Effect of glandular kallikrein on distal nephron HCO<SUP>−</SUP><SUB>3</SUB> secretion in rats and on HCO<SUP>−</SUP><SUB>3</SUB> secretion in MDCK cells

P. Vallés1, S. Ebner2, W. Manucha1, L. Gutierrez1, and M. Marin-Grez2

1 Instituto de Fisiopatología and Instituto de Histología, Facultad de Medicina, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina; and 2 Physiologisches Institut der Universität, Munich D-80336, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Renal kallikrein is localized in the connecting tubule cells and secreted into the tubular fluid at late distal nephron segments. The present experiments were performed to further test the hypothesis that renal kallikrein reduces bicarbonate secretion of cortical collecting duct (CCD). The effect of orthograde injections of pig pancreatic kallikrein (1 or 3 µg/ml) into the renal tubular system was investigated. Urine fractions (Fr) were collected after a 2-min stop flow. Changes in the urine fraction with respect to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/Fr:Ff polyfructosan) increased significantly in the first two urine fractions collected after glandular kallikrein administration (kallikrein, 1 µg/ml, P < 0.05; kallikrein, 3 µg/ml, P < 0.01). HCO<SUP>−</SUP><SUB>3</SUB> secretion of collecting ducts was significantly reduced dose dependently by orthograde and also reduced by retrograde pig pancreatic kallikrein administration. Release of kinins into the fractions was not affected by the retrograde kallikrein injection, even though the kallikrein activity increased considerably (2.26 ± 0.2 vs. 1.55 ± 0.2, P < 0.05). Adequacy of retrograde injections for delivering substances to the CCD was demonstrated by injecting colloidal mercury and detecting the appearance of this mercury in the renal cortex by transmission electron microscopy. The integrity of the renal tissue after a retrograde ureteral injection was confirmed by scanning electron microscopy. These results confirm and extend previous data (M. Marin-Grez and P. Vallés. Renal Physiol. Biochem. 17: 301-306, 1994; and M. Marin-Grez, P. Vallés, and P. Odigie. J. Physiol. 488: 163-170, 1995) showing that renal kallikrein reduces bicarbonate secretion at the CCD, probably by inhibiting HCO<SUP>−</SUP><SUB>3</SUB> transported by a mechanism unrelated to its kininogenase activity. Support for this assessment was obtained in experiments testing the effect of kallikrein on the luminal bicarbonate secretion of a subpopulation of Madin-Darby canine kidney cells capable of extruding the anion. Kallikrein inhibited HCO<SUP>−</SUP><SUB>3</SUB>/Cl- exchange, and the degree of inhibition was dose dependent. This inhibition occurred in the absence of kininogen in the bathing solution.

kallikrein; cortical connecting tubule; cortical collecting duct; bicarbonate; Madin-Darby canine kidney cells

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

SEVERAL OBSERVATIONS OVER the past years have provided evidence for a very specific localization of renal kallikrein. The enzyme has been found to be localized in distal nephron segments. Data indicating that kallikrein is synthesized in the connecting tubule (CNT) cells include: 1) immunohistochemical detection by light microscopy in the distal convoluted tubule (3), 2) detection of kallikrein-like amidolytic activity in the granular portion of microdissected convoluted tubules (i.e., CNT segments) (27), and 3) localization of kallikrein immunoreactivity in the CNT cells by electron microscopy (9).

This localization, along with the fact that kallikrein is found in secretory vesicles close to the tubular lumen (29), suggests that the enzyme's site of action is on the luminal membrane of cells localized downstream from the CNT.

Experimental and clinical evidence for changes of urinary kallikrein activity in conditions of disturbed acid-base balance has been reported (12, 18).

Marin-Grez and Vallés (17, 19) have shown that renal kallikrein is involved in the regulation of acid-base balance, probably by inhibiting the renal secretion of bicarbonate in distal nephron segments.

