Background

Under physiological conditions, the effects of kinins in the kidney are mainly mediated by the bradykinin B2-receptor, whereas the kinin B1-receptor is strongly induced under inflammatory conditions in a variety of tissues. Knowledge of the distribution of the B1-receptor along the nephron is of importance since the B1-receptor might replace B2-receptors under these conditions.

Methods

Using a RT-PCR/Southern blot approach allowing relative quantification of mRNA levels, ten different microdissected rat nephron segments were analyzed for the presence of the B1- and B2-receptor before and after endotoxin treatment to induce experimental inflammation. The functionality of the expressed receptors was assessed by kinin-induced intracellular calcium ([Ca²⁺]_i) mobilization in microdissected nephron segments.

Results

While under physiological conditions no B1-receptor mRNA could be detected, after 18 hours of treatment with bacterial lipopolysaccharide (LPS) the expression of B1-receptor mRNA was strongly induced in the efferent arteriole, the medullary and inner medullary thin limb, and in the distal tubule. Moderate expression was found in the glomerulus, proximal convoluted and straight tubules, and in the medullary thick ascending limb. Small but detectable expression was observed in the cortical collecting duct. The induction of B1-receptor mRNA expression resulted in functional receptor expression, since increases in [Ca²⁺]_i were observed upon B1-agonist stimulation. LPS treatment also increased the expression of B2-receptor mRNA in all nephron segments except in the glomerulus, the inner medullary thin limb and the outer medullary collecting duct. However, no related changes in B2-agonist induced rises in [Ca²⁺]_i were found.

Conclusions

These studies show a functional induction of the B1-kinin receptor along the rat nephron, which should be taken in account to address the effects of kinins under inflammatory conditions in the kidney.

METHODS

Materials

Bradykinin (BK), DBK and angiotensin II (Ang II) were from Sigma and used at a concentration of 0.1 M. B1-receptor antagonist des-Arg⁹-Leu⁸BK was from Sigma and used at a concentration of 1 M and HOE-140 (B2-antagonist) was a generous gift from Prof. B. Schölkens (Hoechtst, Frankfurt, Germany) and was used at concentration of 1 M.

Animals and lipopolysaccharide treatment

Experiments were performed on male Sprague-Dawley rats (Iffa Credo, France), five weeks old and weighing 120 to 140 g. LPS-treatment was performed by one intraperitonal injection of LPS (2 mg/kg; Sigma) 18 hours before sacrifice. LPS was used at a concentration of 10 mg/ml in a 0.9% saline solution. Control animals were injected with 0.9% saline solution only. Fourteen rats were randomly divided into two groups of which 7 were treated with LPS and 7 served as the controls.

Microdissection of nephron segments

Animals were anesthetized with an intraperitonal injection of pentobarbital (60 mg/kg; Sanofi, France). Kidneys were microdissected as previously described³³. The abdominal aorta was cannulated with polyethylene tubing just below the left kidney. The left kidney was perfused with 10 ml of ice-cold solution-1 (135 mM NaCl, 1 mM Na₂SO₄, 1.2 mM Mg SO₄, 5 mM KCl, 2 mM CaCl₂, 5.5 mM glucose, and 5 mM Hepes pH 7.4) followed by perfusion with 10 ml of solution-1 containing 1 mg/ml collagenase (0.38 U/mg collagenase A; Boehringer Mannheim) and 1 mg/ml bovine serum albumin (BSA; Sigma). The left kidney was removed and several coronal sections over the entire cortico-papillary axis were made. These sections were cut into three pieces: cortex, outer medulla, and inner medulla. The different pieces were transferred into individual tubes containing solution-1 with 0.5 mg/ml collagenase and 1 mg/ml BSA. The tubes were incubated for 8 to 15 minutes at 37°C in a shaking water bath and their contents were continuously bubbled with 95% O₂/5% CO₂. These different kidney tissue pieces were dissected on ice under a microscope and transferred to solution-1 containing 10 mM of vanadyl ribonucleotide complex (Sigma), a potent RNase inhibitor, and were placed on ice until further use.

