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Am J Physiol Regul Integr Comp Physiol 275: R439-R447, 1998;
0363-6119/98 $5.00
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Vol. 275, Issue 2, R439-R447, August 1998

RT-PCR study of the distribution of connexin 43 mRNA in the glomerulus and renal tubular segments

Rong Guo, Li Liu, and Luciano Barajas

Department of Pathology, Harbor-University of California Los Angeles Medical Center, Torrance, California 90509

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

An RT-PCR study of the distribution of connexin 43 (Cx43) mRNA in glomeruli and along the rat tubular segments was carried out to establish the differential expression of Cx43 in the different segments of the tubule, in renal regions, in isolated glomerular preparations (IGP), and in microdissected glomeruli. The mRNA level of Cx43 in macrodissected renal regions appeared in the following order: inner papilla > outer papilla and IGP > outer medulla and cortex. Among the microdissected tubules, inner medullary collecting ducts (IMCD) expressed the highest level of Cx43 mRNA, followed by the cortical collecting ducts (CCD). The proximal convoluted tubules and proximal straight tubules expressed significantly less Cx43 than the IMCD, glomeruli, and CCD. Medullary thick ascending limb and distal convoluted tubules showed the lowest level of Cx43 mRNA. The RT-PCR results of the microdissected segments correlate well with those obtained by RT-PCR of the renal regions. The high concentration of Cx43 mRNA in the IMCD together with the observation of abundant punctate immunofluorescence for Cx43 suggests that the IMCD not only expresses Cx43 mRNA but also that the mRNA is translated to Cx43 protein.

reverse transcriptase-polymerase chain reaction; gap junction

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE CONNEXINS (Cx) are the protein constituents of the gap junctions. They are the subunits of the connexons. A connexon is a hexamer of connexin proteins arranged to form a pore. Two connexons, from adjacent cells, are aligned to form an intercellular channel that permits the passage of ions and small molecules between cells. The gap junctions are formed by an array of connexons. Communication between cells by gap junctions contributes to the harmonious integration of cellular activity, which is essential to organ development and function.

The gap junction proteins or connexins constitute a multigene family. The connexins have a basic common structure that consists of four conserved transmembranous domains connected by two, also conserved, extracellular loops and one cytoplasmic loop. The cytoplasmic loop and the COOH-terminus are unique for each connexin while the NH2-terminus is also conserved (4).

Although nine connexins (Cx26, Cx30.3, Cx31, Cx32, Cx37, Cx40, Cx43, Cx45, and Cx46) are known to be expressed in the kidney (6, 20, 21, 29, 30, 40, 43), little is known about their localization in the renal tissues (41). This is in part probably due to the extraordinary histological heterogeneity of kidney that makes it difficult to localize the punctate gap junction expression by light microscopy immunocytochemistry.

Although the distribution of gap junctions in the kidney has been extensively investigated ultrastructurally, knowledge about the distribution of the different connexins in the kidney is very limited. An early immunocytochemical study reported the presence of gap junction immunofluorescence for Cx43 in all tubules, in glomeruli, and in the connective tissue with no reference to any differential expression in different segments of the renal tubule or the presence in the renal vasculature (5). Cx43 was originally found in the heart and has been localized in many vascular tissues (24). The extraglomerular mesangium (EM) is formed by modified vascular smooth muscle cells and has been shown ultrastructurally to contain a high concentration of gap junctions. This finding prompted an immunocytochemical study of the localization of Cx43 in the kidney. With the use of a polyclonal antibody to Cx43 as probe, concentration of punctate immunofluorescence was observed in the renal vasculature and EM (1). In the same study, an intense punctate immunofluorescence was found in the inner medullary collecting duct (IMCD), and very little if any gap junction immunofluorescence for Cx43 was observed in the other tubular segments.

To ascertain the differential expression of Cx43 in the different segments of the renal tubule and to clarify and extend the results obtained by immunocytochemistry, we have applied the method of RT-PCR to macrodissected renal tissues and microdissected tubular segments. The results have been published in abstract form elsewhere (19).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Macrodissection and RT-PCR

Dissection of tissues from the different regions of the kidney and glomerular preparation. Kidneys from male Sprague-Dawley rats weighing ~200 g were removed under anesthesia (Nembutal, 45 mg/kg), and blocks of cortex and medulla were dissected out from slices taken from the midportion of the kidney cut along its longitudinal axis. The arcuate arteries were used as landmarks to separate the cortex from medulla. The renal papilla was cut off from the inner medulla, and it was divided in two sections: the inner and outer papilla. Glomeruli were isolated by passing renal cortex through serial sieves (9).

RT-PCR methods. RT-PCR-related reagents and enzymes, primers (DM151 and DM152), pAW109 RNA, and a model-480 DNA thermal cycler were purchased from Perkin-Elmer. Rat Cx43 cDNA clone, plasmid pCx43, was derived from its original G2 clone (6) and was a gift from Dr. David Paul, Harvard Medical School (Boston, MA).