The present experiments were performed to investigate whether the inhibiting effect of kallikrein on cortical collecting duct (CCD) bicarbonate secretion is dose dependent and to test whether this effect is mediated by kinin release. Exogenous intraluminal glandular kallikrein administration was achieved either by orthograde (intra-arterial) or by retrograde (ureteral) injections. To study the enzyme's effect on bicarbonate transport, bicarbonate concentration was measured in urine fractions obtained after a short ureteral occlusion. To evaluate whether the effect of kallikrein on bicarbonate transport at distal nephron segments depends on local release of kinins from kininogen, the kinin concentration in urine fractions collected after retrograde intraluminal kallikrein administration was also measured. The adequacy of retrograde injections for delivering substances to the CCD was tested by injecting colloidal mercury and analyzing the appearance of this in the renal cortex by optical- and electron-transmission microscopy. Scanning microscopy was performed to assess tissue integrity after retrograde ureteral injection.

Since some cells in wild-type Madin-Darby canine kidney (MDCK) cell monolayers have properties similar to beta -intercalated cells, these were taken as a model to investigate the effect of kallikrein on bicarbonate transport through the luminal membrane of collecting ducts. Stilbene- and acetazolamide-inhibitable intracellular acidification in response to rising luminal chloride concentration was taken to indicate bicarbonate exchange for chloride (Ebner and Marin-Grez, Ref. 8). Intracellular pH (pHi) changes were measured by video-imaging microfluorometry in cells loaded with the pH-sensitive probe 5-(and-6)-carboxy SNAFL-2 (c-SNAFL-2).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The experiments were performed on female Wistar-Kyoto rats weighing 220-290 g. Up to the day of the experiment the animals had free access to tap water and to a commercial rat chow

Orthograde Stop Flow (Experiment I)

Rats (experimental group n = 10, control group n = 10) were anesthetized by intraperitoneal injection of 100 mg/kg body wt thiobutabarbital (Inactin; Byk-Gulden, Constance, Germany). After a tracheostomy was performed, catheters were placed on the right femoral vein for infusing a solution containing 150 mM NaCl, 50 g/l mannitol, and 20 g/l polyfructosan (Inutest; Laevosan, Linz, Austria), adjusted with NaHCO3 to pH 7.40, at a rate of 12 ml/h. A pulled catheter was inserted into the left ureter and advanced as close as possible to the renal papilla without touching it. A catheter was placed in the urinary bladder to allow free flow of urine from the right kidney. The abdominal aorta was freed of adherent tissue above and below the left renal artery. The tip of a catheter introduced into the lower segment of the abdominal aorta was advanced up to the ostium of the left renal artery for intrarenal injections. The left renal vein was freed of adherent tissue close to its caval end. After a 60-min equilibration period, urine was collected under free-flow conditions for a 5-min period for measuring electrolytes, kallikrein, bicarbonate, and polyfructosan. A blood sample was taken from the aorta to assess acid base status. Then, spring-clamping probes were used to occlude the aorta above the left renal artery, the aorta below the renal artery (i.e., around the injection catheter), and the left renal vein. Kallikrein (1 µg, porcine pancreas kallikrein 203 U/mg; Boehringer, Mannheim, Germany) in 2 ml 150 mM NaCl, 50 g/l mannitol, and 80 g/l polyfructosan, or the vehicle was injected into the renal artery. The ureteral catheter was then clamped, and the arterial and venous clamps were released. Urine flow was reestablished 2 min later. An 85-µl fraction (corresponding to the volume of the ureteral catheter) was discarded, and four 140-µl fractions (corresponding to the distal nephron and collecting duct system) were collected into glass capillaries for measuring pH, PCO2, electrolytes, kallikrein, and polyfructosan. Ten minutes later, urine was collected for 5 min in glass capillaries for measuring the same parameters under free-flow conditions. Then, a second kallikrein injection (3 µg in 2 ml vehicle) was given into the renal artery as above. After the injection, four 140-µl urine fractions were obtained in the same way as described above.

