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A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin

John N. Lorenz1 and Eric Gruenstein2

Departments of 1 Molecular and Cellular Physiology, and 2 Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The determination of inulin concentration in nanoliter fluid samples is fundamental to micropuncture investigations of renal function, and this is generally accomplished through the use of radioisotopes. We report here a simple and reliable alternative to the use of radioisotopes that employs FITC-labeled inulin. Samples containing FITC-inulin are stored between oil columns in constant-bore microcapillary tubes, which are then used as cuvettes to determine fluorescence on a microscope fluorometer. Standard curves were generated and found to be linear, with correlation coefficients (R) exceeding 0.99 in every case. Although the fluorescence of FITC-inulin was found to be pH dependent, the pH and fluorescence of each 20- to 40-nl sample could be normalized by the addition of 1 nl of 0.5 M HEPES at pH 7.5. In mice prepared for standard micropuncture, simultaneous measurements of tubular fluid-to-plasma ratios (TF/P) using FITC-inulin and [125I]iothalamate were highly correlated (slope = 0.95, y-intercept = 0.01, R = 0.942), as were whole kidney measurements of glomerular filtration rate (GFR) (slope = 1.25, y-intercept = -53.5 µl/min, R = 0.99). Micropuncture determinations of late-proximal samples from mice before and after treatment with acetazolamide showed expected changes: TF/P of FITC-inulin decreased from 1.89 ± 0.07 to 1.48 ± 0.10; single-nephron GFR (SNGFR) decreased from 9.64 ± 1.1 to 6.65 ± 1.0 nl/min; and fractional fluid reabsorption decreased from 45.3 ± 1.9 to 26.8 ± 5.2%. Measurements of TF/P of FITC-inulin, volume, and SNGFR using this technique were stable for at least 2 wk when samples were stored in the dark at 4°C. These data demonstrate that this simple method for determining inulin clearance represents a viable and accurate alternative to radioactive methods. This approach has the added benefits of being relatively inexpensive and leaving the micropuncture sample intact.

kidney; renal micropuncture; glomerular filtration rate; fluorescein; nanoliter; fluid reabsorption; proximal tubule; mouse

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

DETERMINATION OF RENAL tubular fluid inulin concentration is fundamental to micropuncture investigations seeking to evaluate single-nephron glomerular filtration rate (SNGFR) and tubular fluid reabsorption. Because of the small volume of tubular fluid collections from the mammalian kidney (in the order of 10-50 nl), methods for determining SNGFR and tubular fluid-to-plasma inulin ratios [(TF/P)In] have largely relied upon the use of radiolabeled markers, such as [3H]inulin or [14C]inulin or [125I]iothalamate. Although these markers represent the accepted standard for determination of SNGFR and fluid reabsorption, their use can be inconvenient because of the high levels of radioactivity required and the associated high costs. Furthermore, increasingly stringent radiation safety guidelines have encouraged the search for alternatives to radiochemicals in biological and medical research. A fluorescence-based, chemical assay for tubular fluid inulin was described in 1966 by Vurek and Pegram (9). However, this assay involves labor-intensive pipetting of both sample and reagents, and is subject to significant variability even when high levels of plasma inulin are achieved (3). As an alternative, Sohtell and coworkers (6) proposed a technique for using FITC-labeled inulin as a marker for GFR and demonstrated excellent correlation with radioactive methods. However, their procedure involves multiple pipetting steps, consumes relatively large amounts of sample, and requires that high levels of plasma inulin be maintained. Its subsequent use has therefore been limited. Here, we describe a technique for the use of FITC-inulin for micropuncture which uses widely available fluorescent microscopy techniques to determine fluorescence in tubular fluid samples contained in constant-bore microcapillary tubes as cuvettes. This technique has the advantages of being simple to use, precise, inexpensive, and nonradioactive. In addition, it requires only low plasma levels of inulin, and the analysis does not consume any of the fluid sample. The in vivo experiments reported here were performed in mice because of the renewed interest in micropuncture approaches to evaluate nephron function in genetically altered mouse models.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