To compare relative amounts of Cx43 mRNA in different regions of rat kidney, we extracted RNA as described previously (11) from the macrodissected tissues: outer papilla, inner papilla, renal cortex, outer renal medulla, and glomerular preparations. Given the relatively small amount of RNA present in the renal papilla, total RNA from papilla was prepared from 16 kidneys of 8 rats, and total RNA from the other parts of the kidney was prepared from 8 kidneys of 4 rats. pAW109 RNA was used as an internal control for semiquantitative RT-PCR. A saturation experiment was performed and showed that the RT-PCR was not saturated even when total RNA reached 1 µg in each reaction (data not shown). RT-PCR was carried out as follows: total RNA (0.25 µg) and pAW109 RNA (1 × 104 copies) were reverse transcribed to first-strand cDNA by 2.5 U/µl of recombinant Moloney murine leukemia virus reverse transcriptase in 20 µl of reaction mixture containing 2.5 µM of random hexamer, 1 U/µl of RNase inhibitor, 1 mM of each dNTP, 10 mM Tris · HCl (pH 8.3), 50 mM KCl, and 5 mM MgCl2 at 42°C for 15 min. The reaction was then denatured at 92°C for 5 min and cooled down to 5°C for PCR. PCR was performed by coamplification with two sets of primers (10, 16, 22), one for rat Cx43 mRNA and another for pAW109 RNA. Primer Cx1 (antisense for Cx43) was 5'-TTGTTTCTGTCACCAGTAAC-3', and primer Cx2 (sense for Cx43) was 5'-GATGAGGAAGGAAGAGAAGC-3'. Thus the cDNA amplification product of Cx43 mRNA was predicted to be 588 bp in length.

Coamplification of pAW109 RNA served as an invariant control. The primers for pAW109 RNA resulted in a product of 308 bp and were defined by the following sequence: primer DM152 (antisense for PAW109 RNA), 5'-CATGTCAAATTTCACTGCTTCATCC-3'; primer DM151 (sense for pAW109 RNA), 5'-GTCTCTGAATCAGAAATCCTTCTATC-3'. After preparation of the first-strand cDNA, the reaction solution was mixed with PCR reagents to make a 100-µl reaction solution containing 2.5 units Taq DNA polymerase, 0.15 µM of each primer, 1 mM of each dNTP, 10 mM Tris · HCl, 50 mM KCl, and 2 mM MgCl2. PCR was performed in a Perkin-Elmer PCR thermal cycler by incubation at 95°C for 2 min (initial melt), followed by 30 cycles as follows: 95°C for 1 min (denature), 65°C for 1 min (anneal and extend). PCR was completed for a final extension of 72°C for 7 min. The reaction product was kept at -20°C for further analysis.

Southern blot analysis of the PCR products. Southern blot probe for Cx43 was prepared from a PCR product of plasmid pCx43. pCx43 was amplified by PCR with Cx1 and Cx2 primers. The PCR product of 588 bp was purified with a Sephadex G50 column and labeled with 32P using a method reported previously (13, 14). To identify the RT-PCR product of Cx43 mRNA from total RNA of rat kidney tissues, a portion of the unlabeled PCR product was kept as a size marker (Cx43 size marker). The first-strand cDNA of pAW109 RNA was synthesized by reverse transcription, and the probe for pAW109 RNA was prepared in the same way with primers DM151 and DM152.

RT-PCR products (5 µl) of the RNA from each tissue and Cx43 size marker were fractionated on a 1.5% agarose gel in 1× Tris-acetate/EDTA electrophoresis buffer (TAE) buffer. The gel was stained with ethidium bromide and photographed for semiquantitative analysis. The DNA bands were then denatured, neutralized, and transferred to Hybond N+ membrane (Amersham) by electroblotting with 0.25× TAE (pH 8.3). Blots were baked at 80°C for 10 min to fix the DNA. Prehybridization and hybridization were followed according the method described by Moriyama et al. (28) except equally mixed Cx43 probe and pAW109 probe were used at 2.5 × 105 counts · min-1 · µl hybridization solution-1.

Semiquantitation of Cx43 expression. Semiquantitation of Cx43 was done by densitometer scanning of the ethidium bromide-stained agarose gels. Briefly, 5 µl of RT-PCR products of the RNA from each tissue and Cx43 size marker were electrophoresed on a 1.5% agarose gel in 1× TAE buffer. The gel was stained with ethidium bromide and photographed. The densitometer Scan Analysis program from BioSoft (Cambridge, UK) was applied to gel images using the OneScanner/Ofoto scanning system from Apple Computer (Cupertino, CA). To eliminate the tube effect, the density ratio of the Cx43 RT-PCR band to the pAW109 RT-PCR band was used as a measure of Cx43 mRNA level in the tissue. RT-PCR was performed six times on RNA from each region of the kidney and from the glomerular preparation. The Kruskal-Wallis test comparing all groups, P = 0.0001, was used to determine the statistical significance or the differences between the results of the different groups (35).

Microdissection and RT-PCR

Microdissection of glomeruli and tubular segments. The microdissection methods used in this investigation were based on those described by Wright, Burg, and Knepper (42). Rats weighing 130-180 g were perfused retrogradely through the aorta below the left kidney with 10 ml of cold PBS followed by 10 ml of cold Dulbecco's modified Eagle's medium (DMEM; Sigma; see Ref. 38) containing 1 mg/ml of collagenase (Sigma) and 1 mg/ml of bovine serum albumin (BSA) (Sigma). Kidney slices were incubated for 35 min at 37°C in the perfusion media with 100% O2 and then rinsed with ice-cold DMEM and placed in the same medium and maintained ice-cold during dissection.

