الفهرس 
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Abstract
In The Present Study, The Role Of Blood Coagulation System Activation In The Pathogenesis Of Acute Renal Failure (ARF) Induced Experimentally In Rats By Cisplatin (CP) Was Investigated. In Addition, We Investigated The Possible Protective Role Of Different Anti-Coagulant Drugs In The Protection Against CP-Induced Nephrotoxicity As Compared To The Potent Antioxidant Agent Tempol As A Reference Standard. In This Study, Nephrotoxicity Was Induced By A Single I.P. Dose Of CP Of 6 Mg/Kg.
The Study Was Performed As Two Sets; In The First Set, Time Course Study Was Done In Which Animals Were Classified Into 5 Groups. One group Served As Normal Control, While Groups 2-5 Received CP And Were Sacrificed After 1 Day, 2 Days, 3 Days And 4 Days, Respectively. In The Second Set, To Evaluate The Protective Effect Of selected Drugs Against CP-Nephrotoxicity, Tempol (50 And 100 Mg/Kg/Day), Rivaroxaban (5 And 7 Mg/Kg/Day) And Dabigatran (15 And 25 Mg/Kg/Day) Were Administrated Orally On A Daily Basis Seven Days Prior To CP Administration And Continued For Other 4 Consecutive Days Till The End Of The Experiment. At The End Of The Experiment, Urine, Blood And Tissue Samples Were Collected And Kidney Weight Was Recorded.
Estimation Of Nephrotoxicity Was Performed By Detection Of Serum Markers Of Kidney Functions Including Serum Creatinine (S.Cr) And Cystatin-C (Cyst-C) Levels As Well As Blood Urea Nitrogen (BUN). In Addition, Urinary Markers Of Kidney Functions Including Urine Creatinine (U.Cr), Urine Volume (U.Vol), Urinary Γ-Glutamyl Transferease (GGT) Activity And Glomerular Filtration Rate (GFR). Additionally, We Detected Expression Of Kidney Injury Molecule-1 (KIM-1) And Lipocaline-2 In The Urine As Well As Animal Weight, Relative Kidney Weight And Renal Histological Changes.
Renal Glutathione (GSH) Content, Super Oxide Dismutase (SOD) Activity, MDA Content And Nitric Oxide (Nox) Production Were Determined As Oxidative Stress Markers. Side By Side, Hematological Parameters As Complete Blood Count (CBC) And Coagulation Profile Including Prothrombin Time (PT), Prothrombin Concentration (PC), And International Normalized Ratio (INR) Were Estimated. In Addition, Coagulation Cascade Proteins Including Tissue Factor (TF), Protease Activated Receptor 2 (PAR-2), Thrombin And Fibrin Were Detected In Renal Tissues.
The Main Findings Of The Present Investigation Can Be Summarized As Follow:
A. Time Course Study Of CP- Induced ARF:
1. Effect Of CP On Animal Weight, Relative Kidney Weight And Histological characters:
Administration Of Single I.P. Dose Of CP Affected Animal Weight And Relative Kidney Weigh Significantly As Compared To Normal Control. A Significant Decrease In Animal Body Weight Was Observed After 1 Day Of CP-Administration. This Decrease In Animal Weight Continued Markedly After 2 And 3 Days Till It Peaked After 4 Days Of CP-Administration.
On The Other Hand, CP Caused A Significant Increase In Relative Kidney Weight After 1 And 2 Days As Compared To Normal Control. Relative Kidney Weight Increased Significantly After 3 Days Of CP-Administration And Peaked After 4 Days As Compared To Normal Control, CP 1 Day, CP 2 Days And CP 3 Days.
Histologically, No Effect On Renal Tissue Was Observed After 1 Day Of CP-Administration where Tissue Sections Showed More Or Less Normal Histological Structure Of Renal Glomeruli And Epithelium Layer Lining The Renal Tubules. However, After 2 Days, CP Caused Mild Degenerative Changes Of Renal Epithelial Layer Associated With Mild Desquamation Of Tubular Epithelial Cells.
