Connexin 30.3 Is Expressed in the Kidney But Not Regulated by Dietary Salt or High Blood Pressure

Abstract

Several isoforms of connexin (Cx) proteins have been identified in a variety of tissues where they play a role in intercellular communication, either as the components of gap junctions or as large, nonselective pores known as hemichannels. This investigation seeks to identify the localization and regulation of Cx30.3 in mouse, rat, and rabbit kidney using a Cx30.3^+/lacZ transgenic approach and immunofluorescence. Cx30.3 was detected in all three species and predominantly in the renal medulla. Both the nuclear lacZ staining indicative of Cx30.3 expression and indirect immunohistochemistry provided the same results. Cx30.3 immunolabeling was mainly punctate in the mouse, typical for gap junctions. In contrast, it showed continuous apical plasma membrane localization in certain tubule segments in the rat and rabbit kidney, suggesting that it may also function as hemichannels. In the cortex, Cx30.3 was localized in the intercalated cells of the cortical collecting duct, because the immunoreactive cells did not label for AQP2, a marker for principal cells. In the medulla, dense Cx30.3 staining was confined to the ascending thin limbs of the loop of Henle, because the immunoreactive cells did not label for AQP1, a marker of the descending thin limbs. Immunoblotting studies indicated that Cx30.3 expression was unchanged in response to either high or low salt intake or in spontaneously hypertensive rats. Cx30.3 appears to be constitutively expressed in certain renal tubular segments and cells and its role in overall kidney function remains to be investigated.

Keywords:

• gap junctions
• hemichannel
• immunofluorescence
• intercalculated cells
• loop of Henle

INTRODUCTION

The connexin (Cx) family of proteins is comprised of a group of structurally related, transmembrane proteins found in a diverse range of cell types. Individual Cx proteins form hexameric structures known as connexons. In adjacent cells, these connexons form gap junctions, which participate in intercellular communication between the two cells by allowing the passage of ions, metabolites, and secondary messengers up to 1 to 2 kDa in size (Spray 2006). There is also evidence that Cx proteins may remain in the cell membrane as single hexamers or hemichannels (Ebihara 2003). If the hemichannel is localized to a membrane that does not form a junction with another cell, it allows for the transport of molecules between the cell and the extracellular environment. The opening of the channel is regulated by several factors, including pH, [Ca^{2 +}], and metabolic and mechanical stresses (Trexler et al. 1999; John et al. 1999; Gomes et al. 2005). A number of studies have indicated that hemichannels maybe capable of releasing ATP into extracellular fluid and could therefore participate in purinergic signaling (John et al. 1999; Cotrina et al. 1998).

Several Cx isoforms have been identified in the kidney including Cx30, Cx37, Cx40, Cx43, Cx45 (Arensbak et al. 2001; Barajas et al. 1994; McCulloch et al. 2005; Butterweck et al. 1994), and Cx40 was recently found to play an important role in renal hemodynamics (Wagner et al. 2007). Previous studies have identified Cx30.3 mRNA in the kidney (Tucker et al. 1994); however, Cx30.3 protein localization is yet to be established. Cx30.3 has mostly been associated with the skin disease erythrokeratodermia variabilis (Common et al. 2005). Recent work by Zheng-Fischhöfer utilized a transgenic mouse model where the Cx30.3 gene was replaced with the lacZ reporter gene to study its expression in the skin, olfactory organs as well as in the kidney (Zheng-Fischhöfer et al. 2007). This study found evidence that Cx30.3 was expressed at least in the inner medulla of the kidney, specifically in cells of the thin ascending limb of the loop of Henle. However, detailed renal and intracellular localization of Cx30.3 has not been investigated.

Here we report that Cx30.3 is expressed at both the mRNA and protein levels in the kidney of mice, rats and rabbit, and we characterize in detail its renal localization in these species using both a Cx30.3^+/lacZ transgenic approach and Cx30.3 immunohistochemistry.