With the use of a stereomicroscope (Carl Zeiss) and direct illumination (Fiber-Lite A3200), glomeruli and the following tubular segments were dissected: proximal convoluted tubules (PCT), proximal straight tubule (PST), distal convoluted tubule (DCT), medullary thick ascending limb (MTAL), cortical collecting duct (CCD), and IMCD. The length of the tubule was measured by means of a micrometer. Samples were transferred with a pipette tip precoated with 1% BSA to a petri dish containing DMEM. After two washes, six glomeruli or 3 mm of tubular segment were transferred to an Ependorff tube containing 10 µl of ice-cold PBS in diethyl pyrocarbonate-treated H2O. The PBS solution was then removed from the tube, and 30 µl lysis buffer (containing 2% Triton X-100, 2 U/µl of RNase inhibitor, and 5 mM dithiothreitol) were added. The glomeruli were kept in the lysis buffer for 60 min and the tubules for 30 min. The lysed tissue mix was frozen in liquid nitrogen and stored at -80°C.

RT-PCR of microdissected glomeruli and tubular segments. For RT-PCR, the lysed tissue mix is thawed, and 10 µl were transferred to an RT-PCR tube and were reverse transcribed to first-strand cDNA by 5 U/µl of MuLV reverse transcriptase in 20 µl of reaction mixture containing 0.5 µM of oligo(dT), 1 U/µl of RNase inhibitor, 1 mM of each dNTP, 10 mM Tris · HCl (pH 8.3), 50 mM KCl, and 5 mM MgCl2 at 42°C for 60 min. The reaction was then denatured at 90°C for 5 min and cooled down to 5°C for PCR.

PCR was carried out under conditions similar to that for total RNA from macrodissected tissues except with the following changes. A DNA segment encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) replaced pAW109 cDNA as the internal standard. GAPDH was chosen as the internal standard for microdissection and RT-PCR for quantitative purposes, since GAPDH showed constant expression among different renal tissues (15). The ratio of Cx43 signal to GAPDH signal should be constant for a defined tissue even if the tissue mass for individual RT-PCR varies. This ratio was used as a measure of the relative abundance of Cx43 expression in the microdissected tubular segments and glomeruli. The antisense primer for GAPDH was 5'-AGATCCACAACGGATACATT-3', defined by bases 795-814, and sense primer was 5'-TCCCTCAAGATTGTCAGCAA-3', defined by bases 506-525 (37). These primers were predicted to amplify a cDNA 309 bp in length.

The internal standard GAPDH cDNA and sample Cx43 cDNA were amplified under the same conditions but in two separate tubes. Briefly, each RT reaction (20 µl of total volume) was carried out in two separate PCR reaction tubes, one tube containing 2 µl and the other containing 18 µl. Two microliters of 15 µM Cx43 primers (Cx1 and Cx2) were added to the tube containing 18 µl RT mix for amplification of Cx43 cDNA, and 2 µl of 15 µM GAPDH primers were added to the tube containing 2 µl RT mix for amplification of GAPDH cDNA. Both tubes were brought to a total volume of 100 µl containing 2.5 units of Taq DNA polymerase, 1 mM of each dNTP, 10 mM Tris · HCl (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2. PCR was initiated by 2 min incubation at 95°C (melting), followed by 38 cycles of 95°C for 1 min and 60°C for 1 min. After a final extension at 72°C for 7 min, the reaction mixture was kept at -20°C for further analysis.

The saturation experiment was carried out before the RT-PCR reactions for semiquantitative analysis. Under the conditions described above, no saturation was found in the range of one to six glomeruli or 1 to 3 mm of tubule. Thus two glomeruli or 1 mm of tubule was used in each RT-PCR reaction to get the data for semiquantitative analysis. Two negative controls were carried out by doing the reaction in the same way as in microdissection and RT-PCR except that some components were eliminated. One control had no microdissected tissue in the reaction to check contamination involved in the entire RT-PCR process. Another control had no reverse transcriptase in the reaction to verify that the RT-PCR product of Cx43 was amplified from mRNA, not from genomic DNA.

The RT-PCR product of Cx43 in microdissected tubular segments was characterized by restriction mapping. Ten microliters of RT-PCR reaction mix for microdissected IMCD were digested with Sty I, Hinc II, and the combination of Sty I and Hinc II under the conditions recommended by the manufacturer (New England Biolabs). The digested mixture was electrophoresed on 8% polyacrylamide/Tris-borate/EDTA electrophoresis buffer gel, and the restriction pattern was compared with the predicted restriction map of the Cx43 cDNA segment defined by primers Cx1 and Cx2.

After PCR, the reaction mix for Cx43 and the reaction mix for GAPDH from the same RT reaction were mixed in equal volume and were electrophoresed on 1.5% agarose gel. Band density was measured by densitometric scanning, and the density ratio of Cx43 to GAPDH was used as a measure for Cx43 expression. The conditions for electrophoresis, densitometric scanning, and semiquantitative analysis were the same as described for the RT-PCR products from macrodissected tissues. The results of the microdissection and RT-PCR studies were analyzed statistically by the repeated measures analysis of variance comparing all groups, P = 0.01 or less (12).