Retrograde Stop Flow: Effect of Kallikrein on Bicarbonate Secretion (Experiment II)

Rats (n = 10) were anesthetized by intraperitoneal injection of 100 mg/kg body wt thiobutabarbital sodium. After a tracheostomy was performed, catheters were placed in the right femoral vein for infusion, the right femoral artery for measuring blood pressure and collecting blood samples, and both ureters for injections and urine collections. The animals received intravenous 150 mM NaCl (adjusted to pH 7.40 by addition of 0.5 mM NaHCO3) at a rate of 3 ml/h. After an equilibrium period of 90 min, urine was collected for electrolyte measurements. Porcine pancreas kallikrein (0.1 ml of a 100 mU/ml solution, 203 U/mg; Boehringer) was then injected retrogradely through one ureteral catheter over 5 min. The vehicle (0.1 ml of 300 mM mannitol, 150 mM NaCl, and 80 g/l polyfructosan) was administered through the contralateral catheter. The administration of kallikrein and vehicle were given randomly with a delay of 10 min to either kidney. After injections, the ureteral catheter was occluded, and 10 min later flow was reestablished to collect 125-µl urine fractions into glass capillaries for pH, PCO2, polyfructosan, and electrolytes measurements.

Retrograde Stop Flow: Effect of Intraluminal Administration of Kallikrein on Kinin Release (Experiment III)

The animals (n = 10) were surgically prepared as in experiment II. The rats received intravenous 150 mM NaCl (adjusted to pH 7.35-7.40 by addition of 0.5 mM NaHCO3) at a rate of 3 ml/h. After a 90-min equilibration, 140-µl urine fractions were collected in glass capillaries from both kidneys for urine osmolarity, electrolytes, kallikrein activity, and kinin concentration measurements. Porcine pancreas kallikrein (0.1 ml of a 100 mU/ml solution, as above) was then injected retrogradely through one ureteral catheter over 5 min, the vehicle (0.1 ml of a 300 mM NaCl, polyfructosan 10% solution) was administered through the other. The administrations were given with a delay of 10 min to either kidney in random order. After injections, the ureteral catheter was occluded, and 2 min later flow was reestablished to collect 140-µl urine fractions into glass capillaries for osmolarity, electrolytes, kallikrein activity, and kinin concentration measurements.

Analytical Methods

Urinary electrolytes (Na+ and K+) were measured by flame photometry (Metrolab, 315, Corswant).

Urinary osmolarity was determined by vapor-pressure osmometry (model 5500, Wescor).

Kallikrein activity in urine was measured by incubating appropriately diluted samples with the synthetic peptide D-Val-Leu-Arg-p-nitroanilide (Kabi Diagnostica, Stockolm, Sweden) at 37°C for 120 min. Blanks did not contain the substrate.The reaction was stopped by the addition of 100 µl of 50% acetic acid (5). Polyfructosan was measured by the anthrone method after acid hydrolysis (10). If kallikrein and polyfructosan could not be measured immediately, then samples were stored at -30°C.

It has been reported that an accuracy acceptable in research can be achieved when the urinary ionic strength is taken into consideration for the calculation of the pKa necessary to obtain the bicarbonate concentration (16).

Blood pH and PCO2 were measured immediately after collection by means of electrodes using available equipment (model ABL 30; Radiometer, Copenhaguen, Denmark). The urinary HCO<SUP>−</SUP><SUB>3</SUB> concentration was calculated by using the Henderson-Hasselbalch equation, after correction of the pKa value (14).

Kinin concentration was measured in the urine collected in alcohol (100 µl urine/300 µl ethanol). The final concentration of alcohol was brought to 72% using either water or ethanol as necessary. After centrifugation at 1,000 g for 10 min, the supernatant was saved. The pellet was washed with 1 ml 72% alcohol, and the supernatants were pooled in a polystyrene tube. The alcohol was evaporated by air stream in a water bath at 37°C. The dried extract was kept frozen at -70°C until assayed. Kinins were measured by radioimmunoassay (7) by using a dilution of antibradykinin serum (1:4,000) which bound 40% 125I-labeled [Tyr8]bradykinin (10,000 counts/min). Standards (bradykinin 5-150 pg/ml) or unknown samples (1-20 µl urine) in a final volume of 0.4 ml 0.1 M tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.4, were incubated during 22 h at 4°C. Then dextran-coated charcoal suspension was added to separate bound from free tracer.

Free 125I-[Tyr8]bradykinin adsorbed to the charcoal was precipitated and counted with a gamma counter (model 1240, LKB Wallac). Standard curve was constructed by plotting the fraction bound (B/Bo) versus the logarithm of the corresponding dose of bradykinin standard, where Bo was the counts per minute (cpm) bound in the absence of unlabeled peptide, and B is the cpm bound for each quantity of standard added. Duplicate estimations were made for each point on the curve, and the unknown samples were extrapolated from this plot.