FITC-inulin analysis. Samples of FITC-inulin standards, tubular fluid, plasma, and urine were individually transferred to 1-µl constant-bore capillary tubes (Drummond Scientific, Broomall, PA) containing water-equilibrated mineral oil. The mineral oil was stained with 0.5% Oil Red-O dye (Sigma) to improve contrast, and samples were deposited within the oil, 1-2 mm from the end of the constant-bore capillary, using a pulled glass pipette with a tip diameter of 6-15 µm. Fluid volume was determined by measuring the length of the resulting fluid column on a slide micrometer (Gaertner Scientific, Chicago, IL) and generally ranged from 10-40 nl. Since the fluorescence of FITC is pH dependent, all samples were buffered by the addition of 1 nl of 500 mM HEPES (pH 7.4) using a pulled-capillary volumetric transfer pipette. Samples were allowed to sit for several hours to ensure even mixing of the HEPES buffer. When necessary, appropriate corrections for dilution were made by determining sample volume before and after addition of the HEPES. In a preliminary set of experiments, standards of known FITC-inulin concentration and pH were evaluated before and after buffering with HEPES. Standards were made up in phosphate-buffered saline with the following composition (in mM): 137.0 NaCl, 2.7 KCl, 1.0 NaH2PO4, and 6.4 Na2HPO4. The sample "cuvettes" were then placed on the stage of an epifluorescence microscope (Intracellular Imaging, Cincinnati, OH), equipped with a ×10 Fluor objective (Nikon, Melville, NY) and with excitation and emission wavelengths of 480 and 530, respectively. Images of the fluorescent samples were collected using a model 4910 integrating charge-coupled device camera (Cohu, San Diego, CA), and the fluorescence was analyzed using the InCyt-Im1 image analysis system from Intracellular Imaging. For each cuvette, an identical rectangular region of interest was defined within the central region of the fluorescent area (see Fig. 1), and the average fluorescence per pixel, minus the background, was calculated.


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Fig. 1.   Photomicrographs of Microcap "cuvettes" under bright field (A) and under epifluorescence at 3 different concentrations of FITC-inulin: 0.8 mg/ml (B), 0.4 mg/ml (C), and 0.2 mg/ml (D). The identical rectangular boxes in B-D indicate the area for which fluorescence was determined in each sample. Excitation wavelength was 480 nm, and the emission wavelength was 530 nm.

Micropuncture experiments. Standard micropuncture techniques, adapted for use in the mouse, were used to collect proximal tubular fluid samples. Male NIH-Swiss mice weighing 30-35 g were anesthetized with separate intraperitoneal injections of ketamine (50 µg/g body wt) and Inactin (100 µg/g body wt; Research Biochemicals International, Natick, MA), and placed on a thermally controlled surgical table. Following tracheostomy, the right femoral artery and vein were cannulated with polyethylene (PE) tubing hand-drawn to a fine tip over a flame (OD, 0.3-0.5 mm). The arterial catheter was connected to a fixed-dome pressure transducer (model CDXIII; COBE Cardiovascular, Arvada, CO) for measurement of arterial blood pressure, and the venous catheter was connected to a syringe pump for infusion. The bladder was also cannulated with flared PE-10 tubing for the collection of urine. Blood pressure and heart rate were monitored throughout the experiment using a MacLab 8/e data acquisition system (ADInstruments, Boston, MA) with a sampling rate of 200 samples/s. Body temperature was maintained at 37.5°C, and animals were provided with a steady stream of 100% O2 to breath.

The left kidney was then exposed via a flank incision, carefully dissected free of adherent fat and connective tissue, placed in a Lucite cup, and covered with mineral oil. A maintenance infusion of isotonic saline containing 2.25 g/100 ml bovine serum albumin, 1.00 g/100 ml glucose, and 0.75 g/100 ml of FITC-inulin was then initiated at a rate of 0.25 µl · g body wt-1 · min-1. In those experiments comparing FITC-inulin clearance to [125I]iothalamate clearance (see protocols below), Glofil (Cypros Pharmaceutical, Carlsbad, CA) was also added to this infusion at a concentration of ~20 µCi/ml. After a 30- to 45-min equilibration period, timed collections of urine were made, each lasting 30-60 min. Blood samples (20 µl) were taken in heparinized hematocrit tubes before and after each urine collection period. Timed collections of fluid from random proximal or late-proximal tubule surface segments lasting 4-6 min were performed using glass micropipettes sharpened to a diameter of 6-7 µm.

At the end of each experiment, tubular fluid samples were transferred individually to 1-µl constant-bore Microcaps as described above. Blood samples were centrifuged, and 30- to 40-nl plasma aliquots were pipetted into 1-µl Microcaps in a fashion similar to that of the tubular fluid samples. Urine samples were diluted 1:100 and likewise transferred into 1-µl Microcaps.