Parts of the following segments were isolated: efferent arteriole (EA), glomerulus (Glom), proximal convoluted tubule (PCT), proximal straight tubule (PST), medullary thin descending limb (MTL), inner medullary thin limb (IMTL), medullary thick ascending limb (MTAL), distal tubule (DT), and cortical and outer medullary collecting ducts (CCD and OMCD, respectively).

Morphometry of nephron segments

The surface of the microdissected nephron segments was determined using photographs of freshly isolated nephron segments at 63 magnification, using an image-morphometry system (BIOSTAT™, Toulouse, France). The determination of the surface of each segment was repeated five to seven times.

Reverse transcription

For reverse transcription (RT), one glomerulus (surface 480 80 m²) or a total surface of 480 89 m² of each renal tubule segment was transferred to a reaction tube with 10 l of an ice-cold solution containing 1 U/l of human placental RNase inhibitor (Pharmacia) and 5 mM dithiothreitol (DTT; Gibco-BRL). Genomic DNA was degraded in the following way. Reaction tubes containing the different nephron segments were centrifuged for one minute (10,000 rpm). The RNase-inhibitor solution was replaced by 9 l of a solution containing 2% Triton X-100, 1 U RNase inhibitor, 5 mM DTT and 3 U RNase-free DNase (Promega) was added and incubated at 37°C for 30 minutes to permeabilize the cells and to digest the genomic DNA. For first-strand cDNA synthesis the following components were added (final reaction volume 100 l): 0.5 g pd(T)15, 32 U RNase inhibitor, 25 mM DTT, 5 mM dNTPs, RT buffer and 200 U reverse transcriptase (M-MLV; Gibco BRL). After RNA denaturation (7 min, 70°C), the reaction was carried out at 42°C for 50 minutes in a Perkin Elmer Thermo Cycler 480, heated to 95°C for five minutes and chilled on ice.

Polymerase chain reaction

For the polymerase chain reaction (PCR), 10 l of each cDNA preparation was amplified. PCR amplification was performed in Taq-buffer (Appligene) in a final volume of 100 l by adding 140 pM of each oligonucleotide, and 2 U Taq polymerase (Appligene) and 0.5 mM dNTPs.

For B1-receptor cDNA amplification, samples were denatured for two minutes and 30 seconds at 93°C. Then the PCR was performed for 35 cycles (1 min at 95°C, 1 min and 30 seconds at 62°C, 1 min at 72°C) followed by incubation for 10 minutes at 72°C. The sequences of the oligonucleotides used for B1-receptor cDNA amplification were designed to be located in the coding region of the rat B1-receptor (Genbank, accession number U66107). The sequence of the sense oligonucleotide was 5'-CTACAGGCTCCTGGTATACC-3' (bases 435 to 454, relative to B1 start codon) and of the antisense oligonucleotide 5'-CTCAGGGAGGCCAGGATGTG-3' (bases 700 to 719).

As described previously³⁴, the B2-receptor cDNA samples were denatured for two minutes and 30 seconds at 95°C, then the PCR was performed for 35 cycles (1 min at 95°C, 1 min and 30 seconds at 59°C, 1 min at 72°C) followed by 10 minutes at 72°C. The B2-receptor oligonucleotides were designed based on the 3' untranslated region of the cDNA from the sequence of the rat gene⁶. The sense oligonucleotide was defined by bases 2955-2975 (relative to B2 start codon, 5'-ACCAGAGATAGGATAGCCTTC-3') and the antisense oligonucleotide was defined by bases 3451-3473 (5'-ACAGTGTGTTAGCCTCAGAAGC-3').

For glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) amplification, samples were denatured for three minutes at 94°C, then PCR was performed for 27 cycles (1 min at 95°C, 1 min and 30 seconds at 57°C, 1 min at 72°C) followed by 10 minutes at 72°C. The GAPDH oligonucleotides were designed from the rat gene. The sequences of the GAPDH oligonucleotides were defined by bases 439 to 458 (relative to GAPDH start codon, 5'-AATGCATCCTGCACCACCAA-3', sense) and by bases 889 to 909 (5'-GTCATTGAGAGCAATGCCAGC-3', antisense).

The predominant cDNA amplification products were predicted to be 285 bp and 518 bp in length for the B1-receptor and B2-receptor, respectively. The expected size of the product for GAPDH is 470 bp. The PCRs for the B1- and B2-receptors and GAPDH were performed on the same cDNA. The PCR was carried out using a Perkin Elmer Thermal Cycler (model 480).

Southern blot analysis

Thirty microliters of each PCR reaction were electrophorized through a 1.8 to 2% agarose gel. The gel was denatured, neutralized and blotted overnight onto a nylon membrane (HybondTM-N; Amersham) with 20 SSC (sodium chloride/sodium citrate) as the transfer buffer. The DNA was fixed to the membrane under UV radiation (254 nm) in an UV-crosslinker (Stratagene). The membranes were hybridized with nested B1- and B2-receptor probes. The sequences of these B1- and B2-receptor probes were: 5'-CCTTGCAAAGTGCCAAGC-3' and 5'-CGTTAAGTTTATCACAGG-3', respectively. The probes were end-labeled with [³²P]-ATP (4500 Ci/ml) by the use of T4 polynucleotide-kinase (Promega). Membranes were prehybridized overnight at 42°C in 50 mM phosphate buffer pH 6.5 containing 50% deionized formamide, 5 SSC, 5 Denhardt's reagent (Sigma), and 50 g/ml of denaturated salmon sperm DNA (Sigma). Hybridization was performed overnight at 42°C in 20 mM phosphate buffer pH 6.5 containing 50% deionized formamide, 5 SSC, 1 Denhardt's reagent, and 50 g/ml of denatured salmon sperm. After hybridization, the membrane was washed with 2 SSC/0.1% SDS once at room temperature and twice at 65°C for 30 minutes. Autoradiography was performed for five hours at -80°C using Kodak XAR-5 films and intensifying screens.

Relative quantification

The densitometric values of each band were calculated using the Personal Densitometer SI of Molecular Dynamics. B1- and B2-receptor signals were corrected for differences in loading by normalization with GAPDH. Preliminary experiments have shown a linear non-saturating increase in amplification products between cycles 25 and 40 for the B1-receptor and between cycles 20 and 35 for GAPDH (not shown). These relationships were determined as described previously for the relationship between the quantity of B2-receptor mRNA and that of its amplification product³⁴. Thus, performing RT-PCR/Southern blot experiments within these amplification boundaries with GADPH as the loading control allows a relative quantitative evaluation of the expression of B1- and B2-receptor mRNA.

Measurement of the intracellular Ca²⁺ mobilization

The intracellular calcium ([Ca²⁺]_i) mobilization measurements were performed as described previously³³. In brief, the microdissected renal tubule segments were individually transferred onto a thin glass microscope coverslip in 1 ml of solution-1 containing 1% agarose (Sigma; type IX) at 37°C. Then the agarose was jellied by cooling the coverslip for one minute on ice. The samples embedded in agarose were loaded with 5 M fura-2 acetylmethoxy ester (fura-2/AM) at room temperature for one hour. For fluorescence measurements, each sample was placed on the stage of an inverted microscope and was continuously perfused at a rate of 0.6 ml/min at 37°C with solution-1, which could be replaced at any time by the solutions to be tested. Fura-2 was alternatively excited at 340 and 380 nm using a 75W xenon light source, filters, and a chopper (PTI Photoscan II System, Kontron). The illumination path included a 40 objective (Nikon) and an ultraviolet dichroic mirror. Emitted signals passed through a 480 20 nm bandpass filter before detection by a photomultiplier and were recorded every 2 seconds. The fluorescence intensities (S at 340 nm and L at 380 nm) were recorded from about 15% of the total glomerulus or tubule segment area and controlled by an adjustable window diaphragm. Correction for autofluorescence was performed as described³⁵. [Ca²⁺]_i was calculated from the equation of Grynkiewicz et al³⁶. [Ca²⁺]_i = K_d [(R - R_min)/(R_max - R)] (L_max/L_min) where K_d (= 224 nM) is the dissociation constant of fura-2 for calcium, r = S/L, R_min and R_max are values of R at 0 and saturating calcium concentrations, respectively, and L_max/L_min is the ratio of L at 0 and saturating calcium concentrations. R_min, R_max, L_max and L_min are constant parameters that depend on the optical system used. Under our conditions these values were: R_min = 0.96, R_max = 9.21, L_max/L_min = 6.38.