MATERIALS AND METHODS

Animals

New Zealand white rabbits (500 g; Irish Farm, Norco, CA) were maintained on standard diet with normal water. Sprague-Dawley rats, spontaneously hypertensive rats (SHRs) and C57Bl/6 mice were age and weight matched for all experiments. Transgenic mice with the coding region of the Cx30.3 gene replaced by the lacZ reporter gene with a nuclear localization signal (NLS-lacZ) under the control of the Cx30.3 promoter were developed and previously described by Zheng-Fischhöfer et al. (2007). The physiological parameters of the SHR animals, including blood pressure measurements have been previously published (Yang et al. 2007). All animal protocols have been approved by the Institutional Animal Care and Use Committee at the University of Southern California.

Salt-Adjusted Diet

Male Sprague-Dawley rats (200 g; Harlan, Madison, WI) were fed standard chow (0.3% NaCl), high salt (TD 92012: 8% NaCl; Harlan) or low salt (TD 90228: 0.01% NaCl; Harlan) rodent diet for 1 week. Rats fed the high salt diet also received 0.45% NaCl (w/v) containing drinking water.

M1 Cells

The M1 cell line was previously characterized (Fejes-Toth et al. 1992) and have a phenotype representative of both the intercalated and principal cells of the collecting duct. Cells were purchased from American Type Culture Collection (Manassas, VA).

Antibodies

Rabbit polyclonal anti-Cx30.3 antibodies were purchased from Zymed Laboratories (San Francisco, CA). A mouse monoclonal anti-β-actin antibody was purchased from Abcam (Cambridge, MA) and a mouse monoclonal anti-villin antibody was purchased from Immunotech (Chicago, IL). The mouse monoclonal anti-aquaporin 2 (AQP2) antibody, kindly provided by Dr. Mark Knepper, was from the original batch of immunization and was characterized in a previous publication (Nielsen et al. 1993). A mouse monoclonal antibody against aquaporin 1 (AQP1) was purchased from Novus Biologicals (Littleton, CO).

Genotyping of Transgenic Mice

DNA was extracted from mouse tail tips using the ZR Genomic DNA II kit according to the manufacturer's protocol (Zymo Research, Orange, CA). Two microliters of purified DNA was amplified by PCR using a master mix containing Taq polymerase (Platinum PCR kit, Invitrogen, Carlsbad, CA) and the following primers: Cx30.3 wild-type sense: 5′-GGCCAAGGTTCAAGACCACCTGTG-3′; LacZ sense 5′-AACGACGGGATCATCGCGAGCCAT-3′Cx30.3; antisense (shared): 5′-CCCCTCTTCTTGCTCAGGTTGCTG-3′. Primers sequences were previously published (Zheng-Fishhöfer et al. 2007). The PCR reaction was carried out for 45 cycles of the following: 94°C for 30 s, 60°C for 45 s, and 72°C for 60 s. The PCR product was analyzed on a 2% agarose gel stained with ethidium bromide to identify fragments of approximately 672 bp for the wild-type allele and 359 bp for the mutant allele.

Immunoblotting of Mouse and Rat Tissue

Mice and rats were anesthetized with 100 mg/ml Inactin and kidneys were perfused retrogradewith ice-cold phosphate-buffered saline (PBS) to remove blood. Slices of whole kidney (mouse) or cortex (rat) were manually dissected and tissue was homogenized with a rotor-stator homogenizer in a buffer containing 20 mM Tris-HCl 1 mM EGTA, pH 7.0, and a protease inhibitor cocktail (BD Bioscience, San Jose, CA). Samples were centrifuged at low speed to pellet cellular debris and supernatant was collected and assayed. 40 μ g of protein were separated per lane, along with a protein standard molecular weight marker (BioRad, Hercules, CA) on a 4% to 20% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then transferred to a polyvinyledene difluoride membrane (Millipore, Billerica, MA). After blocking the membrane in blocking buffer (Li-Cor, Lincoln, NE), immunoblotting was performed with the rabbit polyclonal antibodies to Cx30.3 (Zymed) at a dilution of 1:2500 overnight. A mouse monoclonal antibody to β-actin (Abcam) (1:2500 dilution) was used for a loading control for salt-adjusted samples. A mouse monoclonal anti-villin antibody (Immunotech) (1:1000 dilution) was used as a loading control for SHR samples as has been previously published (Yang 2007). Reactivity of the primary antibodies was detectedby either IR680-labeled goat anti-rabbit or IR800-labeled goat anti-mouse (1:15,000 dilutions; Li-Cor) secondary antibodies. Blots were imaged and quantified using Odyssey Infrared ImagingSystem (Li-Cor) and accompanying software. The data were then normalized against the control sample and an average for each group was calculated. Statistical significance was tested using an unpaired t test and data are shown as mean ± SE.