Immunocytochemistry

The immunocytochemical methods used in this study have been reported in detail elsewhere (1). Briefly, adult Sprague-Dawley rats were fixed by transcardial perfusion with 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Kidneys were removed and kept in the same fixative for 4 h. Ten-micrometer-thick frozen sections were obtained with a cryostat microtome and mounted on gelatin-coated slides. Sections were processed for fluorescence immunostaining with a polyclonal antibody raised against synthetic peptides representing unique portions (COOH-terminus of Cx43; amino acids 252-271) of the rat Cx43 gap junction protein (kindly provided by Dr. E. Beyer of Washington University, St. Louis, MO; see Ref. 5) at 1:100 dilution in PBS. Control sections were incubated with normal rabbit serum at 1:100 and omitting the primary antibody. The sections were observed and photographed with a Zeiss photomicroscope equipped with epifluorescent illumination. Photographs were taken with Kodak TMZ 3200 film.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Semiquantitative RT-PCR of Macrodissected Renal Regions

The different regions of the kidney sampled for RT-PCR analysis are shown in Fig. 1a. Representative results from agarose gel electrophoresis and Southern blot hybridization of RT-PCR products of Cx43 mRNA are shown in Fig. 1b. Two bands were visualized in the gel. Band A was assigned to RT-PCR product of Cx43 by the expected molecular mass of 588 bp and by its comigration with Cx43 size marker (Fig. 1b, top). It was further verified to be an RT-PCR product of Cx43 by Southern blot analysis with Cx43 probe (band A, Fig. 1b). Band B has the same molecular mass as the pAW109 RNA size marker and was identified as an RT-PCR product of pAW109 RNA.


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  		Fig. 1.   Macrodissection and RT-PCR. a: Diagram of kidney illustrating the regions of kidney analyzed by RT-PCR. C, cortex; M, medulla; OP, outer papilla; IP, inner papilla. b: Coamplification of connexin 43 (Cx43; 588 bp) with pAW109 (308 bp) for semiquantitative analysis of Cx43 mRNA expression in cortex, medulla, outer papilla, inner papilla, and glomerular preparation. Top: ethidium bromide-stained agarose gel; bottom: autoradiogram of corresponding Southern blot. G, glomerular preparation. c: Results from densitometer scanning. Data shown as means ± SD. Data analyzed by Kruskal-Wallis test pairwise comparisons showed significant differences at P = 0.05 or less between different groups in the following order: outer medulla < cortex < glomerular preparation < outer papilla < inner papilla. y-Axis: ratio (Cx43/pA W109). S, molecular size marker; S1, Cx43 size marker; S2, pAW109 size marker; band A, Cx43; band B, internal standard.

The relative amount of Cx43 mRNA in the total RNA from isolated outer papilla, inner papilla, cortex, outer medulla, and glomerular preparation was estimated from their RT-PCR products. Coamplification of Cx43 mRNA from tissue RNA and pAW109 RNA was performed in the exponential phase to avoid the "tube effect" of PCR (17). As shown in Fig. 1b, top, Cx43 mRNA expression in inner papilla was most prominent among the regions studied. The semiquantitative analysis of Cx43 expression was done by densitometric scanning of the Cx43 bands and pAW109 bands, and the density ratio of the Cx43 band to the pAW109 band was used as a measure for Cx43 mRNA expression. Inner papilla showed the highest level of Cx43 mRNA (11.1 ± 3.12). Outer papilla and glomerular preparation ranked second (7.11 ± 1.86 and 6.51 ± 1.74), whereas the cortex and medulla expressed the least (1.89 ± 0.57 and 1.32 ± 0.79; Fig. 1c). Controls performed without tissue RNA, using procedures identical to those for RT-PCR of tissue RNA, gave no Cx43 band and verified the absence of contamination in the experiments.

RT-PCR for Microdissected Glomeruli and Tubular Segments

Figure 2a shows a typical pattern of the agarose gel analysis of the RT-PCR products from microdissected glomeruli and tubular segments. Band A was identified as a Cx43 RT-PCR product by restriction mapping. Band A was ~588 bp in length. When digested by Hinc II, two fragments of 521 and 67 bp appeared; digestion by Sty I generated fragments of 233 and 355 bp; mixed digestion by the two enzymes resulted in three fragments of 67, 288, and 233 bp (Fig. 2c). The restriction pattern is identical to the predicted restriction map of the Cx43 RT-PCR product schemed in Fig. 2d. Band B (Fig. 2a) was 309 bp in length, the same as the predicted length of the segment defined by the PCR primers for GAPDH, and was characterized as the RT-PCR product of GAPDH. The two negative controls, one lacking reverse transcription and the other containing no tissues, showed no RT-PCR products (data not shown), verifying that the PCR products were amplified from mRNA, not from genomic DNA, and the microdissection and RT-PCR were not contaminated.