Protocol 1: Comparison of FITC-inulin and [125I]iothalamate. Micropuncture experiments were performed in which animals received both FITC-inulin and [125I]iothalamate, and each sample of proximal tubule fluid, urine, and plasma was analyzed for both fluorescence and radioactivity. After determining the fluorescence of each sample on the microplate fluorometer, we dispensed the contents of each Microcap into 0.5 ml of water and counted for 120 min on a 10-channel gamma counter (Cobra Auto-Gamma model 5010; Packard Instrument, Downers Grove, IL).

Protocol 2: Effect of acetazolamide. Carbonic anhydrase inhibition with acetazolamide has been thoroughly investigated and is known to inhibit fluid reabsorption in the proximal tubule. To verify that FITC-inulin measurements using this methodology could detect such changes, we determined the values of (TF/P)In, SNGFR, and proximal tubule fluid reabsorption before and after treatment with acetazolamide. Late proximal tubule surface segments were identified by injecting a small volume of stained saline (0.25 g/100 ml Fast green, Sigma) into a random surface tubule using a 3- to 4-µm pipette. Collections were first made during a 1-h collection period as described above. A 75-µl bolus of 20 mg/ml acetazolamide in 0.15 M NaHCO3 was then given, followed by a steady infusion of 10 mg/ml acetazolamide at 3 µl/min. After a 30-min equilibration period, late proximal fluid collections were repeated.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

The photomicrographs in Fig. 1 show sample microcapillary "cuvettes" under bright field and under epifluorescence illumination at three different concentrations of FITC-inulin. Since the pH of tubular fluid varies significantly along the length of the nephron, resulting in micropuncture samples with different pH, we designed an initial experiment to evaluate the linearity and pH sensitivity of FITC-inulin fluorescence. Four sets of standards, ~40 nl each, were constructed with known FITC-inulin concentrations of 0, 0.05, 0.1, 0.2, 0.4, and 0.8 mg/ml. Two of the sets were made up in phosphate-buffered saline at a pH of 7.4, whereas the other two were made up in phosphate-buffered saline at pH 6.0. One set of standards from each of these pH groups was then buffered with an additional 1 nl of 0.5 M HEPES buffer at pH 7.4, while the other was left untreated. Results from the microscope fluorometer are shown in Fig. 2. Fluorescence intensity was found to be a linear function of FITC-inulin concentration at both pH values, but there was a substantially lower slope at the lower pH. Addition of 1 nl 0.5 M HEPES buffer to ~40 nl of the FITC-inulin standards returned the slope of the pH 6.0 phosphate-buffered standard curve close to that of the pH 7.4 phosphate-buffered solutions. Addition of HEPES buffer to the phosphate-buffered solution already at pH 7.4 had little effect on fluorescence, indicating that HEPES itself is not a significant factor. Although we were not able to measure the resultant pH in these nanoliter samples, when we repeated this dilution on a larger scale (1 ml), we were able to confirm that the pH was the same in all HEPES-buffered samples (data not shown). The correlation coefficient of each of these curves, as well as all others generated with this method, exceeded 0.99. 


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Fig. 2.   Representative standard curves for FITC-inulin fluorescence showing pH dependence. Two sets of standards were made up in PO4 buffer at pH 7.4 (bullet , open circle ), and two sets were made up in PO4 buffer at pH 6.0 (black-triangle, triangle ). Approximately 40 nl of each standard was transferred into oil-filled Microcaps as described in the METHODS. One nanoliter of 0.5 M HEPES buffer at pH 7.4 was added to each sample, represented by filled symbols (bullet , black-triangle).

To evaluate the variability of this assay, 15 separate measurements were made on each of two prediluted urine samples containing ~0.1 mg/ml and 0.2 mg/ml FITC-inulin. The coefficient of variation for these two samples was 1.48% (0.11 ± 0.002 mg/ml) and 1.11% (0.21 ± 0.002 mg/ml, mean ± SD), respectively. In a separate series of experiments, the recoveries of FITC-inulin from five plasma samples at added concentrations of 0.1 and 0.2 mg/ml were 91.5 ± 2.6% and 96.1 ± 4.7%. Recoveries from five prediluted urine samples at added concentrations of 0.2 and 0.4 mg/ml were 97.1 ± 1.5% and 96.5 ± 1.5%.

