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3 protocols using para aminohippuric acid pah

1

Measuring Renal Blood Flow with PAH

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Para-amino hippuric acid (PAH) (Sigma-Aldrich, Gillingham, UK) was used to determine renal blood flow. Micro-catheters were inserted in the left jugular vein and left ureter. A primed, constant 1 mM PAH infusion was infused in the jugular vein at a rate of 0.08 ml h−1 after a prime infusion of 0.032 ml (100 g)−1 of 50 mM PAH to reach a steady state. The final concentrations of PAH have no influence on renal ammonia excretion43 (link). An equilibration period of 30 min was observed before blood and urine collection.
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2

Immunofluorescence Staining of CRB3 Protein

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2,4-Dichlorophenoxyacetic acid (2,4-D) (CAS no. 94-75-7), para-aminohippuric acid (PAH) (CAS no. 61-78-9), triclopyr (CAS no. 55335-06-3), trimethylsilyldiazomethane (TMSD) (CAS no. 18107-18-1), N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) (CAS no. 77377-52-7), ethyl acetate, toluene, lucifer yellow, pilocarpine, and other general laboratory chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CRB3 rat primary monoclonal antibody was purchased from MyBioSource (Cat # MBS588104) and goat anti-rat IgG secondary antibody-Alexa Fluor 488 conjugate was purchased from Invitrogen (Cat # A11006).
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3

Renal Function Assessment in Rats

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After housing in a metabolic cage, the rats were i.p. anesthetized with thiopental sodium (60 mg/kg, Cristália, Asuncion, Brazil). The body temperature of each animal was maintained at 37 • C using a servo-controlled heating blanket (LE 13206, LSI Letica Scientific Instruments, Woonsocket, RI, USA), and tracheostomy was performed to reduce airway resistance. A polyethylene catheter (PE-50 tubing) was implanted in the femoral artery for recording BP. A PE-50 catheter was surgically implanted into the right carotid artery for blood sample collection. Urine was collected through a 24G needle (3/4" 0.7 × 19 mm) syringe implanted in the right unclipped kidney. A solution containing 5 mg/min/kg of inulin (Sigma Aldrich, St Louis, MO, USA) and 1.33 mg/min/kg of para-aminohippuric acid (PAH) (Sigma Aldrich, St Louis, MO, USA) was continuously infused via the femoral vein using a pneumatic pump at a rate of 1.2 mL/h (KDS 100, KD Scientific, Holliston, MA, USA). Three samples of urine and blood were collected at 30 min intervals. GFR was determined through inulin clearance, and RPF was determined by PAH clearance. The values obtained from inulin and PAH represent the means of the three collection periods. The GFR and RPF were normalized per gram (g) of kidney tissue.
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