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  		Fig. 2.   Microdissection and RT-PCR. a: Typical microdissection and RT-PCR results shown on ethidium bromide-stained agarose gel. PCT, proximal convoluted tubule; PST, proximal straight tubule; DCT, distal convoluted tubule; MTAL, outer medullary thick ascending limb; CCD, cortical collecting duct; IMCD, inner medullary collecting duct. b: Results from densitometer scanning. Data shown as means ± SD. Data analyzed by repeated-measures analysis of variance showed significant differences at P = 0.01 or less between different groups in the following order: IMCD and G > CCD > PCT and PST > MTAL and DCT. y-Axis: ratio (Cx43/GAPDH). c: Ethidium bromide-stained polyacrylamide gel showing restriction mapping of Cx43 RT-PCR product from microdissected IMCD. S1, Cx43 RT-PCR product; Hinc II, digested by restriction enzyme Hinc II; Sty I, digested by Sty I; Hinc II + Sty I, digested by the 2 enzymes. d: Predicted fragments of Cx43 RT-PCR product digested by restriction enzymes. 1: Cx43 PCR fragment; 2: digested by Hinc II; 3: digested by Sty I; 4: digested by Hinc II and Sty I.

It is shown clearly in Fig. 2a that the CCD and IMCD did express Cx43 to a significant level. To estimate the expression level in the microdissected tissues, the bands in Fig. 2a were scanned in a densitometer, and the data from five different experiments were averaged and graphed in Fig. 2b. Briefly, glomeruli expressed a moderate amount of Cx43. (Ratio of Cx43 to GAPDH is 3.94 ± 0.49.) In PCT, PST, DCT, and MTAL, nondetectable to minimum expression of Cx43 was evidenced (ratios of Cx43 to GAPDH: 0.66 ± 0.29 for PCT; 0.72 ± 0.49 for PST; 0.25 ± 0.16 for DCT; 0.34 ± 0.30 for MTAL). The CCD and IMCD expressed a significantly higher amount of Cx43 mRNA compared with other tubules (ratios of Cx43 to GAPDH: 5.39 ± 1.65 for IMCD and 1.54 ± 0.59 for CCD).

Immunocytochemistry

The punctate immunofluorescence characteristic of gap junction (GJI) was observed in all major regions of the kidney. It was observed in the renal vasculature. In the renal cortex, GJI is observed in the wall of the interlobular arteries and glomerular arterioles. There is a concentration of GJI in the EM (Fig. 3a). In the outer medulla, the efferent arterioles of the juxtamedullary nephrons branch into the vascular bundles where the GJI is particularly abundant (Fig. 3b). Abundant punctate immunofluorescence was observed in the medullary collecting ducts (Fig. 3, c and d). It was strikingly abundant in the IMCDs. Punctate immunofluorescence analogous to that seen in the vasculature was not seen in other tubules. Although fluorescence was seen in occasional proximal tubules, it appeared less intense than that interpreted as GJI. In any event, the presence of GJI in tubules other than the collecting ducts could not be entirely excluded. The epithelium lining the pelvic surface of the papilla was also rich in punctate immunofluorescence as were the inner layers of the pelvic epithelium. No definite punctate immunofluorescence was seen in the superficial cell layer.


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  		Fig. 3.   a: Punctate gap junction immunofluorescence for Cx43 in the extraglomerular mesangium (arrow). b: Gap junction immunofluorescence in the vascular bundles. c: Punctate immunofluorescence for Cx43 in the collecting ducts in outer papilla. d: Punctate immunofluorescence in the inner papillary collecting ducts (ducts of Bellini) and papillary epithelium.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Very little is known about the distribution of the different connexins in the kidney. In one of the few studies available, Cx43 immunofluorescence has been shown in the rat to be present in all tubules, and some immunofluorescence was seen in the glomeruli and in the connective tissue (5). More recently, abundant Cx43 GJI localization in the wall of the renal arterial and arteriolar vasculature, EM, vascular bundles, and medullary collecting ducts and pelvic epithelium showed punctate immunofluorescence consistent with gap junctions (1). In the same study, spots of fluorescence could be observed in some proximal tubules, but they were limited to occasional tubules, were not as intense as that characteristic of GJI, and were not consistently seen. Thus interpretation of the proximal tubular fluorescence in those preparations was difficult.

The discrepancy between the results of different immunocytochemical investigations of the localization of Cx43 in the kidney led to this study of the distribution of Cx43 mRNA in microdissected renal structures. Correlation between immunocytochemical and RT-PCR results was used to clarify the distribution of Cx43 in the structures of the kidney. The relatively high concentration of Cx43 RT-PCR product in glomerular preparations and microdissected glomeruli correlates with reported histochemical localization for Cx43 and ultrastructural data, indicating a high concentration of gap junctions in the EM of the juxtaglomerular apparatus (JGA). The EM contains no capillaries, and its location in the JGA makes it a required pathway for the macula densa message to reach the vascular effector component in the tubuloglomerular mechanism in the control of glomerular filtration rate or in the macula densa control of renin secretion. The absence of capillaries in the EM is consistent with the observation of large numbers of gap junctions in avascular structures such as the lens or the ovarian follicles where they are thought to have a role in maintaining the homeostasis (4). In the EM they may play such a role in addition to that of transmitting signals between the components of the JGA. The EM extends into the glomerulus, it is continuous with the glomerular mesangium, and gap junctions have been shown ultrastructurally between the EM and glomerular tuft (7, 8, 25, 32). A high concentration of gap junctions has been observed ultrastructurally in both sections of the mesangium and in the JGA (7, 8, 23, 36). The borders separating the extraglomerular from the intraglomerular mesangium are arbitrary and impossible to establish even by electron microscopy.