To determine whether micropuncture results obtained with FITC-inulin compared favorably to those obtained with a well-accepted marker, we performed micropuncture and clearance collections in mice that were simultaneously given FITC-inulin and [125I]iothalamate. The results of micropuncture collections are shown in Fig. 3. The resulting scatter plot of TF/P ratios is linear and near unity and demonstrates a close correlation between the TF/P ratios of FITC-inulin [(TF/P)FITC-In] and of [125I]iothalamate (R = 0.942, P < 0.0001). The inset of Fig. 3 shows that plasma levels of FITC-inulin and [125I]iothalamate closely paralleled each other throughout the experiment. Measurements of whole kidney FITC-inulin and [125I]iothalamate clearance are shown in Fig. 4. Again, determinations of GFR using these two markers show a high degree of correlation and linearity (R = 0.99; P < 0.0001), with only a slight deviation of the slope from unity.


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Fig. 3.   Scatter plot comparing tubular fluid-to-plasma ratios (TF/P) for FITC-inulin to those for [125I]iothalamate. Animals were infused with both markers simultaneously, and micropuncture and plasma samples were first evaluated for fluorescence and then for radioactivity as described in METHODS. The dotted line represents unity. Inset: correlation of plasma levels of FITC-inulin (in arbitrary fluorescence units) and [125I]iothalamate (in cpm/µl) for 1 experiment.


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Fig. 4.   Scatter plot comparing whole kidney values of glomerular filtration rate (GFR) determined with FITC-inulin to those determined simultaneously with [125I]iothalamate. Urine samples were diluted 1:100 and transferred to Microcaps for the determination of both fluorescence and radioactivity. The dotted line represents unity.

To evaluate SNGFR and fluid reabsorption before and after treatment with the carbonic anhydrase inhibitor acetazolamide, a separate experiment was conducted in which FITC-inulin was measured in micropuncture samples taken from late proximal tubule segments. As expected, (TF/P)FITC-In ratio, SNGFR, and fractional proximal tubular fluid reabsorption decreased following administration of acetazolamide (Fig. 5).


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Fig. 5.   Values for TF/P (left), fractional reabsorption of fluid (middle), and single-nephron GFR (SNGFR, right), determined with FITC-inulin, before and after treatment with acetazolamide. Micropuncture samples were obtained from late-proximal tubules during a 1-h control period. Mice were then given a 1.5-mg bolus of acetazolamide, followed by an infusion of 1.8 mg/h. After 30 min, a second 1-h micropuncture period was initiated. Open circles are means (error lines are ± SE) of the values indicated by the solid circles.

Although the fluorescence of micropuncture samples was usually determined within 24 h after collection, we wanted to evaluate the effect of long-term storage of the samples on the measured fluorescence. Proximal and distal tubular fluid samples and plasma samples from two experiments were measured as described, then stored at 4°C in the dark for 2 wk and reevaluated. As shown in Fig. 6, measurements of (TF/P)FITC-In, volume flow, and SNGFR were nearly identical to those taken 2 wk earlier.


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Fig. 6.   Scatter plots illustrating the stability of volume and fluorescence measurements over a 2-wk period. Initial measurements of (TF/P)FITC-In (left), flow rate (middle), and SNGFR (right) were determined within 24 h of the micropuncture experiment (x-axis). Then, measurements were repeated 2 wk later on the same samples ( y-axis). Samples were stored in their Microcap cuvettes in the dark at 4°C.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

We report here the practical use of FITC-inulin as a marker for single nephron function. (TF/P)In ratios have long been used as a hallmark of tubular function, and accurate measurements of this ratio are essential for micropuncture evaluation of renal fluid handling. Radiolabeled markers have been widely used in this regard, but they have a number of disadvantages associated with cost and safety issues that are inherent to the use of radioisotopes. The data presented here demonstrate that FITC-inulin can be conveniently and accurately used as a marker of GFR, with very high correlation to measurements obtained with [125I]iothalamate at both the single nephron and whole kidney level. Our approach is based on the use of readily available constant-bore microcapillary tubing both as a fluorometer cuvette and as a storage vial. Furthermore, this method provides the distinct advantage of not consuming the micropuncture sample, leaving it intact for other analyses. Finally, FITC-inulin can be used for a small fraction of the cost of radioactive methods. In our experience with mice, a typical FITC-inulin experiment costs approximately $2.50, compared with $35 for [125I]iothalamate.