Statistical analysis

Results are given as mean SEM. The differences were tested using the non-parametric Mann-Whitney U-test which was used for comparison between two unpaired variables. P < 0.05 was considered significant.

RESULTS

Distribution of B1-receptor mRNA along the nephron from rats treated with LPS

While in microdissected nephron segments of nontreated control rats no B1-receptor mRNA could be detected (data not shown), 18 hours of LPS (2 mg/kg) treatment induced in all but one segment B1-receptor mRNA expression Figure 1a. B1-receptor mRNA expression was studied by RT-PCR followed by Southern blot analysis using a nested B1-receptor probe. Moreover, to verify that the samples were not contaminated by genomic DNA, RNA samples were submitted to the RT-PCR amplification without the reverse transcriptase enzyme. In those samples no bands were observed except those of the primers at the bottom of the gel (not shown). Except in the OMCD, a single band of predicted size (285 bp) was found in all microdissected nephron segments. In order to perform a relative quantification of the expression of B1-receptor PCR products and to analyze the integrity of the mRNA, we amplified in parallel a housekeeping gene, that of GAPDH, and analyzed its expression in ethidium bromide stained gels Figure 1b. The amplification product of GAPDH was found in all renal structures at the predicted size of 470 bp. After densitometric analysis, B1-receptor mRNA expression was corrected using GAPDH and expressed as the percentage of the B1-receptor mRNA expression in the efferent arteriole in which the largest B1-receptor mRNA signal was observed Figure 2. As evaluated by our relative quantitative method, large signals for B1-receptor mRNA were detected in the EA, MTL, IMTL and DT. Moderate signals were found in the Glom, PCT, PST, and MTAL. A small, but detectable signal was found in the CCD.

Figure 1.

B1-receptor and GAPDH mRNA expression in nephron segments of lipopolysaccharide (LPS)-treated rats. Rats were treated for 18 hours with 2 mg/kg LPS followed by nephron segment isolation using microdissection. Chromosomal DNA was degraded and samples were in parallel subjected to RT-PCR analysis for GAPDH mRNA expression and RT-PCR/Southern blot analysis for B1-receptor mRNA expression. (A) B1-receptor mRNA expression (revealed by means of a nested B1-receptor probe). (B) GAPDH mRNA expression (revealed by ethidium bromide staining). One representative experiment is shown.

Full figure and legend (50K)

Figure 2.

Relative quantification of B1-receptor mRNA expression. One glomerulus (surface 480 80 m²) or a total surface of 480 89 m² of each renal tubule segment was used. Band intensities were scanned (4 times) using a densitometer. B1-receptor mRNA expression was corrected using GAPDH, amplified in parallel, and expressed as the percentage of B1-receptor mRNA expression in the efferent arteriole (EA). Values are means SEM of seven experiments.