In contrast to the high concentration of RT-PCR product observed in the glomerular preparations, the concentration of RT-PCR product was relatively low in the total cortex. This finding is also consistent with the finding of very low levels of RT-PCR products in the microdissected renal tubules except for the CCD and IMCD. The renal mass in the cortex is formed predominantly by proximal renal tubules, which showed little Cx43 GJI and RT-PCR products. A similar case can be made for the low concentration of Cx43 RT-PCR products in the outer medulla where glomeruli are absent and tubules formed the majority of the tissue mass. Cx43 GJI was found only in the vascular bundles and arterial and arteriolar vessels.

The high concentration of Cx43 RT-PCR product in the papilla and the IMCD correlated well with the immunocytochemical observations showing abundant punctate immunofluorescence for Cx43 in the collecting ducts (1). In the outer papilla where the collecting ducts are interspersed with abundant ascending loop limbs, the concentration of Cx43 RT-PCR products was less than in the inner papilla, which is mainly populated by large collecting ducts interspersed with only a sparse number of loop limbs (23). The presence of Cx43 and GJI in the IMCD could be related to its homogeneous cellular composition and suggests that IMCD function is more integrated and homogenous than that of the rest of the collecting duct, which contains a heterogenous cell population. It is also consistent with the finding of a high concentration of the same type of punctate immunofluorescence in the pelvic epithelium. This similarity may be related to their anatomical continuity and their common embryological origin.

A significant result from the microdissection and RT-PCR experiments is that they showed convincingly that IMCD expressed Cx43 mRNA at the most abundant level among the microdissected tubular segments and glomeruli. This finding is consistent with the reported localization in the collecting duct of mRNA for Cx43 by in situ hybridization (27). Together with the immunocytochemical data (1) it also supports the notion that the collecting duct not only expresses Cx43 mRNA but that the mRNA is also translated to Cx43 protein. Most tubules, such as PCT, PST, DCT, and MTAL, express much less Cx43 than the collecting duct, showing that the amount of expression of Cx43 mRNA is tissue specific. The high concentration of Cx43 RT-PCR product in the IMCD explains the origin of the abundant Cx43 mRNA expression in the papilla from macrodissection experiments in this study.

The presence of gap junctions in the collecting ducts has not been, in spite of extensive freeze-fracture and transmission electron microscopy studies, conclusively demonstrated ultrastructurally (23, 31). There is at least one report of gap junctions seen in the collecting ducts. They were observed in association with tight junctions (34). However, no ultrastructural illustrations of gap junctions in the collecting ducts are, to our knowledge, available in the literature. This association has been reported to exist in a variety of tissues, such as the epithelial cells of the lung, where small clusters of particles interpreted as connexons have been shown in close association with tight junctions (2, 3, 18). Some authors, however, investigating the effects of taurine and nitrogen dioxide injury, using freeze-fracture electron microscopy, in the alveolar epithelia of the lung have been unable to observe gap junctions in the alveolar epithelium of the normal lung (18). It is of interest that, in the lung alveolar epithelium as in the collecting duct, no gap junctions have been seen by transmission electron microscopy. In spite of the morphological uncertainties, evidence of gap junction communication and the expression of a number of connexins have been shown in alveolar epithelial cells in culture (25). The abundance of both punctate Cx43 GJI and Cx43 mRNA in the IMCD permits the speculation that collecting ducts may contain Cx43 connexons as part of gap junctions. These gap junctions could be particularly small and obscured by their association to the highly developed tight junctions seen in the collecting ducts. They perhaps could be transient and, as a result, have escaped ultrastructural identification. Another possibility to explain this apparent discrepancy is that Cx43 in the collecting duct may be present but not incorporated into gap junctions.

In summary, this study shows a high concentration of Cx43 mRNA by RT-PCR in the microdissected glomeruli and IMCDs relative to the concentration of Cx43 mRNA in other parts of the renal tubule. These results correlate well with those obtained by macrodissection of renal regions and the immunocytochemical observations of Cx43 in the kidney.

Perspectives

The immunocytochemical and RT-PCR data demonstrate a high concentration of Cx43 GPI in the EM and of Cx43 mRNA in the microdisected glomeruli and glomerular preparations. This result suggests that Cx43 is a likely participant in the function of the JGA and consequently in tubuloglomerular feedback control of the glomerular filtration rate and in the control of renin secretion. The role of that connexin in the function of the IMCD is harder to explain, since, at this time, gap junctions have not been convincingly demonstrated ultrastructurally in that part of the tubule. The presence of punctuated immunofluorescence for Cx43 and high concentration of Cx43 mRNA suggest that the IMCD not only expresses Cx43 mRNA but that the mRNA is translated to protein. The reason for the difficulty in demonstrating gap junctions in this part of the tubule remains unclear. However, this situation has been found to exist in other organs (18, 26), and the various explanations offered for this discrepancy remain to be investigated. Little is known about the distribution of the other connexins in the kidney. Immunocytochemical studies indicate that Cx32 and Cx26 are preferentially located in the tubules (5, 33, 39). The localization of the rest of the connexins expressed in the kidney is unknown. The results reported here represent the first investigation, to our knowledge, of the distribution of a connexin using RT-PCR of microdissected renal structures. They show that the use of this method provides valuable information as to where the connexins are preferentially located in the kidney. Application of this method to the localizations of the other connexins present in the kidney is in progress.