LacZ Staining of Kidney Tissue

Kidneys from Cx30.3^+/lacZ mice and wild-type littermates were frozen on dry ice, embedded in Tissue-Tec (Sakura, Zoeterwoude, The Netherlands), sectioned (10 to 20 mm) on a cryostat (MICROM HM500 OM), and transferred onto superfrost plus slides (Menzel, Braunschweig, Germany). Sections were fixed with 0.2% glutaraldehyde in PBS, rinsed three times in lacZ washing buffer (0.1M phosphate buffer, pH 7.4, 1.25 mM MgCl₂, 5 mM EGTA, 0.2% Nonidet P-40, 0.01% sodium deoxycholate), and stained in lacZ substrate buffer overnight (lacZ washing buffer supplemented with 0.4 mg/ml X-Gal [5-brom-4-chloro-3-indolyb-d-galactopyranoside], 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide) at 37°C. Sections were then washed in PBS, stained in 0.1% eosin for 5 min, rinsed in water, and mounted in Entellan (Merck, Darmstadt, Germany).

Immunofluorescence Labeling of Kidney Tissue

Kidneys were fixed in situ by perfusion of 4% paraformaldehyde. Coronal kidney sections containing all kidney zones were then post-fixed overnight at 4°C in 4% paraformaldehyde and then embedded in paraffin. Subsequently, 4-μ m sections of the paraffin block were deparaffinized in toluene and rehydrated through graded ethanol. To retrieve antigens, slides were heated for 2 × 10 min in a microwave with medium heat in PBS and allowed to cool for 40 min. Sections were then incubated for 30 min with 5% normal goat serum in PBS to block nonspecific binding. An additional block with goat anti-rabbit Fab immunoglobulin G (IgG) for rabbit tissue (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA) for 40 min was used to reduce nonspecific binding with rabbit polyclonal antibodies. Rat and rabbit sections were then incubated with Cx30.3 antibodies overnight at a 1:50 dilution and washed in PBS. Sections were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and enhanced with Alexa Fluor 594–labeled tyramide signal amplification (TSA) according to the manufacturer's instructions (Molecular Probes, Eugene, OR). Some rat tissue sections were double-labeled with an anti-AQP2 monoclonal antibody overnight at a 1:50 dilution and the secondary and TSA steps were repeated as above, except using a HRP-conjugated goat anti-mouse IgG and Alexa Fluor 488 TSA (Molecular Probes). A similar method was employed for mouse sections, however primary antibody incubation was limited to one hour and TSA amplification was not utilized. Additionally, some mouse sections were double-labeled with an anti-AQP1 monoclonal antibody for 1 hour (1:100 dilution). All antibody dilutions and incubation times were experimentally determined. Following a final wash step, all sections were mounted with Vectashield mounting medium containing the nuclear stain DAPI (Vector Laboratories) and examined with a Leica TCS SP2 confocal microscope. All sections were labeled in parallel.

Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)

Total RNA was purified from whole mouse kidney samples or confluent M1 cells using a Total RNA Mini Kit in accordance with manufacturer's instructions (Biorad, Hercules, CA). RNA was then quantified using spectrophotometry and reverse-transcribed to single-stranded cDNA using avian reverse transcriptase and random hexamers according to manufacturer's instructions (Thermoscript RT-PCR system, Invitrogen). Two microliters of cDNA was amplified using a master mix containing Taq polymerase (Invitrogen) and the following primers: Connexin 30.3 sense, 5′GGCCAAGGTTCAAGACCACCTGTG-3′; connexin 30.3 antisense, 5′-CCCCTCTTCTTGCTCAGGTTGCTG-3′; β-actin sense, 5′-GGTGTGATGGTGGGAATGGGTC-3′, β-actin antisense 5′-ATGGCGTGAGGGAGAGCATAGC-3′; each at a final concentration of 200 μ M. Connexin 30.3 and β-actin oligonucleotides were based on previously published primer sequences (Zheng-Fischhöfer et al. 2007; McCulloch et al. 2005). The PCR reaction was carried out for 45 cycles of the following: 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The PCR product was analyzed on a 2% agarose gel stained with ethidium bromide to identify fragments of approximately 672 bp for connexin 30.3 and 350 bp for β-actin.

RESULTS

Detection of Cx30.3 mRNA and Protein in the Kidney

RNA was isolated from whole mouse kidney or from a mouse renal cell line (M1; mixed phenotype with both the principal and intercalated cells of the collecting duct) and using RT-PCR, the presence of Cx30.3 mRNA was detected (Figure 1A). The amplification of β-actin served as a positive control. We observed bands of the predicted size of 672 bp for Cx30.3 and 350 bp for β-actin in both tissue and cell culture samples.

Figure 1 Detection of connexin 30.3 (Cx30.3) mRNA (A) and protein (B) in the kidney. (A) mRNA isolated from mouse whole kidney (lane 1) or from a mouse renal collecting duct cell line (M1, lanes 3, 5) was amplified by RT-PCR to detect Cx30.3 and β-actin (lanes 2, 4, 6). Bands approximately at the predicted sizes of 672 and 350 bp, respectively, were observed in both samples. (B) Western blotting with Cx30.3 antibodies produced a band of approximately 37 kDa in wild-type (WT) mouse kidney homogenates, while tissue homogenates from Cx30.3 knockout mice (KO; n = 4 each) failed to produce a signal. Incubation with a β-actin antibody produced a band of approximately 42 kDa and confirmed equal protein loading amounts. (C) Western blotting with Cx30.3 antibodies produced a single band of approximately 37 kDa in wild-type mouse, rat and rabbit kidney homogenates (n = 2 each).

Immunoblotting kidney homogenate from wild-type mice with Cx30.3 antibodies produced a band around 37 kDa, as was expected. Tissue from the Cx30.3 knockout mouse was used as a negative control and when probed with the same antibodies it failed to produce detectable bands (Figure 1B). β-Actin was used as a control for loading (Figure 1B). Additional immunoblotting of mouse, rat, and rabbit kidney samples produced a single band around 37 kDa confirming specificity of the Cx30.3 antibodies in all three species studied.

Nuclear lacZ Reporter Gene Expression in Transgenic Cx30.3^+/lacZ Mouse Kidneys

The intrarenal localization of Cx30.3 was analyzed first using sections of heterozygous Cx30.3^+/lacZ adult mouse kidneys by staining for nuclear lacZ reporter gene expression (Figure 2). Wild-type kidney samples served as negative controls (Figure 2B, Figure 2F). LacZ staining was specific and most intense in the inner medulla (IM) followed by the weakly labeled outer medulla (OM) (Figure 2A). LacZ staining in the renal cortex was very weak (Figure 2A). Higher magnification revealed that the dense nuclear lacZ staining in the IM region was confined to long, thin-walled tubular structures reminiscent of the loop of Henle. Most cells of the large collecting ducts were not labeled (Figure 2C). Only a few, similar thin tubular structures showed lacZ staining in the OM region (Figure 2D). In the cortex, only a very few, select cells of branching tubular structures, suggestive of the collecting duct were positive for lacZ. Other tubules, the vasculature and glomeruli were negative (Figure 2E).