Full figure and legend (12K)

B1-agonist-induced intracellular calcium mobilization [Ca²⁺]_i

To verify whether induction of B1-receptor mRNA is translated into functional B1-receptors, the ability of B1-agonist DBK to mobilize intracellular calcium in the microdissected nephron segments was examined. Although with our method we were unable to detect B1-receptor mRNA in nephron segments of control rats, DBK-induced calcium mobilization was observed in the MTAL and the CCD (Figure 3, insets; Table 1), which was prevented by the B1-antagonist des-Arg⁹-Leu⁸BK (not shown). The other nephron segments of control rats did not respond to DBK (Table 1).

Figure 3.

Representative recordings of intracellular calcium mobilization in response to DBK along the nephron of LPS-treated rats (18 hr, 2 mg/kg). Shown are traces stimulated with DBK (0.1 M) without (a) and with (b) B1-antagonist des-Arg⁹-Leu⁸BK (1 M, added 5 min before and during DBK stimulation). Insets in the MTAL and CCD show the responses to 0.1 M DBK in segments of control rats (treated with vehicle only). In the other parts of the nephron no responses to DBK were obtained in control rats. Mean peak values are shown in Table 1.

Full figure and legend (50K)

Table 1 - Effect of DBK (0.1 M) on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the different microdissected nephron segments obtained from untreated (control) and treated rats (LPS) 18 hours with LPS 2 mg/kg.

Full table (38K)

Except for PCT and PST, all LPS-treated segments in which an increase of B1-receptor mRNA was detected, responded to an addition of DBK (0.1 M) by mobilization of intracellular calcium (Figure 3, traces marked a; Table 1). However, the amplitudes of the calcium responses after DBK-addition in the MTAL and the CCD were not significantly different between control and LPS-treated rats (Table 1). The shapes and amplitudes of the calcium responses obtained in the nephron segments were different. The [Ca²⁺]_i increase was large in the glomerulus, MTL, IMTL and DT, and smaller in the EA, MTAL and CCD. The EA, glomerulus, IMTL and DT showed a biphasic response composed of a sharp peak followed by a sustained phase. In the MTL and CCD, the sustained phase was less pronounced. In the MTAL the biphasic response was characterized by a small initial peak followed by a pronounced sustained phase. In LPS-treated rats, basal [Ca²⁺]_i levels were around 90 nM with the lowest value in the EA (40 5 nM) and highest in the MTL (125 25 nM). Peak values were between 133 11 nM in the EA and 536 34 nM in IMTL (Table 1). The B1-agonist induced intracellular calcium mobilization was specific for the B1-receptor, since it could be abolished by B1-antagonist des-Arg⁹-Leu⁸BK (1 M) pretreatment (added 5 min before and during B1-agonist (0.1 M) stimulation; Figure 3, traces marked b). The absence of DBK-induced calcium responses in the PCT and PST is not specific for the B1-receptor and might be related to the experimental conditions used. Angiotensin II (Ang II), which is known to induce a calcium response along the rat nephron³⁷, was used as a control and was effective in all the microdissected nephron segments except in the PCT and PST (not shown).

Effect of LPS on the expression of B2-receptor mRNA along the nephron

Figure 4 shows a typical pattern of the distribution of the B2-receptor mRNA along the nephron of control rats Figure 4a and LPS-treated (18 hr) rats Figure 4c. A single band of 518 bp was found in the EA, Glom, PCT, PST, MTL, IMTL, MTAL, DT, CCD and OMCD. Interestingly, using our RT-PCR/Southern blot approach, we have localized B2-receptor mRNA expression in the EA, MTL and OMCD, three nephron structures where to our knowledge the presence of B2-receptor mRNA has never been described. As described above for the B1-receptor, samples were analyzed for contaminating genomic DNA by performing the RT-PCR amplification without the reverse transcriptase enzyme. In those samples no bands were observed except those of the primers at the bottom of the gel (not shown). As described for B1-receptor mRNA, GAPDH mRNA expression amplified in parallel was detected in all renal structures Figure. 4b and 4d. The evaluation of the relative variation between B2-receptor mRNA expression of control and LPS treated animals is shown in Figure 5. The densitometric values are presented as the percentage of EA value obtained in LPS treated rats. Whereas no significant variation in B2-mRNA expression was observed between control and LPS treated rats in the Glom and OMCD, surprisingly, we found a significant increase in B2-receptor mRNA expression in the EA, PCT, PST, MTL, MTAL, DT and CCD. On the contrary, a net decrease in B2-receptor mRNA expression was observed in the IMTL segment.