Our findings are consistent with an earlier report by Sohtell and co-workers (6) that TF/P measurements made with FITC-inulin accurately reflect those using well-accepted radioactive markers. Although these earlier experiments demonstrated the feasibility and accuracy of using FITC-inulin in renal micropuncture, their proposed methodology had significant disadvantages, all of which are corrected in our method. First, relatively high levels of the marker were required to obtain a satisfactory fluorescence signal with infusate concentrations of 40 mg/ml and plasma levels ranging from 0.15 to 2 mg/ml (6). In contrast, our infusate concentration was 7.5 mg/ml, which resulted in plasma levels ranging from 0.1 to 0.4 mg/ml. Therefore, the approach proposed here avoids the potential osmotic effect that can accompany the higher urine concentrations of inulin in earlier methods (6, 9). The maximum urine concentration of FITC-inulin in our experiments was ~40 mg/ml, representing a solute concentration of no more than 20 mosmol/kgH2O. Second, the previous method required volumetric pipetting of 5 nl of sample to an oil-filled cuvette chamber. In addition to introducing an additional source of error, this approach is labor intensive and consumes the sample, reducing the opportunity to perform further analyses. Third, this earlier method did not account for the pH dependence of FITC fluorescence. Tubular fluid pH varies from a maximum value of ~7.4 in glomerular filtrate to 7.0 in the late proximal tubule, and can be as low as 6.5 in the early distal tubule (4). This drop in pH is primarily due to the reabsorption of HCO-3 from the proximal tubule and loop of Henle. Our data demonstrate that over this range, the fluorescence of FITC-inulin can vary considerably and illustrates the need to buffer each fluid sample to a standard pH. Addition of a small amount of 0.5 M HEPES to each sample effectively normalizes both pH and fluorescence.

Although we performed this assay in the 0.05 to 0.8 mg/ml concentration range, we found that the fluorescence of FITC-inulin samples was linear up to at least 8 mg/ml, which would allow this assay to be used at much higher plasma concentrations of inulin. Although this linearity could also permit urine samples to be evaluated without dilution, it is important to note that urine is very acidic compared with tubular fluid samples and that greater buffering is required to bring urine samples to a normal pH (up to 100 mM HEPES). In our case, this potential problem was obviated by prediluting all urine samples in phosphate-buffered saline, which provided the dual advantage of normalizing pH and bringing all samples to a similar location on the standard curve. In all of our assays, there was no evidence of autofluorescence of plasma or urine blanks. We did find, however, that the Fast green dye used to identify tubules can result in signal quenching, if it is present in the tubular fluid samples. For this reason, it is imperative that no dye be allowed to leak into the tubule during the collection period.

Although the correlation between our FITC-inulin-based assay and the [125I]iothalamate assay was found to be very good overall (see Figs. 3 and 4), small, systematic variations in TF/P and GFR values were observed. We do not know the reason for these differences, but there are several possibilities. Quenching of the fluorescence by endogenous plasma chromagens, or autofluorescence in tubular fluid samples could lead to the slight overestimation of the TF/P ratio seen in some of the values in Fig. 3. Although we saw no evidence of autofluorescence in plasma or tubular fluid sample blanks, we observed a slight tendency to underestimate the concentration of FITC-inulin in "spiked" plasma samples (see RESULTS). It is also possible that unbound FITC may contribute to systematic variation of TF/P values. However, we have dialyzed the FITC solution extensively against distilled water (molecular weight cutoff = 1,000), and found no trace of unbound FITC. Another possible source of variation is the pipetting and volume measurements that must be performed when evaluating [125I]iothalamate clearance. Since very small samples, ranging from 10 nl to 1 µl, must be accurately dispensed into counting vials, a potential random source of error is introduced that is not encountered in the FITC-inulin method. Thus, although both the FITC and radioactive methods have advantages and disadvantages in terms of potential error, it is evident that both methods yield reasonable and similar estimates of GFR.

We found the fluorescence of the fluid samples and standard curves to be stable when stored in the dark at 3°C. Although fluorescence measurements were normally performed within 12 h of the completion of the micropuncture experiments, we found that the samples could be stored for up to 2 wk in the refrigerator without appreciable changes in the (TF/P)FITC-In ratio, sample volume, or SNGFR. Although baseline SNGFR values ranging from 8 to 12 are quite low compared with normal values in the rat, they are consistent with previously reported values in the mouse using [125I]iothalamate (5) or [3H]inulin (7).

