium staining, Life-Death kit confocal microscopy).
Pathology:
Heart and aortic morphology: Structural examination of mouse heart muscle cells on half-thin tissue sections using point counting stereology. Digital measurements of the intima thickening in mouse aorta.
Organ perfusion: Perfusion of the organs in order to acquire samples for histology and molecular biology.
Standard histochemical stainings: Staining of the paraffine embedded samples using PAS, hematoxylin-eosin, Trichrome, Sudan IV, Masson, Oil red O etc.
Immunohistology: C3, fibrotic markers (collagen, fibronectin), and TGF-beta detection on samples using immunohistological methods.