Figure 4.

B2-receptor and GAPDH mRNA expression in nephron segments of control and LPS-treated rats. Rats were treated for 18 hours with 2 mg/kg LPS or with vehicle only followed by nephron segment isolation using microdissection. Chromosomal DNA was degraded and samples were in parallel subjected to RT-PCR analysis for GAPDH mRNA expression and RT-PCR/Southern blot analysis for B2-receptor mRNA expression. B2-receptor mRNA expression (revealed by means of a nested B2-receptor probe) in control (A) and LPS-treated rats (C). GAPDH mRNA expression (revealed by ethidium bromide staining) in control (C) and LPS-treated rats (D). One representative experiment is shown.

Full figure and legend (90K)

Figure 5.

Relative quantification of B2-receptor mRNA expression. B2-receptor mRNA expression was corrected using GADPH, amplified in parallel, and expressed as the percentage of B2-receptor mRNA expression in the efferent arteriole (EA) of LPS-treated rats. Abbreviations are in the Appendix. Symbols are: () B2, control nephron segments; () LPS-treated nephron segments. Values are means SEM of seven experiments.

Full figure and legend (20K)

Intracellular calcium mobilization induced by B2-receptor agonist

Bradykinin induced an increase in [Ca²⁺]_i in all segments except in the PCT and PST (Table 2). As described previously³⁸, BK induced a biphasic calcium response consisting of a transient phase followed by a sustained phase. A typical profile obtained from an IMTL segment of control rats is shown in Figure 6 (control, trace a). The observed calcium mobilization induced by BK-stimulation is due to the activation of B2-receptors since the calcium response was abolished with HOE-140 a specific B2-receptor antagonist Figure 6, control, trace b) and not by the specific B1-antagonist des-Arg⁹-Leu⁸BK (not shown). The basal calcium level in individual segments was between 39 12 nM in the EA and 135 11 nM in the MTAL and the peak values were between 62 15 nM in the EA and 443 28 nM in the IMTL (Table 2). Since LPS treatment led to B2-receptor mRNA variations in some nephron segments, we have examined if those variations were associated with any modification in the [Ca²⁺]_i response induced by BK-stimulation. As shown in Table 2, no variations in the calcium response were observed after LPS treatment. Moreover, the decrease in B2-mRNA expression observed in the IMTL segments is not associated to a significant decrease in the calcium response Figure 6; LPS, trace a).

Figure 6.

Typical recordings of intracellular calcium mobilization in response to 0.1 M BK in control and LPS-treated rats in the inner medullary thin limb (IMTL). Shown are traces stimulated with BK without (a) and with (b) B2-antagonist HOE-140 (1 M, added 5 min before and during BK stimulation). Thin line is 5 minutes of BK; heavy line is ten minutes of HOE-140. Mean peak values are shown in Table 2.

Full figure and legend (14K)

Table 2 - Effect of BK (0.1 M) on intracellular [Ca²⁺]_i concentration in the different microdissected nephron segments.

Full table (33K)