Figure 2 Renal Cx30.3 expression analyzed by staining for nuclear lacZ reporter gene expression (blue) in heterozygous Cx30.3+/lacZ adult mouse kidneys (A, C–E). (A, B) Cross section through the whole kidney. (A) The most intense lacZ staining was found in the inner medulla (IM). Labeling was weak in the outer medulla (OM) while the renal cortex (C) appeared to be devoid of lacZ staining. (B, F) Wild-type kidney samples served as negative controls. (C–F) High magnification of the IM (C), OM (D, F), and C (E) kidney regions. (C) In the IM, cell nuclei of thin tubular structures stained positive, whereas cells of the large collecting ducts (*) were not labeled. (D) In the OM, only a few tubular structures showed lacZ staining (*), but the dominant structure of the region, the medullary thick ascending limbs (mTAL) were not labeled. (E) In the cortex, only select cells of branching tubular structures, reminiscent of the collecting duct (*) were positive for lacZ. Other tubules, the vasculature and glomeruli (G) were negative. (F) High magnification of the OM region in a wild-type kidney shows no labeling. Bars: 100 μ m.

Immunolocalization of Cx30.3 in the Mouse Kidney

Immunofluorescence studies were conducted in kidney sections from both wild-type and Cx30.3 knockout mice to confirm the localization of Cx30.3 in the kidney and to further test the specificity of the Cx30.3 antibodies in immunohistochemical techniques. Specific labeling was detected in wild-type mouse sections (Figure 3A), because Cx30.3 knockout tissues were negative for antibody labeling (Figure 3C). In the mouse tissue, the pattern of Cx30.3 immunofluorescence was predominately punctate and intracellular (Figure 3A). In some tubules, however, Cx30.3 did appear to localize to the apical plasma membrane (Figure 3B). Consistent with the lacZ expression data above, the majority of the renal cortex, the vasculature, glomeruli, and proximal tubules were devoid of labeling. Only select cells of the cortical collecting duct were Cx30.3-positive (Figure 3A, Figure 3B). Significantly more structures were immunoreactive in the renal medulla, all of which appeared as thin-walled tubules with no red blood cells in their lumen. These tubules were later identified as the thin ascending limb of the loop of Henle, based on their lack of labeling for the water channel aquaporin 1 (AQP1), a marker of the descending thin limbs (Figure 3D). No other major structures were labeled in the renal medulla.

Figure 3 Detection of Cx30.3 in the mouse kidney by immunofluorescence (red). (A, B, D) Specific staining was observed in wild-type mouse sections labeled with Cx30.3 antibodies. (C) Labeling of Cx30.3 knockout mouse tissue failed to produce a signal. Mostly punctate and cytosolic labeling was observed in only a few cells in the renal cortex, in select cells of the cortical collecting duct (CCD) (A). Occasionally, some staining resembling apical membrane localization was also observed (B). Other cortical structures including the proximal tubule (PT) were devoid of staining. In the renal medulla, dense labeling of thin-walled tubular structures was found. The Cx30.3-positive tubules (*) did not label with AQP1 (green), a marker of the descending thin limb of the loop of Henle (#). Nuclei are stained with 4′,6′-diamidino-2-phenylindole (DAPI; blue). Bars: 10 μ m.

Immunolocalization of Cx30.3 in the Rat and Rabbit Kidney

Localization of Cx30.3 in the rat and rabbit kidney was studied by immunofluorescence of paraformaldehyde-fixed, paraffin-embedded kidney sections. The localization and pattern of Cx30.3 immunolabeling was essentially identical in these two species. Similar to the mouse, staining for Cx30.3 was observed mainly in the inner medulla. However, the pattern of labeling was different from the one observed in the mouse kidney. In addition to some punctate staining which is typical for gap junctions, majority of the Cx30.3 immunolabeling was continuous and localized to the apical plasma membrane of certain tubular segments. Like in the mouse, the renal cortex was mainly negative with the exception of a few, select cells of the cortical collecting duct (Figure 4A). These select Cx30.3-positive cells of the collecting duct were found in both the cortex and medulla and were later identified as intercalated cells, because they did not label for the apical membrane water channel aquaporin 2 (AQP2), a marker of the principal cells of the cortical collecting duct (CCD) (Figure 4B). Cx30.3 immunolabeling was highest in the inner medulla, and localized in thin-walled tubular structures (Figure 4C). Higher magnification with differential interference-contrast (DIC) overlay revealed continuous apical membrane labeling in these tubules (Figure 4D). Figure 5 summarizes the localization of Cx30.3 expression in different renal tubular segments/cells.