    ACKNOWLEDGEMENTS

We express special thanks to Dr. Qi-Feng Ma for many suggestions during the performance of this work.

    FOOTNOTES

These studies were supported by National Heart, Lung, and Blood Institute Grant Ro1 18340.

This paper was presented at the 30th Annual Meeting of the American Society of Nephrology, San Antonio, TX, November 2-5, 1997, and has been published as an abstract (J. Am. Soc. Nephrol. 8: 346, 1997).

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: L. Barajas, Dept. of Pathology, Harbor-UCLA, Medical Center, 1000 West Carson St., Torrance, CA 90509.

Received 23 February 1998; accepted in final form 31 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Barajas, L., L. Liu, and M. Tucker. Localization of connexin 43 in rat kidney. Kidney Int. 46: 621-626, 1994[Medline].

2.   Bartels, H. The air-blood barrier in the human lung, a freeze-fracture study. Cell Tissue Res. 198: 269-285, 1979[Medline].

3.   Bartels, H., H.-J. Oestern, and G. Voss-Wermbter. Communicating-occluding junction complexes in the alveolar epithelium. Am. Rev. Respir. Dis. 121: 1017-1024, 1980[Medline].

4.   Beyer, E. C. Gap junctions. Int. Rev. Cytol. 137C: 1-37, 1993.

5.   Beyer, E. C., J. Kistler, D. L. Paul, and D. A. Goodenough. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J. Cell Biol. 108: 595-605, 1989[Abstract/Free Full Text].

6.   Beyer, E. C., D. L. Paul, and D. A. Goodenough. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J. Cell Biol. 105: 2621-2629, 1987[Abstract/Free Full Text].

7.   Biava, C. G., and M. West. Fine structure of normal human juxtaglomerular cells. Am. J. Pathol. 49: 679-721, 1966[Medline].

8.   Boll, H.-U., W. G. Forssmann, and R. Taugner. Studies on the juxtaglomerular apparatus. Cell Tissue Res. 161: 459-469, 1975[Medline].

9.   Burlington, H., and E. P. Cronkite. Characteristics of cell cultures derived from renal glomeruli. Proc. Soc. Exp. Biol. Med. 142: 143-149, 1973[Medline].

10.   Chelly, J., J. C. Kaplan, P. Maire, S. Guatron, and A. Kahn. Transcription of the dystrophin gene in human muscle and non-muscle tissues. Nature 333: 858-860, 1988[Medline].

11.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

12.   Dunn, O. J., and V. A. Clark. Applied Statistics: Analysis of Variance and Regression. New York: Wiley, 1986.

13.   Feinsberg, A. P., and B. Vogelstein. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13, 1983[Medline].

14.   Feinsberg, A. P., and B. Vogelstein. A technique for radiolabeling DNA restriction endonuclease fragments in high specific activity. Anal. Biochem. 137: 266-267, 1984[Medline].

15.   Fort, P., L. Marty, M. Picchaczyk, S. E. Sabrouly, C. Dani, P. Jeanteur, and J. M. Blanchard. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate dehydrogenase multigene family. Nucleic Acids Res. 13: 1431-1442, 1985[Abstract/Free Full Text].

16.   Fuqua, S. A. W., S. D. Fitzgerald, and W. L. McGuire. A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts. Biotechniques 9: 206-211, 1990[Medline].

17.   Gilliland, G., S. Perrin, K. Blanchard, and H. F. Bunn. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA 87: 2725-2729, 1990[Abstract/Free Full Text].

18.   Gordon, R. E., R. F. Heller, J. R. DelValle, and R. F. Heller. Membrane perturbations and mediation of gap junction formation in response to taurine treatment in normal and injured alveolar epithelia. Exp. Lung Res. 15: 895-908, 1989[Medline].

19.   Guo, R., L. Liu, and L. Barajas. A RT-PCR study of the distribution of connexin 43 mRNA in the glomerulus and renal tubular segments (Abstract). J. Am. Soc. Nephrol. 8: 346, 1997.

20.   Haefliger, J. A., R. Bruzzone, N. A. Jenkins, D. J. Gilbert, N. G. Copeland, and D. L. Paul. Four novel members of the connexin family of gap junction proteins; molecular cloning, expression, and chromosome mapping. J. Biol. Chem. 267: 2057-2064, 1992[Abstract/Free Full Text].

21.   Hennemann, H., H.-J. Schwarz, and K. Willecke. Characterization of gap junction genes expressed in F9 embryonic carcinoma cells: molecular cloning of mouse connexin31 and -45 cDNAs. Eur. J. Cell Biol. 57: 51-58, 1992[Medline].