DISCUSSION

All components of the kallikrein-kinin system are present in the kidney¹ and it is well established that kinins contribute to renal hemodynamics and excretory function³⁹. Since all kinin-effects are mediated by specific receptors, the precise localization of kinin-receptors along the nephron allows a better understanding of the actions of BK in the kidney. B2-receptor distribution along the nephron has been extensively studied since this receptor mediates the larger part of the kinin-responses under physiological conditions^{24,25,27,28,29}. Under physiological conditions the B1-receptor is not expressed at significant levels, but its expression is often induced during inflammation. For example, studies on B1-receptor function in the cardiovascular and pulmonary systems have revealed a clear link between B1-receptor induction and the cytokine network¹⁰. In this respect an increase in inflammatory factors (chemokines, cytokines) released from resident or infiltrating cells such as monocyte/macrophages, neutrophils and T cells^40,41 is observed in a number of renal diseases (glomerulonephritis, lupus nephritis, renal transplant rejection, ischemia, HIV nephropathy and acute hypertensive nephritis). Although it remains to be demonstrated, the B1-receptor might be induced in these renal diseases. Despite this potential importance of the B1-receptor under inflammatory conditions in the kidney, nothing is known about the renal localization of the B1-receptor. Therefore, in an attempt to better understand the role of the renal B1-receptor we studied the precise localization of the B1-receptor along the rat nephron under physiological and inflammatory conditions using microdissected nephron segments. Experimental inflammation was induced by 18 hours of LPS treatment, which is usually sufficiently long to induce B1-receptor induction in different tissues¹⁰.

Our data suggest differences in B1-receptor coupling and translational control along the different parts of the nephron. In the MTAL and the CCD of control rats a calcium response to DBK was observed, although no corresponding B1-receptor mRNA was detected under our experimental conditions. This indicates that under normal conditions in these segments, the small basal number of B1-receptors are efficiently coupled to the PLC-transduction pathway that was not changed by the induction of new B1-receptor mRNA after LPS treatment. In the other segments, de novo B1-receptor mRNA induction is directly coupled to a de novo functional B1-receptor response (calcium mobilization). However, there is no direct relationship between B1-receptor mRNA levels and the amplitude of the calcium response, again indicating this segmental regulation. Detailed studies are necessary to decorticate these segmental control mechanisms, but are out of the scope of this study. The absence of both DBK and Ang II-induced calcium responses in the proximal tubules might be due to the microdissection conditions used. Microdissection in the presence of collagenase, indispensable for nephron segment preparation, can result in irreproducible calcium responses in the proximal tubules³⁷. Omitting collagenase during microdissection restores this response for Ang II³⁷, but interferes in the comparison between different segments.

Whereas this study was not designed to determine the pathophysiological significance of the presence of the B1-receptor in the different parts of the nephron, it shows that the effects of kinins may pass through this receptor-subtype under inflammatory conditions. In this respect, using isolated perfused rat kidneys, conditions (that is, ischemia and surgery) known to favor B1-receptor induction in other tissues¹⁰, Guimares et al have reported that the B1-agonist DBK increased renal vascular resistance⁴². The authors have postulated that B1-kinin receptor responses may be of importance in the generation and/or the maintenance of renal vasoconstriction in disease states that lead to renal failure. In this context, the increased vascular resistance might originate from the vasoconstriction of the EA induced by the presence of B1-receptors under these conditions. This is consistent with our observation that experimental inflammation induces a high-level B1-receptor mRNA expression in the EA. Furthermore, it has been hypothesized that under pathological conditions, the B1-receptor could amplify or replace the function of the B2-receptor¹⁰, which, unlike the B1^31,32, desensitizes upon prolonged B2-agonist stimulation⁴³. The induction of B1-receptor mRNA at the glomerular level is of particular interest since it has been reported that in primary culture of mesangial cells the B1-agonist stimulates cell proliferation³⁸. The presence of B1-receptors on these cultivated cells without LPS treatment can be explained by the fact that the mesangial cell has an inflammatory character in culture⁴⁴, conditions that favor the induction of the B1-receptor in other tissues. Moreover, DBK is able to stimulate the formation of collagen in cultured human lung fibroblasts⁴⁵. Such effects have to be related to renal pathologies where fibrotic processes are observed.