Recovery of exogenously added bradykinin (10 ng/100 µl) before drying was 86.6 ± 2.1% (n = 3).

Cell Culture and Video Microscopy

MDCK cells were cultured, and pHi was measured as previously described (8). Briefly, high-resistance wild-type MDCK cells were grown to confluence on cell culture dishes (Falcon, Becton-Dickinson) containing Dulbecco's modified Eagle's medium supplemented with 10% basal medium support (BMS; Biochrom, Berlin, Germany). The cells were loaded with the pHi indicator by incubating them with 10 µM c-SNAFL-2 diacetate (Molecular Probes, Eugene, OR) for 30-60 min at 37°C and 5% CO2 in the cell culture incubator. pHi was measured by the two excitation wavelength ratio imaging method. A xenon lamp was used as the light source (Osram, Berlin, Germany), and the band-pass filters (520 ± 2, 550 ± 2 nm) were positioned by means of a step motor-powered filter wheel and focused on the stage of an inverted microscope (Nikon Diaphot). Fluorescence images were taken through a long-pass filter (580 nm) with an intensified charge-coupled device camera connected to a frame grabber (Matrox; Electronic Systems, Dorval, Quebec, Canada). Filter positioning, image capture, background subtraction, and division of the gray value intensity of the 550-nm and 520-nm images were performed by a personal computer. The nigericin method was used to calibrate the fluorescence intensity of the ratio image to the corresponding pHi (26). The cytosolic pH was measured in the cytosolic region of each of 10 cells/culture dish. Immediately prior to an experiment, the cell monolayer was carefully rinsed four or five times with Dulbecco's phosphate-buffered saline (Biochrom, Berlin, Germany) to eliminate traces of exogenous proteins. The effect of extracellular chloride concentration changes on pHi was investigated by replacing the control bathing solution (in mM: 5 glucose, 140 Na+, 5 K+, 1 MgSO4, and 1 calcium lactate) by the appropriate solution at room temperature. Chloride was replaced isosmotically by gluconate in the concentration range 0-140 mM. Porcine pancreatic kallikrein (Medor, Herrsching, Germany) was added to a final concentration of 0.5 or 1.0 µg/ml (500 U/mg) into the experimental dishes. Images were taken 5 min after each solution change. Two experiments were performed to investigate 1) the effect of 1.0 µg/ml kallikrein on the intracellular acidification in response to rising extracellular chloride concentrations at 30 mM intervals in individual dishes and 2) the effect of 0.5 µg kallikrein on the pHi response to a change in extracellular chloride from 0 to 110 mM in additional dishes.

Statistical Analysis

The results of experiment I were assessed by one-way analysis of variance for comparisons within groups and two-way analysis of variance for comparisons between groups. Significance of difference was estimated by the test of Duncan (within a group) or test of Scheffé (between groups). Student's test was used to compare the means when the experimental design consisted of two samples (experiments II and III). Statistical significance was assessed by Student's paired t-test to compare control and experimental values within the same group of rats. P < 0.05 was considered to be significant. Results are means ± SE.

Histological Studies

Surgery. Female Wistar-Kyoto rats (n = 6) weighing 250-280 g were anesthetized with 100 mg/kg thiobutabarbital. After a tracheostomy was performed, catheters were placed in the left femoral vein, in the descending aorta, and in the left ureter.

Colloidal mercury injection. The animals were infused with 150 mM NaCl intravenously at a rate of 2 ml/h for 60 min. Then, after the arterial flow to the kidney was interrupted by clipping the aorta above the renal artery and the left renal vein was slit, the kidney was rinsed free of blood by infusing 150 mM NaCl through the descending aorta. Colloidal mercury, 0.1 ml (10 g/l) in 150 mM NaCl, was slowly (10 min) injected retrogradely through the left ureter. The control group received a retrograde injection of 150 mM NaCl. Then, the left kidney was excised, cut into 1-mm3 cubes, fixed in 50 g/l glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.40) for 5 h, and washed in the same buffer for 12 h. The kidney cortex was cut along the corticopapillary axis. Kidney slices from the outer cortex were washed with 0.1 ml cacodylate buffer (pH 7.4). After fixing the slices in 1% osmium tetroxide in cacodylate buffer, these were dehydrated through acetone and embedded in plastic Spurr medium. Ultrathin sections (600-900 Å) were stained with lead citrate and uranyl acetate. The slices were observed by transmission electron microscopy (Elmiskop I-A) at a magnification of ×5,000-15,000.

