EXPERIMENTAL PROTOCOL
                       RENIN STUDIES IN THE RAT

1) APPROXIMATELY 6 DAYS PRIOR TO EXPERIMENT, WEIGH AND DETERMINE BLOOD 
PRESSURE [Mean Arterial Pressure] OF SIX NORMAL RATS. THE PREFERRED WEIGHTS
OF THESE BEING 200-250 GRAMS (8-10 WEEKS OLD). THE BLOOD PRESSURE MAYBE
DETERMINED USING THE TAIL CUFF APPERATUS OR BY CANULATION OF THE CAROTID
ARTERY AND MEASURING IT DIRECTLY WITH A BLOOD PRESSURE MONITOR.
RECORD THESE IN RECORD BOOK.

2) ANESTHETIZE RAT WITH Na-PENTABARBITOL ACCORDING TO WEIGHT SPECIFICATIONS.
PROCEED TO PLACE CLIP ON RENAL ARTERY OF THE LEFT KIDNEY. 
THIS IS DONE BY CUTTING A MIDLINE INCISION AND EXPOSING THE LEFT KIDNEY WITH
EASY ACCESS TO THE RENAL PEDICLE. THE RENAL ARTERY LIES OVER THE RENAL VEIN.
THEIR PROXIMITY REQUIRES GREAT CARE NOT TO PERFORATE EITHER OF THE TWO. ONCE
THE ARTERY IS ISOLATED, ONE CAN OBSERVE CHANGES OF COLOR AND TEXTURE IN THE 
KIDNEY ACCORDING TO GRADE OF OCCLUSION INDUCED BY THE BLUNT SIDE OF A TWEEZER.

3) PREPARATION OF CLIP IS PRESENTLY AT A STAGE THAT THE TECHNICIAN MUST 
ESTIMATE THE GIRTH OF THE RENAL ARTERY AND BEND A SMALL WIRE AROUND IT'S 
CIRCUMFERENCE. THE WIRE SHOULD CLAMP ON TO THE ARTERY AND SHOULD REDUCE 
BLOOD FLOW BY 70%. THIS REQUIRES SPECIAL CARE NOT TO COMPLETELY OCCLUDE ARTERY 
FOR IT MAY PRODUCE RENAL NECROSIS. THE MATERIAL USED AS THE WIRE CLIP HAS NOT
YET BEEN ESTABLISHED AS TO WHICH IS THE IDEAL.

4) PRIOR TO COMPLETING SURGERY, MAKE SURE THAT PROPER ASEPTIC CONDITIONS ARE 
MAINTAINED AND THAT THE RATS ABDOMINAL CAVITY IS PROPERLY LUBRICATED WITH 0.9% 
SALINE. THEN CLOSE INCISION.

5) APPROXIMATELY 6 DAYS AFTER SURGERY, MEASURE  BLOOD PRESSURE [MAP] TO VERIFY 
THAT CLIP HAS INDUCED HYPERTENSION. WEIGH RAT AND RECORD THIS IN RECORD BOOK. 

6) PREPARATION FOR GLOMERULAR ISOLATION (METHODS AND MATERIALS):
        A) DAY BEFORE EXPERIMENT PRE-WIEGH PHOSPHATE BUFFER SOLUTION.
                (REFER TO PHOSPHATE BUFFER PREP)
        B) PREPARE 6 CANULAS PE 50 WITH 23G 1 1/2 INCH NEEDLES. USED FOR 
                RENAL PERFUSION WITH BUFFER.
        C) HEMATOCRIT CAPILLARY TUBES FOR BLOOD COLLECTION. USED TO DETERMINE
                PLASMA RENIN ACTIVITY. (PREPARE EPPENDORF 1.5 ml TUBES WITH 10 
                ul OF EDTA KEPT ON ICE, PRIOR TO EXCISING KIDNEYS)
        D) MAKE SURE THAT ALL FILTERS AND STEEL WIRE MESH FOR BOTH LEFT AND 
                RIGHT KIDNEYS ARE SOAKING IN BUFFER. SIZES 250, 210, 105, 62.
        E) USE ALL PLASTIC VIALS, SYRINGES AND TEST-TUBES TO PREVENT 
                GLOMERULAR ADHESION. (LABEL TUBES FOR IDENTIFICATION: RENIN 
                AND PROTEIN) 
        F) MAKE SURE THAT SHAKER BATH IS TURNED ON WITH TEMPERATURE SETTING 
                SET TO 37øC. (SHOULD BE CHECKED WITH THERMOMETER)
        G) WEIGH-OUT DRUGS TO BE USED IN INCUBATION OF GLOMERULI (ACCORDING TO 
                SPECIFICATIONS GIVEN BY YOUR SUPERVISOR).
7) EACH RAT SHOULD BE ANESTHETIZED WITH 0.1CC Na-PENTABARBITOL/100 g OF TOTAL 
        BODY WEIGHT. MAKE INCISION AT SAME LOCATION OF PRIOR INCISION, 
        CHECKING FOR SYMPTOMS OF INFECTION AND OTHER POSSIBLE COMPLICATIONS.
8) ISOLATE THE AORTA ABOVE AND BELOW THE BRANCHING OFF OF THE RENAL ARETERIES 
        SO THAT THEY CAN BE TIED OFF JUST PRIOR TO PERFUSION WITH BUFFER.PLACE 
        A 0-0-0 SILK SUTURE ABOVE AND BELOW THE BRANCHES BUT DO NOT TIE. FILL 
        SYRINGES WITH 20cc OF BUFFER AND FEED THROUGH PREPARED CANULA. TAKE 
        BULLDOG CLAMP AND CLAMP AORTA ABOVE THE TWO RENAL ARTERIES AND  
        CANULATE THE AORTA BELOW THE TWO ARTERIES. RELEASE CLAMP AND TAKE 
        SAMPLES OF BLOOD IN CAPILLARY TUBES (3 HEMATOCRIT CAPILLARY TUBES 
                          PER SAMPLE).
9) TIE-OFF AORTA POSTERIOR TO CANULA AND ANTERIOR TO BRANCHING-OFF OF RENAL 
        ARTERIES. THE CANULA IN PLACE AND COMMENCE PERFUSION OF KIDNEYS WITH
        APPROXIMATELY 20cc OF BUFFER. EXCISE KIDNEYS AND INJECT RAT WITH 1cc
        OF EUTHANASIA. REPEAT SAME PROCEDURE FOR ALL SIX RATS.
        
10) ONCE KIDNEYS ARE SEPARATED ACCORDING TO LEFT AND RIGHT, SPECIAL CARE  
        SHOULD BE TAKEN NOT TO MIX THEM.