Compared to other work on the B2-receptor, based either on binding or immunohistological studies^25,27,29, our approach, RT-PCR/Southern blot analysis allowing relative quantification of B2-receptor expression in microdissected nephron segments, appears to be more sensitive and permits complementary information, since we have been able to detect B2-receptor mRNA expression in three new nephron segments: the EA, the MTL and the OMCD. BK is known to reduce both afferent and efferent glomerular arteriolar tone, with the most pronounced decreases in the afferent arteriole⁴⁶, thus suggesting the presence of B2-receptors in these structures. The presence of B2-receptors in the afferent arteriole was recently confirmed²⁷, and here we provide a molecular basis for the observed actions of BK in the efferent arteriole: the presence of functional B2-receptors. The functions of BK in the medullary thin limb and outer medullary collecting duct are not yet known, but the presence of B2-receptors suggest contribution of these segments to the natriuretic and diuretic effects of BK. Whereas it has been reported that BK increases [Ca²⁺]_i in cultured proximal tubular cells⁴⁷, no BK- or Ang II-induced [Ca²⁺]_i increase in the proximal tubule segments could be observed under our experimental conditions. As discussed for the B1-receptor above, the absence of any receptor specific response is most likely due to the microdissection conditions.

Alhough based on a relatively quantitative approach, the increased B2-receptor mRNA expression in the EA, PCT, PST, MTL, MTAL, DT and CCD, and decreased B2-mRNA expression in the IMTL after LPS treatment are worth being discussed. Whereas no previously published data were available on the effects of LPS on B2-receptor expression in renal cells, it has been reported that synovial cells that are insensitive to BK stimulation under basal conditions became responsive to BK (stimulation of PGE₂ release) after pretreatment with the cytokine IL-1⁴⁸. This effect was abrogated by cycloheximide and actinomycin D, strongly suggesting a de novo B2 receptor synthesis following cytokine treatment⁴⁸. In our experiments, the changes in B2-receptor mRNA expression were not accompanied by a changed functional response as determined by the mobilization of intracellular calcium. As described above for the B1-receptor, these data suggest differences in B2-receptor translational control and receptor coupling along the nephron segments. Furthermore, up-regulated B2-receptors can be specifically coupled as we have shown recently in rat mesangial cells in which cAMP induces B2-receptor expression that is coupled to the PLA₂ pathway and not to the PLC pathway⁴⁹. Therefore, selective up- or down-regulation of only one B2-receptor transduction pathway remains a possibility to be investigated to account for a segmental regulation of B2-receptors along the nephron.

From a therapeutical point of view, the precise distribution of the B1- and B2-receptor along the nephron should be of great help in extending our knowledge of the mechanism of the protective effect of the angiotensin converting enzyme inhibitors (ACE-inhibitors), which not only decrease Ang II formation but also retard kinin catabolism. Chronic administration of ACE-inhibitors markedly attenuates the development of glomerular lesions in different models of progressive glomerular sclerosis⁵⁰. In subtotal nephrectomized rats that were performed to mimic a loss of renal mass, we demonstrated that ACE-inhibitors delay selectively the evolution of tubular hypertrophy in the proximal tubule⁵¹. Whether a kinin mechanism is involved in these anti-hypertrophic effects of ACE-inhibitors cannot be demonstrated at present. In this respect, we have recently reported that the B1-agonist induced the expression of its own receptor in human lung fibroblast cells⁵². Thus, ACE-inhibitor treatment, which increases the kinin concentration in several tissues including kidney⁵³, can lead to increased B1-receptor expression. Indeed, pretreatment of rabbits with ACE-inhibitors made these animals responsive to DBK, and it was suggested that potentiation of endogenous kinins would 'prime' the expression of B1-receptors⁵⁴.

In conclusion, our studies show, to our knowledge for the first time, the localization of bradykinin B1-receptors along the rat nephron. Although absent under physiological conditions, functional B1-receptors were found along the nephron under inflammatory conditions. This suggests that in inflammatory renal diseases, the effects of kinins may pass through B1-receptors.

References

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Acknowledgments

M.E. Marin-Castaño is supported by a grant from the French Nephrology Society. J.P. Schanstra is a recipient of a 'Poste Vert' from INSERM.