Iothalamate measured by capillary electrophoresis is a suitable alternative to radiolabeled inulin in renal micropuncture.
BACKGROUND
Inulin remains the gold standard for measurements of fluid reabsorption (Jv) and single nephron glomerular filtration rate (SNGFR) in micropuncture experiments. However, the method used to measure cold inulin in nanoliter samples is time-consuming, while the use of radiolabeled inulin is disadvantaged by possible radioactive contamination, disposal of radioactive material and cost of the isotope. It has been reported that non-radiolabeled iothalamate may be a suitable alternative for estimation of whole kidney GFR. The present study tested whether iothalamate can be used to measure Jv in microperfusion and free-flow micropuncture experiments.


METHODS
Superficial loops of Henle (LOH) were perfused from late proximal to early distal tubules with an end-like proximal solution. In the first set of experiments, the perfusate contained both iothalamate (1.9 mmol/L) and 3H-methoxy-inulin (50 microCi. mL(-1)). To test if iothalamate was able to detect changes in Jv, two additional sets of experiments were performed: (1) mannitol (61 mmol/L) was added to the perfusate to partially replace NaCl, a condition known to inhibit Jv; (2) LOH of remnant kidneys were perfused, which in previous experiments we showed to have a higher Jv. Lastly, free-flow micropuncture experiments were performed by infusing iothalamate IV at 18.3 mg. h(-1). Iothalamate analysis in nanoliter samples of renal tubular fluid obtained in vivo was performed by capillary electrophoresis (CE).


RESULTS
In the first set of experiments, liquid scintillation counting of 3H-methoxy-inulin versus iothalamate analysis with CE resulted in almost identical calculated perfusion rates (20.4 +/- 0.6 vs. 20.6 +/- 0.7 nL. min(-1), N = 20) and tubular fluid/perfusate ratios (TF/P; 1.35 +/- 0.04 vs. 1.36 +/- 0.04) and thus also Jv (5.17 +/- 0.50 vs. 5.38 +/- 0.59 nL. min(-1)). In the mannitol experiments, iothalamate measurements showed that the addition of mannitol significantly reduced Jv from 4.98 +/- 0.40 (N = 19) to 0.72 +/- 0.58 nL. min(-1) (N = 33; P < 0.0001). Iothalamate determinations by CE were able to detect a significant increase in Jv in LOH of remnant rats perfused at 40 nL. min(-1)[from to 8.40 +/- 0.73 (N = 20) in sham-operated to 17.8 +/- 2.9 nL. min(-1) (N = 6) in remnant animals; P < 0.0001]. In free flow micropuncture experiments the ratio of tubular fluid to plasma iothalamate (TF/P) along the proximal tubule was 1.62 +/- 0.10 (N = 15).


CONCLUSIONS
These data demonstrate that iothalamate can replace inulin to measure Jv in microperfusion and free-flow micropuncture experiments. Since iothalamate analysis by CE technique is a fast, easy and highly reproducible technique, it may become the gold standard method for the detection of fluid reabsorption in microperfused nephron segments.
Capasso
Giovambattista
G
Chair of Nephrology, Department of Biochemistry, Faculty of Medicine, Second University of Naples, Padiglione 17 Policlinico Nuovo, Via Pansini 5, 80131 Naples, Italy. gb.capasso@unina2.it
Unwin
Robert J
RJ
Pica
Angelo
A
Quagliuolo
Lucio
L
Giovane
Alfonso
A
D000818:Animals; D003287:Contrast Media / Q000032:analysis*; D019075:Electrophoresis, Capillary; D005919:Glomerular Filtration Rate; D007444:Inulin; D007483:Iothalamic Acid / Q000032:analysis*; D008138:Loop of Henle / Q000502:physiology; D008297:Male; D009399:Nephrons / Q000502:physiology*; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D014316:Tritium
D003287:Contrast Media; D014316:Tritium; D007483:Iothalamic Acid; D007444:Inulin