For scanning electron microscopy, the slices were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 15 min, washed with the same buffer and then post fixed with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer. After dehydration in 100% ethanol, the dry critical point method was performed. After vaporization and sputter coating, the slices were observed in a Siemens Autoscan electron microscope. Exposure time was 1 min.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Orthograde Stop Flow (Experiment I)

The body weight of rats in the kallikrein-injected group (experimental) did not differ from that of those in the control group. Blood pressure of both groups was similar before the first stop flow and before starting the second stop flow. Acid-base parameters were normal in both groups during the administration of 1 and 3 µg of kallikrein (Table 1).

                              
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Table 1.   Orthograde stop-flow (experiment I)

Intraluminal administration of kallikrein (1 and 3 µg/ml) increased renal kallikrein activity (Fr:Ff kk/Fr:Ff polyfructosan) in the first fraction (140 µl) of urine collected 2 min after ureter occlusion. Bicarbonate secretion (Fr:Ff HCO<SUP>−</SUP><SUB>3</SUB>/Fr:Ff polyfructosan) was lower in the first fraction (140 µl) from kidneys to which kallikrein (1 µg/ml and 3 µg/ml) had been administered (Table 2). An increase of 44% and 51% on kallikrein activity, in the first urine fractions collected after intraluminal administration of the enzyme, induced a dose-dependent decrease on bicarbonate secretion (Fig. 1).

                              
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Table 2.   Orthograde stop-flow


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Fig. 1.   Orthograde stop flow (experiment I): intraluminal administration of 1 and 3 µg of porcine pancreatic kallikrein. Progressive increase on the enzyme activity (top) and the corresponding decrease on bicarbonate secretion (bottom) in the first and second urine collected fractions (F1 and F2) are evident. Data are shown as percent; n = 10 for experimental group, and n = 10 for control group.

Retrograde Stop Flow: Effect of Kallikrein on Bicarbonate Secretion (Experiment II)

The mean blood pressure of these rats was 103 ± 2 mmHg.

Acid-base balance was normal at H+ = 42 ± 0.7 nM, PCO2 = 42.8 ± 1.5 mmHg, and HCO<SUP>−</SUP><SUB>3</SUB> = 24.7 ± 0.8 mM. Na+ and K+ concentrations in urine ([Na+]u and [K+]u, respectively) collected from experimental and control kidneys did not differ significantly ([Na+]u, control 47.7 ± 8.2 and experimental 36.8 ± 4.1 mM; [K+]u, control 114.2 ± 13.3 and experimental 97.3 ± 10.6 mM).

The HCO<SUP>−</SUP><SUB>3</SUB>/polyfructosan concentration ratio (µeq:ml/mg:ml) of the fractions collected from the kallikrein-injected kidneys was significantly lower than that measured in the fractions obtained from the vehicle-injected kidneys (Fig. 2A).


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Fig. 2.   Retrograde stop flow (experiments II and III). A: ratio of bicarbonate concentration to polyfructosan concentration (HCO<SUP>−</SUP><SUB>3</SUB>/Polyf) in 125-µl urine fractions collected at the end of a short urinary stop flow. Effect of a retrograde kallikrein injection (solid bars) or the vehicle (open bars) into the collecting ducts of anesthetized rats (n = 10). B: kallikrein (KK) activity of urine fractions collected after stop flow. C: kinin concentration (BK, i.e., bradykinin) was measured in the same fraction. Values are means + SE. * P < 0.05 vs. control (Student's t-test).