In The Third Day We Observed That CP Caused A Moderate Epithelial Degeneration Of Lining Epithelium And Glomeruli Tufft Showed Mild To Moderate Glomerulonephrosis And Also Focal Leuckocytic Infiltration Could Be Detected In The Interstitial Area. Finally, After 4 Days, CP Caused Severe Degenerative Changes And Necrosis Associated With Desquamation Of Renal Lining Epitheliums. Moreover Glomeruli Tuft Are Showing Severe Degenerative Changes.
2. Effect Of CP On Kidney Function Tests:
It Took 2 Days To Start A Significant Increase In S.Cr, Cyst-C And BUN Levels where No Changes Were Observed In These Markers After 1 And 2 Days Of CP-Administration As Compared To Normal Control. In The Third Day, S.Cr, Cyst-C And BUN Began To Significantly Increase As Compared To Normal Control, CP 1 Day And CP 2 Days. Finally, A Marked Increase Was Induced In The Levels Of S.Cr, Cyst-C And BUN After 4 Days Of CP-Administration As Compared To Normal Control, CP 1 Day, CP 2 Days And CP 3 Days.
On The Other Side, The Decrease In U.Cr And GGT Started To Be Observed After 1 Day Of CP-Administration As Compared To Normal Control. After 3 Days, CP Caused Significant Decrease In U.Cr And GGT Activity As Compared To CP 1 Day And CP 2 Days. The Decrease In U.Cr Content And GGT Activity Reached To The Lowest Level After 4 Days Of CP-Administration As Compared To CP 1 Day, CP 2 Days And CP 3 Days. In This Study, We Also Found That CP Caused Significant Increase In U.Vol And GFR After 1 Day Of Administration But This Increase Was Followed By Significant Decrease In Their Values After 2 Days Of CP-Administration. The Decrease In U.Vol And GFR Was Continued After 3 Days And Reached To The Lowest Level After 4 Days Of CP-Administration.
We Also Observed That CP Caused A Significant Increase In U. KIM-1 Level After 1 Day As Compared To Normal Control. The Level Of U. KIM-1 Decreased After 2 And 3 Days But Its Level Increased Significantly Again After 4 Days Of CP-Administration As Compared To CP 1 Day, CP 2 Days And CP 3 Days. In-Contrast, No Changes Were Observed In U. Lipocaline-2 Till The Fourth Day, Which Recorded A Significant Increase In U. Lipocaline-2 Level As Compared To Normal Control.
3. Effect Of CP On CBC And Coagulation Profile:
Several Changes Were Observed In CBC And Coagulation Profile Of Rats Through The Four Days Of CP-Administration. After 1 Day, CP Did Not Cause Any Significant Change In CBC And Platelets Count Except A Significant Decrease In Monocytes. In Addition, CP Caused Significant Increase In PT, PTT And INR With Significant Decrease In PC After 1 Day Administration.
On The Other Hand, After 2 Days Of CP-Administration, We Observed Significant Decrease In The Count Of Wbcs, Lymphocytes And Monocytes Without Change In Granulocytes And Platelets Count. Moreover, CP Caused Significant Increase In PT And INR With Significant Decrease In PC, While No Change In PTT Was Observed.
On The Other Side, After 3 Days Of CP Injection, We Observed That Count Of Wbcs, Lymphocytes, Monocytes And Granulocytes Started To Be Elevated But Not Significantly Without Change In Platelets Count. In Addition, Significant Increases In PT, PTT And INR With Significant Decrease In PC Were Also Observed. In The Fourth Day, CP Significantly Increased Count Of Wbcs, Lymphocytes, Monocytes And Granulocytes With Significant Decrease In Platelets Count. Moreover, Significant Increases In PT, PTT And INR With Significant Decrease In PC Were Observed.