Figure 4 Immunofluorescence labeling of Cx30.3 in rat (A, B) and rabbit (C, D) kidney sections. In the rat cortex (A), apical membrane localization was apparent in select cells of the cortical collecting duct (CCD). No other cortical structures were labeled including the proximal tubule (PT). (B) In the rat outer medulla, the Cx30.3-positive cells of the CCD (arrow) did not label with AQP2 (green, arrowheads), a marker of the principal cells of the CCD. Fluorescent labeling was highest in the inner medulla, and localized in thin-walled tubular structures in the loop of Henle (C). Higher magnification with DIC overlay revealed apical membrane labeling in these tubular structures (*) (D). Most parts of the large medullary collecting ducts (#) were devoid of staining (C, D). Nuclei are stained with DAPI (blue). Bars: 10 μ m.

Figure 5 Schematic representation of Cx30.3-expressing nephron segments (bold line) in both the mouse, rat, and rabbit kidney. G: glomerulus; PT: proximal tubule; DTL: descending thin limb; ATL: ascending thin limb; TAL: thick ascending limb of the loop of Henle; DT: distal tubule; CNT: connecting tubule; CCD: cortical collecting duct; MCD: medullary collecting duct. Cx30.3 labeling was found in the ATL and in select (intercalated) cells of the CCD and MCD.

Cx30.3 Expression in Rats with Salt-Adjusted Diets

The effects of changes in dietary salt intake on Cx30.3 protein expression were examined in rat kidneys to help determine a functional role for Cx30.3. Animals were fed salt-adjusted diets and cortical kidney tissue homogenate samples from each rat experimental group were immunoblotted for Cx30.3 (Figure 6A). The amount of protein loaded was confirmed by concurrently probing the blots with β-actin (Figure 6B). Band intensity was quantified using densitometric analysis and normalized against protein loading amounts, as determined by β-actin levels (Figure 6C). The percent change from control was as follows: high salt: 1.6%; low salt: 16.8% (n = 4 rats per group). There was no statistically significant difference in Cx30.3 expression levels between the control group and either of the salt-adjusted diet groups.

Figure 6 Immunoblotting analysis of Cx30.3 expression in the rat kidney under various dietary salt conditions. (A) Representative Cx30.3 blots of rat cortical tissue from animals fed a control, high, or low salt diet (n = 4 rats per experimental group). (B) Blots were probed with β-actin to demonstrate even loading. Specific bands for Cx30.3 and β-actin were detected around 37 and 42 kDa, respectively. (C) Densitometric analysis of Cx30.3 expression. No significant difference between control and high salt (p = 0.92) or control and low salt groups was observed (p = 0.09). Shown is mean ± SE of four rats per experimental group.

Cx30.3 Expression in Sprague-Dawley Rats and SHRs

To determine whether hypertension had an effect on the expression of Cx30.3 in the rat kidney, samples from SHR rats were compared to Sprague-Dawley control samples (Figure 7A). Villin was used to demonstrate even protein loading (Figure 7B) before calculating Cx30.3 expression levels using densitometric analysis (Figure 7C). The percent change in Cx30.3 expression in SHR compared to SD rats was 5.3% (n = 6 per group). This difference was not statistically significant.