22.   Innis, M. A., G. H. Gelfand, J. J. Sninsky, and T. J. White. PCR Protocol: A Guide to Methods and Applications. San Diego, CA: Academic, 1990, p. 60-69.

23.   Kaissling, B., and W. Kriz. Structural analysis of the rabbit kidney. In: Advances in Anatomy Embryology and Cell Biology, edited by A. Brodal, W. Hild, J. Van Limborgh, R. Ortmann, T. H. Schiebler, G. Töndury, and E. Woff. New York: Springer-Verlag, 1979, vol. 56, p. 1-123.

24.   Larson, D. M., C. Haudenschild, and E. C. Beyer. Gap junction messenger RNA expression by vascular wall cells. Circ. Res. 66: 1074-1080, 1990[Abstract/Free Full Text].

25.   Lee, Y.-C., C. E. Yellowley, Z. Li, H. J. Donahue, and D. E. Rannels. Expression of functional gap junctions in cultured pulmonary alveolar epithelial cells. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L1105-L1114, 1997[Abstract/Free Full Text].

26.   Li, Z., Z. Zhou, and E. E. Daniel. Expression of gap junction connexin 43 and connexin 43 mRNA in different regional tissues of intestine in dog. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G911-G916, 1993[Abstract/Free Full Text].

27.   Micevych, P. E., and L. Abelson. Distribution of mRNAs coding for liver and heart gap junction proteins in the rat central nervous system. J. Comp. Neurol. 305: 96-118, 1991[Medline].

28.   Moriyama, T., H. R. Murphy, B. M. Martin, and A. Garcia-Perez. Detection of specific mRNAs in single nephron segments by use of the polymerase chain reaction. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1470-F1474, 1990[Abstract/Free Full Text].

29.   Paul, D. L. Molecular cloning of cDNA for rat liver gap junction protein. J. Cell Biol. 103: 123-143, 1986[Abstract/Free Full Text].

30.   Paul, D. L., L. Ebihara, L. J. Takemoto, K. I. Swenson, and D. A. Goodenough. Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J. Cell Biol. 115: 1077-1089, 1991[Abstract/Free Full Text].

31.   Pricam, C., F. Humbert, and A. Perrelet. A freeze-etch study of the tight junctions of the rat kidney. Lab. Invest. 30: 286-291, 1974[Medline].

32.   Pricam, C., F. Humbert, A. Perrelet, and L. Orci. Gap junctions in mesangial and lacis cells. J. Cell Biol. 63: 349-354, 1974[Free Full Text].

33.   Sainio, K., S. F. Gilbert, E. Lehtonen, M. Nishi, N. M. Kumar, N. B. Gilula, and L. Saxen. Differential expression of gap junction mRNAs and proteins in the developing murine kidney and in experimentally induced nephric mesenchymes. Development 115: 827-837, 1992[Abstract].

34.   Shibata, Y., T. Izumi, and T. Yamamoto. Tissue-specific granularity of gap junction cytoplasmic surfaces revealed by rapid-freeze, deep-etch replicas. Anat. Rec. 223: 113-120, 1989[Medline].

35.   Sprint, P. Applied Non-parametric Statistical Methods. New York: Chapman and Hall, 1989, p. 112-115.

36.   Taugner, R., A. Schiller, B. Kaissling, and W. Kriz. Gap junctional coupling between the JGA and the glomerular tuft. Cell Tissue Res. 186: 279-285, 1978[Medline].

37.   Tereda, Y., K. Tomita, H. Nonoguchi, and F. Marumo. PCR localization of angiotensinogen mRNAs in rat kidney. Kidney Int. 43: 1251-1259, 1993[Medline].

38.   Todd-Turla, K. M., J. Schnermann, G. Fejes-Toth, A. Naray-Fejes-Toth, A. Smart, P. D. Killen, and J. P. Briggs. Distribution of mineralocorticoid 1993 receptor mRNA along the nephron. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F781-F791, 1993[Abstract/Free Full Text].

39.   Traub, O., J. Look, R. Dermietzel, F. Brummer, D. Hulser, and K. Willecke. Comparative characterization of the 21-lD and 26-kD gap junction proteins in murine liver and cultured hepatocytes. J. Cell Biol. 108: 1039-1051, 1989[Abstract/Free Full Text].

40.   Tucker, M., and L. Barajas. Rat connexins 30.3 and 31 are expressed in the kidney. Exp. Cell Res. 213: 224-230, 1994[Medline].

41.   White, T. W., R. Bruzzone, and D. L. Paul. The connexin family of intercellular channel forming proteins. Kidney Int. 48: 1148-1157, 1995[Medline].

42.   Wright, P. A., M. B. Burg, and M. A. Knepper. Microdissection of kidney tubules segments. In: Methods in Enzymology: Biomembranes, edited by S. Fleisher, and B. Fleisher. New York: Academic, 1990, vol. 101, pt. 5, p. 226-231.

43.   Zhang, J.-T., and B. J. Nicholson. Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J. Cell Biol. 109: 3391-3401, 1989[Abstract/Free Full Text]. Legends

Am J Physiol Regul Integr Compar Physiol 275(2):R439-R447
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


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