4. Effect Of CP On Oxidative Stress:
Oxidative Stress Parameters Affected Gradually Through The 4 Days Of CP-Administration. After 1day, CP Caused Significant Decrease In Renal GSH Content With Significant Increase In Lipid Peroxidation And Nox Production, While No Effect On Renal SOD Activity Was Observed. In Addition, After 2 Days Of CP-Administration, Significant Decreases In Renal GSH Content And SOD Activity With Significant Increases In Lipid Peroxidation And Nox Production Were Observed.
Sequentially, Significant Decreases In Renal GSH Content And SOD Activity With Significant Increase In Lipid Peroxidation And Nox Production Were Observed 3 And 4 Days After CP-Administration As Compared To Normal Control.
5. Effect Of CP On Coagulation Proteins Expression:
The Expression Of Coagulation Proteins Including TF, Thrombin, PAR-2 And Fibrin Began To Up-Regulate Significantly After 1 Day Of CP-Administration. PAR-2 Exceptionally, Began To Increase Significantly After 2 Days Of CP-Administration. All Coagulation Protein’s Expression Up Regulated In Renal Tubular Cells Gradually After 2 Days And 3 Days Till Peaked After 4 Days Of CP-Administration.
B. Protective Effect Of selected Drugs Against CP-Induced ARF:
1. Effect Of Tempol Against CP-Induced ARF:
Pretreatment Of Rats With Tempol In Small Or Large Doses Significantly Decreased Animal Weight Loss And Relative Kidney Weight As Well As Ameliorated The Histological characters Of Renal Tissue Of Diseased Rats. We Observed That Animal Pretreated With Small Dose Of Tempol Showed Moderate Degenerative Changes Of Renal Epithelium And Granular Deposits In Lumens. Meanwhile, Tissue Sections Obtained from Rats Pretreated With High Dose Of Tempol Showed Mild Glomerulonephrosis Only.
In Addition, Tempol In Both Doses Improved Renal Function Tests where They Significantly Decreased S.Cr, Cyst-C And BUN Levels. In Addition, They Increased Urinary GGT Activity, Urine Volume And GFR Significantly. Tempol Also Decreased U. KIM-1 And U. Lipocaline-2 Levels Significantly.
Pre-Treatment Of Animals With Tempol Significantly Decreased Count Of Wbcs, Monocytes And Granulocytes With Significant Increase In Platelets Count. Moreover, Significant Decreases In PT, PTT And INR Were Observed.
Tempol Also Improved Oxidative Stress Parameters where Both Doses Of Tempol Significantly Increased Renal GSH Content And SOD Activity, As Well As They Significantly Decreased Lipid Peroxidation And Nox Production. In Addition, Both Doses Of Tempol Significantly Down Regulated The Expression Of Coagulation Proteins Including; TF, Thrombin, PAR-2 And Fibrin.
2. Effect Of Rivaroxaban Against CP-Induced ARF:
Pre-Treatment Of Animals With Small Or High Doses Of Rivaroxaban Significantly Decreased Relative Kidney Weight And The Loss In Animal Weight Induced By CP. In Addition, Both Doses Of Rivaroxaban Improved The Histological characters Deteriorated By CP. Rats Treated With Rivaroxaban 5 Mg/Kg Or 7 Mg/Kg Showed Moderate Degenerative Changes Of Renal Epithelium Associated With Mild Glomerulonephrosis.
In Addition, Rivaroxaban In Small Or High Doses Improved Renal Function where They Caused Significant Decrease In S.Cr, Cyst-C And BUN Levels. On The Other Hand, They Caused Significant Increase In Urinary GGT Activity, U.Vol. And GFR. Moreover, Both Doses Of Rivaroxaban Decreased U. KIM-1 And Urinary Lipocaline-2 Levels Significantly.