Retrograde Stop Flow: Release of Kinin by Intraluminal Injection of Kallikrein (Experiment III)

The urinary osmolarity of fractions collected after kallikrein administration was higher than the osmolarity measured in the preinjected control samples (experimental, 1,451 ± 97 vs. 929 ± 73 mosmol/l; P < 0.01). The same holds for the vehicle injected kidneys (1,357 ± 87 vs. 916 ± 75 mosmol/l; P < 0.01). No difference was observed between groups. The urine [Na+]u and [K+]u concentrations did not differ significantly after kallikrein or vehicle injections ([Na+]u, experimental 30.4 ± 4.2 vs. control 30.3 ± 1.7 mM; [K+]u, experimental 17.5 ± 1 vs. control 17.9 ± 0.8 mM). Kallikrein activity in the urine fraction collected after stop flow was significantly higher in kidneys that had received kallikrein injection compared with kidneys with vehicle injection (P < 0.05). By contrast, there were no differences in kinin concentration in the 100-µl urine fraction collected after kallikrein administration (Fig. 2, B and C).

Effect of Kallikrein on Bicarbonate Secretion of beta -MDCK Cells (Experiment IV)

Some cells in a MDCK monolayer display high fluorescence intensity when loaded with carboxy SNAFL-2 diacetate. These cells secrete bicarbonate in exchange for extracellular chloride. The anion exchange can be assessed by the pHi decrease brought about by a rise of chloride in the luminal solution (8).

Figure 3A (open symbols) depicts the decrease in cytosolic fluorescence ratio (decrease in pHi) of untreated beta -MDCK cells loaded with SNAFL in response to an increase in extracellular chloride (n = 7 cells in 1 dish). The response to 140 mM [Cl-] was taken to represent 100%. The rise of extracellular chloride concentration from 0 to 110 mM reduced the fluorescence intensity ratio from 0.69 ± 0.02 to 0.40 ± 0.04, corresponding to a reduction of pHi from 7.45 ± 0.04 to 6.78 ± 0.09. The pHi change obtained in these cells was similar to previously published data (8).


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Fig. 3.   Effect of kallikrein on intracellular pH (pHi) of confluent wild-type MDCK cells to extracellular chloride concentration ([Cl-]o) changes. A: ordinate expresses the pHi decrease in percent of that obtained to a chloride concentration change from nominal 0 to 140 mM; solid symbols, 1 µg/ml kallikrein; open symbols, control. Percent decrease is inversely related to the ratio changes. B: effect of 0.5 and 1.0 µg/ml kallikrein (solid bars) on the fluorescence ratio reduction to 110 mM extracellular chloride (open bars). Each point or bar is the mean ± SE of the measurements obtained by video imaging in 7 cells.

A dose of 1 µg/ml kallikrein completely prevented the intracellular acidification induced by the rising extracellular chloride. The effect of the enzyme was investigated in seven cells in separate dishes for each chloride concentration step (Fig. 3A, solid symbols).

A dose of 0.5 µg/ml kallikrein only partially inhibited the acidification due to the increase in extracellular chloride from 0 to 110 mM, whereas the inhibition due to 1 µg/ml kallikrein was almost complete. This indicates that the effect of kallikrein on luminal chloride/bicarbonate exchange is dose dependent.

Histological Studies

Substances injected retrogradely through the ureter in a volume of 100 µl solution reached the CCD. This was demonstrated by detecting the presence of colloidal mercury particles on the luminal space and on the cytoplasmic membranes of CCD cells (Fig. 4). Transmission electron microscopy confirmed this finding, since electron dense particles were found on the luminal space of CCD of rats injected with the colloidal mercury but not in controls. (Fig. 5, A and B) and also in intercalated cells (Fig. 6). No damage of these structures could be found by inspection with an scanning electron microscopy (Fig. 7).


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Fig. 4.   Optic micrograph of rat cortical collecting ducts (CCD) after retrograde injection of 100 µl colloidal mercury (1%). Arrow, presence of colloidal mercury particles in the luminal space of CCD. Magnification, ×280.


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Fig. 5.   A: transmission electron micrograph of rat CCD after retrograde injection of 100 µl of 150 mM NaCl (control). Magnification, ×11,400. B: transmission electron micrograph from rat CCD after retrograde injection of 100 µl colloidal mercury (1%). Colloidal mercury particles are present in the luminal space of CCD. Magnification, ×11,400.


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Fig. 6.   Transmission electron micrograph from rat CCD after retrograde injection of 1% colloidal mercury. Arrow, presence of particles of colloidal mercury on the cytoplasmic membrane of intercalated cells. Magnification, ×6,500.