Figure 7 Immunoblotting analysis of Cx30.3 expression in the kidneys of normotensive (Sprague-Dawley, control) and hypertensive (SHR) rats. (A) A representative blot of control and SHR samples probed with Cx30.3. (B) The same blot probed with villin antibodies to demonstrate even loading. Specific bands for Cx30.3 and villin were detected around 37 and 92 kDa, respectively. (C) Densitometric analysis of Cx30.3 expression. No statistically significant difference in Cx30.3 expression was observed between the two groups (p = 0.85). Shown is mean ± SE of six rats per experimental group.

DISCUSSION

Here we report on the expression and localization of Cx30.3 in the kidneys of mice, rats, and rabbits. The protein was found in the kidney predominantly in the renal medulla, with the same level of expression and localized to the same nephron segments in each species (Figure 5). Cx30.3 immunolabeling was mainly punctate in the mouse, a pattern of labeling was expected, because connexins predominantly form gap junctions. In contrast, it showed continuous apical plasma membrane localization in certain tubule segments in the rat and rabbit kidney. Because this part of the cell membrane interfaces with the lumen of the tubule and is nonjunctional, these findings raise the possibility that Cx30.3 may also function as hemichannels.

Both the Cx30.3^+/lacZ transgenic and immunohistochemical approaches found essentially the same results, confirming their specificity. Both techniques identified the ascending thin limb of the loop of Henle and the intercalated cells of the collecting duct as the only two cell types within the kidney which express Cx30.3 (Figure 2, Figure 3, Figure 4). Specificity of the Cx30.3 antibody was also confirmed by the lack of immunoreactive signals in Cx30.3 knockout kidneys using either western blotting or immunohistochemistry (Figure 1 and Figure 3).

The apical membrane localization was found in the loop of Henle and the collecting duct, tubular segments that are both active in salt and water reabsorption, suggesting that Cx30.3 may contribute to this function. Cx30.3 was consistently present in intercalated cells, suggesting that it may be involved in acid/base homeostasis. To help ascertain a renal function for Cx30.3, kidney homogenate was first obtained from rats treated with high-or low-salt diets. Neither group showed a significant difference from control in the level of Cx30.3 expression, which was surprising, since then Cx30.3–expressing nephron segments, the loop of Henle and the collecting duct control salt reabsorption. This also marked a deviation from the renal expression seen with Cx30 (McCulloch et al. 2005), which showed similar expression patterns, but did show altered expression due to altered salt intake. Because the same modifications to dietary salt did induce changes in Cx30 expression, it appears that the unaltered expression of Cx30.3 is not due to a lack of sensitivity in our approach. We also examined kidney samples from SHRs because other groups have proposed a role for other connexins in hypertension and have noted changes in expression levels of some Cxs as a result (Rummery et al. 2002; Li et al. 2002, Wagner et al. 2007). Again, no significant deviation from the levels of Cx30.3 expression found in the control rat was observed. These results, however, do not exclude the possibility of functional changes in Cx30.3. One limitation of this technique is that whole cell immunoblots cannot detect changes in synthesis, processing, and transport of proteins within cells. It is possible that translocation of Cx30.3 between cytosolic and cell membrane pools could result in functional changes.

Although it is easy to suggest that Cx30.3 plays a role in renal physiology based on its localization, determining the mechanisms through which this occurs remains a challenge. One possible mechanism of action is through purinergic signaling, which is implicated in several renal functions, including ion transport (Unwin et al. 2003). For example, luminal levels of ATP are known to be high in the proximal part of the nephron including the loop of Henle (Vekaria et al. 2006). Given the significant amount of nucleotidase activity found along the apical membrane (Le Hir et al. 1993; Leipziger et al. 2003), it follows that ATP must be secreted along the luminal membrane to produce detectable levels in the tubular fluid (Vekaria et al. 2006). In CCDs, ATP has been shown to inhibit the small-conductance potassium (SK) channel of principal cells (Lu et al. 2000). Because purinergic signaling occurs in an autocrine/paracrine manner (Schwiebert et al. 2001), it follows that ATP would be released from neighboring intercalated cells. However, the exact mechanism of ATP release in the tubules has not yet been elucidated, although several candidates exist (Schwiebert et al. 2001). Among them are connexin hemichannels, which have been shown to release ATP in other cell types (Gomes et al. 2005; Cotrina et al. 1998). Given its localization in the ascending thin limb of the loop of Henle and the collecting ducts, Cx30.3 hemichannels could be involved in ATP release there.