We Also Found That, Pre-Treatment Of Animal With Both Doses Of Rivaroxaban Significantly Decreased Count Of Wbcs And Granulocytes With Significant Increase In Platelets Count. In Addition, They Significantly Decreased Monocytes With No Effect On Lymphocyte Count. Meanwhile, High Dose Of Rivaroxaban Caused Significant Decrease In Lymphocytes And Significant Increase In Monocytes Count. Side By Side, Significant Decreases In PT, PTT And INR Were Observed After Treatment Of Animals With Both Doses Of Rivaroxaban.
Pre-Treatment Of Animals With Rivaroxaban Ameliorated Oxidative Stress Markers where They Increased Renal GSH Content And SOD Activity Significantly As Well As It Decreased Lipid Peroxidation And Nox Production Significantly. Furthermore, Both Doses Of Rivaroxaban Significantly Down Regulated The Expression Of Coagulation Proteins Including TF, Thrombin, PAR-2 And Fibrin.
3. Effect Of Dabigatran Against CP-Induced ARF:
Pre-Treatment Of Animals With Small Or High Doses Of Dabigatran Significantly Increased Animal Weight And Decreased Relative Kidney Weight. Moreover, Both Doses Of Dabigatran Improved The Histological characters Deteriorated By CP. Dabigatran Caused Moderate Degenerative Changes Of Renal Epithelium Associated With Mild Glomerulonephrosis.
Dabigatran In Small Or High Doses Caused Significant Decrease In S.Cr, Cyst-C And BUN Levels. On The Other Hand, Both Doses Of Dabigatran Caused Significant Increase In Urinary GGT Activity, U. Vol And GFR. Moreover, They Caused Significant Decrease In Urinary KIM-1 And Urinary Lipocaline-2 Levels.
Pre-Treatment Of Animal With Both Doses Of Dabigatran Significantly Decreased Count Of Wbcs, Monocytes And Granulocytes With Significant Increase In Platelets Count. In Addition, Significant Decreases In PT, PTT And INR Were Observed After Treatment Of Animals With Both Doses Of Dabigatran.
Dabigatran In Both Doses Caused Significant Increase In Renal GSH Content And SOD Activity. On The Other Hand, Both Doses Caused Significant Decrease In Lipid Peroxidation And Nox Production. Furthermore, Both Doses Of Dabigatran Significantly Down Regulated The Expression Of Coagulation Proteins Including TF, Thrombin, PAR-2 And Fibrin.
Depending On The Previous Findings, It Could Be Concluded That:
1. Cisplatin Administration In A Single Dose Of 6 Mg/Kg, I.P. Is A Successful Model For Induction And Development Of ARF In Rats. Activation Of Blood Coagulation Plays A Crucial Role In The Pathophysiology Of CP-Induced Renal Failure. Oxidative Stress And Release Of Inflammatory Mediators Have Important Role In The Progression Of CP Nephrotoxicity.
2. The High Expression Of Coagulation Cascade Proteins Prior To The Nephrotoxicity Induced By CP May Elucidate The Role Of Activation Of Coagulation System In The Initiation Of Nephrotoxicity. Thrombocytopenia, Prolongation Of PT And PTT And Leukocytosis Are Considered The Main Blood Disorder Accompanied To CP Nephrotoxicity.
3. Tempol Has A Protective Role Against CP-Induced Nephrotoxicity Through Anti-Oxidant And Anti-Inflammatory Activity. The Ability Of Tempol To Down Regulate The Expression Of TF, PAR-2, Thrombin And Fibrin Elucidate The Possible Anti-Coagulant Activity Of Tempol.
4. Inhibition Of Coagulation Cascade By Rivaroxaban (Fxa Inhibitor) Or Dabigatran (Thrombin Inhibitor) May Protect Against CP Nephrotoxicity.
5. These Drugs Seem Promising For Protection Against CP Nephrotoxicity But Clinical Trials Are Needed To Prove These Findings On Human.