In several experiments, we compared whole kidney clearance measurements obtained by our microfluorometric method to measurements obtained on the same samples with a conventional fluorometer. Three-microliter samples of plasma or diluted urine were dispensed into 197 µl of 10 mM HEPES buffer in 96-well microplates. The fluorescence was read using Labsystems Fluoroskan II microplate fluorometer with an excitation wavelength of 485 nm and an excitation wavelength of 538 nm. Results obtained from these two methods were very similar (data not shown).

The primary pathway for fluid reabsorption in the proximal tubule is mediated by a process requiring carbonic anhydrase activity (1). Inhibition of carbonic anhydrase by either acetazolamide or benzolamide inhibits proximal tubule reabsorption in the rat by 50% or more (2, 8). In addition, these earlier investigations demonstrated that carbonic anhydrase inhibition results in a decrease in SNGFR of ~30% (8), a change that was attributed to activation of the tubuloglomerular feedback mechanism. Our methodology using FITC-inulin as a GFR marker provided results that were consistent with these earlier findings: proximal fluid reabsorption was decreased by 41% and SNGFR by 31% in mice treated with acetazolamide. These data further support the conclusion that microanalysis of FITC-inulin clearance provides measurements that accurately reflect GFR and fluid reabsorption.

In summary, we have described a new methodology for determining FITC-inulin concentration in nanoliter volumes of fluid which is based on the use of a constant-bore capillary tube as a measuring cuvette. Measurements obtained with this approach accurately reflect determinations obtained with more commonly used radioisotopic methods. The method is simple to use, has a high degree of precision, and is relatively inexpensive. In our experiments, we used a microscope fluorometer equipped with a digital image analysis system, but the approach can be easily tailored to use any commonly available microscope fluorometer.

    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50594.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: J. N. Lorenz, Dept. of Molecular & Cellular Physiol., Univ. of Cincinnati, P.O. Box 670576, Cincinnati, OH 45267-0576.

Received 1 June 1998; accepted in final form 17 September 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Berry, C. A., and F. C. Rector, Jr. Mechanism of proximal NaCl reabsorption in the proximal tubule of the mammalian kidney. Semin. Nephrol. 11: 86-97, 1991[Medline].

2.   Cogan, M. G., D. A. Maddox, D. G. Warnock, E. T. Lin, and F. C. Rector, Jr. Effect of acetazolamide on bicarbonate reabsorption in the proximal tubule of the rat. Am. J. Physiol. 237 (Renal Fluid Electrolyte Physiol. 6): F447-F454, 1979[Medline].

3.   Conger, J. D., H. N. Rhoads, S. N. Christie, and T. J. Burke. A modification of the fluorescence method for micro-inulin determinations. Kidney Int. 8: 334-337, 1975[Medline].

4.   Hamm, L. L., and R. J. Alpern. Cellular mechanisms of renal tubular acidification. In: The Kidney: Physiology and Pathophysiology, edited by D. W. Seldin, and G. Giebisch. New York: Raven, 1992, p. 2581-2626.

5.   Schnermann, J. B., T. Traynor, T. Yang, Y. G. Huang, M. I. Oliverio, T. Coffman, and J. P. Briggs. Absence of tubuloglomerular feedback responses in AT1A receptor-deficient mice. Am. J. Physiol. 273 (Renal Physiol. 42): F315-F320, 1997[Abstract/Free Full Text].

6.   Sohtell, M., B. Karlmark, and H. Ulfendahl. FITC-inulin as a kidney tubule marker in the rat. Acta Physiol. Scand. 119: 313-316, 1983[Medline].

7.   Sonnenberg, H., U. Honrath, C. K. Chong, L. J. Field, and A. T. Veress. Proximal tubular function in transgenic mice overexpressing atrial natriuretic factor. Can. J. Physiol. Pharmacol. 72: 1168-1170, 1994[Medline].

8.   Tucker, B. J., R. W. Steiner, L. C. Gushwa, and R. C. Blantz. Studies on the tubuloglomerular feedback system in the rat. The mechanism of reduction in filtration rate with benzolamide. J. Clin. Invest. 62: 993-1004, 1978[Medline].

9.   Vurek, G. G., and S. E. Pegram. Fluorometric method for the determination of nanogram quantities of inulin. Anal. Biochem. 16: 409-419, 1966.


Am J Physiol Renal Physiol 276(1):F172-F177
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society