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Fig. 7.   Scanning electron micrograph of rat CCD, after retrograde injection of 0.1 ml 150 mM NaCl. Note integrity of principal and intercalated cells. Magnification, ×6,600.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Since renal kallikrein is mainly secreted toward the lumen of the renal tubules, a primary role for the enzyme outside the kidney is unlikely. The release of tissue kallikrein into the circulation has been suggested, a possibility supported by the finding of a kallikrein-like enzyme in a enriched fraction in basolateral membranes of tubular cells (32) and also by the report of the presence of kallikrein in the venous effluent, albeit only in studies performed with isolated perfused kidneys (20). It has been suggested that kidney kallikrein could reach the circulation through the lymphatic vessels. However, very small amounts of tissue kallikrein have been detected in the renal lymph (11). Several experimental studies have demonstrated that under conditions known to affect renal kallikrein, the kinin concentration in renal vein is found unchanged (15) and that urinary kinins and kallikrein changes are, more frequently than not, unrelated to each other (21, 22). Thus it seems quite unlikely that this enzyme normally has systemic or vascular effects.

It has become evident that renal kallikrein is secreted into the tubular lumen of the CNT (2), supporting the previous finding of the enzyme in secretory vesicles that are in contact with the lumen of the CNT cells (29). Because of this specific localization, the functional role of renal kallikrein is related to its rate of secretion into the tubular fluid, exerting its effects downstream from the site of secretion.

Previously, Marin-Grez and Vallés (17) have demonstrated that the bicarbonate concentration in urine fractions obtained from the collecting duct system after whole kidney stop flow in rabbits is inversely related to kallikrein activity. In addition, Marin-Grez et al. (19) demonstrated inhibition of bicarbonate secretion into the collecting ducts of rats injected with kallikrein. Furthermore, aprotinin infusion through the renal artery induces inhibition of kallikrein excretion and increases bicarbonate concentration in urine fractions (19). Those experiments have shown that a rise of intraluminal kallikrein activity reduces the tubular bicarbonate secretion.

In the present study, performed on rats in normal acid-base balance, an increase of 44% and 51% of kallikrein activity in the first fractions collected after intraluminal administration of kallikrein induced a dose-dependent decrease in bicarbonate secretion, i.e., a 60% reduction by 1 µg/ml kallikrein and a 79% reduction by 3 µg/ml kallikrein (experiment I).

A lower effect on kallikrein activity was observed in the second urine collected fractions after the administration of the enzyme. This is probably due to the ramification of the collecting ducts, which implicates a dilution of the fraction that reaches the cortical segment. However, a progressive decrease on bicarbonate secretion was observed. An increase on intercalated beta -cells in cortical segments could be taken into consideration for this effect.

Injection of kallikrein into the lumen of the collecting ducts by an retrograde route (experiment II) also reduced significantly the bicarbonate secretion into the tubular fluid.

In these experiments, we administered porcine pancreatic kallikrein. Both porcine (4) and rat tissue kallikrein (24) have the same key amino acids in the substrate-binding pocket. Consequently, porcine kallikrein is able to hydrolyze rats substrates. Thus porcine kallikrein is appropriate to study the effect of exogenous kallikrein on rat renal tubular functions. To confirm that enzyme injected by a retrograde route actually reached the CCD, colloidal mercury was injected in the same volume by this way. The arrival of this substances to the CCD was detected by histological techniques.

Although kinins have been found both in the tubule (23) and in the urine (1), the origin of renal kinins is unknown. Some evidence permits to postulate that renal kallikrein cannot effectively interact with kininogen at the distal tubule. The concentration of kinin-containing kininogen in urine is about three orders of magnitude below the Km for the reaction with kallikrein, and the luminal pH at the site at which kallikrein is secreted is far from the pH optimum for kinin release. Not surprisingly, there are several reports indicating that kallikrein and kinin excretions are mostly unrelated to each other (6, 15, 22, 28).

Marin-Grez et al. (19) have previously demonstrated that retrograde injection of bradykinin into the collecting ducts did not decrease the bicarbonate concentration in CCD fractions, indicating that the physiological effect of renal kallikrein on bicarbonate transport is unrelated to kinin release. In the present study, after kallikrein injection, we found no difference in concentration of kinins, nonetheless, bicarbonate was reduced.