Another criterion in determining whether Cx30.3 hemichannels are involved in renal physiology is the regulation of their conformation. It is clear that hemichannels cannot be constitutively open, but instead must be gated. If hemichannels do function in the kidney, it needs to be determined whether they can be opened under the (patho)physiological conditions. Several factors have been proposed to open hemichannels, including metabolic and mechanical stress (John et al. 1999; Gomes et al. 2005). Both factors are potentially at work in the kidney and it is interesting to speculate what influence they may have in the regions where we have found Cx30.3. The intercalated cells of the CCD have been shown to respond to mechanical stress from increased flow with changes in the [Ca^{2 +}]_i and it is known that purinergic signaling can increase [Ca^{2 +}]_i through P2Y receptors (Liu et al. 2003). Luminal release of ATP through connexin hemichannels in intercalated cells may provide the link between increased flow in the CCD and increased intracellular Ca^{2 +}. Another finding that suggests hemichannel versus gap junction function of Cx30.3 in the collecting duct intercalated cells is that electron microscopy was not able to detect classical gap junctions in this part of the nephron (Liu et al. 2003). The presence of a luminal ATP channel in the loop of Henle and the collecting duct would be very consistent with the purinergic autocrine and/or paracrine regulation of salt and water reabsorption or perhaps with acid/base homeostasis. Supporting evidence is the colocalization of ATP-degrading enzymes, the ecto-5′-nucleotidase, and purinergic receptors, particularly at the luminal membrane of intercalated cells (Le Hir et al. 1993, Leipziger et al. 2003, Schwiebert et al. 2001). These cells have been suggested to be capable of producing large changes in cell volume (Le Hir et al. 1993), which may stimulate ATP release (Schwiebert et al. 2001). We find it very intriguing that it is exactly one of the only two cell types in the kidney, the intercalated cell apical membrane where Cx30.3 was localized in all species studied. The unusual and interesting apical membrane localization of Cx30.3 and the overlap of its expression with those of ATP degrading enzymes further necessitate the exploration of Cx30.3 function in renal (patho)physiology in future work. Because of the lack of selective inhibitors, Cx30.3 hemichannel function could be best studied using the recently established Cx30.3 knockout mouse model (Zheng-Fischhöfer et al. 2007).

In the medulla, a previous study identified Cx30.3 in the ascending thin limb (Zheng-Fischhöfer et al. 2007). The rhythmic contractions of the pelvic muscles, which cause periodic squeezing (compressions) of the renal pelvis, could serve as mechanical stimulus for the opening of Cx30.3 hemichannels strongly expressed in the ascending thin limb. Tubuloglomerular feedback-induced tubular flow oscillations are also transmitted to this nephron segment and beyond (Kang et al. 2006), which could also provide mechanical stimulus for hemichannel opening.

In summary, this study described the detailed localization of Cx30.3 in mouse, rat, and rabbit kidneys. Cx30.3 was found in the ascending thin limb of the loop of Henle and in the intercalated cells of the collecting duct. Labeling was mostly punctate in the mouse (typical of gap junction localization); however, apical cell membrane localization was evident in the rat and rabbit kidneys, which may suggest hemichannel function. Cx30.3 expression was unchanged in response to either high or low salt intake or in spontaneously hypertensive rats (SHRs). Cx30.3 appears to be constitutively expressed in certain renal tubular segments and cells and its role in overall kidney function remains to be resolved.

These studies were supported by grant DK64324 from NIH, and by an Established Investigator Award 0640056N from the American Heart Association to J.P.P. Work in the Bonn laboratory was supported by grants of the German Research Association (SFB 645, B2, and Wi270/29-1) to K.W.