Ebner and Marin-Grez (8) have previously demonstrated that some cells in monolayers of wild-type high-resistance MDCK cells are capable of secreting bicarbonate in response to increasing luminal chloride concentration. MDCK cells have many properties of the CCD and are used as a model for the CCD. The bicarbonate-secreting MDCK cells may serve as a model for beta -intercalated cells. A kallikrein concentration in the range normally found in rats and human urine (to our knowledge the urine kallikrein concentration in dog urine is unknown) markedly or completely inhibited bicarbonate secretion by these dog-derived cells after a 5-min incubation at room temperature. Thus it seems likely that at the lower concentration of kallikrein expected in the CCD (but higher incubation temperature), this may well be a physiological regulator of bicarbonate secretion. Furthermore, experiments are in progress to determine the mode of action of kallikrein on the anion exchanger. These results, along with the demonstration that renal kallikrein secretion is regulated by extracellular pH (18), suggest that kallikrein is part of a negative feedback loop regulating acid base balance.

It has been shown that renal (or tubular) kallikrein activity or immunoreactivity is increased in animals given high potassium intake for prolonged periods (13, 30).

This leads to the suggestion that the enzyme might be involved in the metabolism of this cation; furthermore, it is known that abnormalities of the potassium balance lead to changes of the acid-base balance (25). Since the latter was not investigated in the previous papers, it cannot exclude the fact that the increased kallikrein in response to high-potassium diet was related to a concomitant acidosis rather than to the hyperkalemia.

It has been assumed for decades that the physiological role of renal kallikrein is related to its kininogenase activity. The experiments presented here suggest that kallikrein has a role unrelated to kinin release. Kallikrein could only release kinins if it were to come into contact with kininogen in a context appropriate for the "substrate-enzyme" interaction. Kallikrein is almost exclusively secreted into the tubular lumen. Kallikrein and kininogen could interact if kininogen were present in the same vesicles in CNT cells; however, this has never been shown. Alternatively, kallikrein could be taken up by distally located target cells into the same vesicles as kininogen. Again, this has never been shown. In addition, the evidence indicating that low-molecular-weight (LMW) kininogen is actually synthesized in the kidney is not very strong. All hitherto published data relating to kinins released from kidney homogenates after incubation with trypsin, immunohistochemistry using polyclonal antibodies against purified LMW-kininogen, and Northern blots at low stringency using probes allegedly recognizing specifically LMW-kininogen could be interpreted to reflect the local synthesis of T-kininogen. The base sequence of the cDNA of this kinin-containing molecule, which is not a substrate for renal kallikrein, is more than 70% homologous to that of LMW-kininogen (24). Thus it appears that renal kallikrein and LMW-kininogen could, at the most, react with each other in the tubular lumen. However, it is not known at what level in the nephron kininogen is added to the tubular fluid. It could be, as it appears to be the case for the tubular appearance of kinins (23), that this occurs in the terminal portion of the medullary collecting duct. Needless to say, the incubation conditions in the lumen of the collecting duct are far from adequate; the intratubular pH is at least 2.5 U below optimum, and the sodium concentration is in the inhibiting range. Even a local release of kinins could be without any physiological relevance; to date there is no evidence that the luminal membrane of collecting duct cells contains kinin receptors. In summary, there is no convincing evidence indicating that renal kallikrein interacts with kininogen in the kidney, that kinins are locally released, that the peptides could be taken up luminally and have intracellular actions, or that bradykinin may act on the luminal membrane of collecting duct cells.

These results strongly support the suggestion from Marin-Grez et al. (17, 18) that renal kallikrein is part of a negative feed-back loop which controls extracellular acid-base balance by regulating bicarbonate secretion directly, i.e., by a mechanism unrelated to kinins.

    ACKNOWLEDGEMENTS

This work was performed with the financial support provided by Consejo de Investigaciones, Universidad Nacional de Cuyo, Mendoza, Argentina and by the German Research Foundation.

    FOOTNOTES

Address for reprint requests: P. Vallés, Instituto de Fisiopatología, UNC, Casilla de Correo 33, 5500 Mendoza, Argentina.

Received 1 April 1997; accepted in final form 15 July 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Renal Physiol 273(5):F807-F816
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