Localization of protein inhibitor of neuronal nitric oxide synthase in rat kidney

Agnes Roczniak2, David Z. Levine1,2, and Kevin D. Burns1,2

Departments of 1 Medicine and 2 Cellular and Molecular Medicine, University of Ottawa and Ottawa Hospital, Ottawa, Ontario, Canada K1H 8L6


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have recently demonstrated that in rats with 5/6 nephrectomy (5/6 Nx), renal cortical and inner medullary neuronal NOS (nNOS) expression is downregulated, associated with decreased urinary excretion of nitric oxide (NO) products. Recently, a novel 89-amino acid protein [protein inhibitor of nNOS (PIN)] was isolated from rat brain and shown to inhibit nNOS activity. The present studies localized PIN in the rat kidney and determined the effect of 5/6 Nx on PIN expression. By RT-PCR, PIN mRNA was detected in the kidney cortex and inner medulla. Immunohistochemistry revealed staining for PIN in glomerular and vasa rectae endothelial cells. PIN was also localized to the apical membranes of inner medullary collecting duct (IMCD) cells. Two weeks after 5/6 Nx, inner medullary PIN expression was significantly upregulated (sham, 0.18 ± 0.07 vs. 5/6 Nx, 0.58 ± 0.13 arbitrary units; n = 6, P < 0.02), as determined by Western blotting. In summary, our data show that PIN, a specific inhibitor of nNOS activity, is expressed in the IMCD, a site of high nNOS expression in the kidney. PIN expression is upregulated in the inner medulla of 5/6 Nx rats. Inhibition of nNOS activity by PIN may have important implications for the regulation of NO synthesis in the IMCD of normal and remnant kidneys.

inner medulla; chronic renal failure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) is generated in the kidney by the conversion of L-arginine to L-citrulline, in a reaction catalyzed by members of the NO synthase (NOS) family (15). The neuronal NOS (nNOS) is highly expressed in the macula densa and inner medullary collecting duct (IMCD) of the kidney (3, 23). NO generated by nNOS in the macula densa has been implicated in the regulation of tubuloglomerular feedback and renin secretion (16), whereas nNOS in the inner medulla may be involved in the long-term regulation of arterial blood pressure (20).

The activity of nNOS is regulated by Ca2+/calmodulin binding, phosphorylation, and feedback inhibition by NO (15). Another potential mechanism of nNOS regulation has recently been identified. Using the yeast two-hybrid system, Jaffrey and Snyder (11) isolated a novel 89-amino acid protein termed "protein inhibitor of nNOS" (PIN) from rat hippocampus. PIN is a highly conserved protein across species, and shares 100% homology with the light-chain component of dynein (5, 8). In HEK-293 cells, PIN inhibits calcium ionophore-stimulated cGMP formation and nNOS activity, in a concentration-dependent manner (11). The mechanism of PIN-induced inhibition of nNOS activity involves its binding to a 17-amino acid sequence of nNOS, causing destabilization of the dimeric structure of nNOS (6). Inhibition of nNOS activity by PIN appears to be highly specific, since the PIN binding domain of nNOS (amino acid residues 161-245) is absent from both the endothelial and inducible NOS (ecNOS and iNOS, respectively) (6).

Chronic renal failure (CRF) is characterized by decreased intrarenal NO production, which may contribute to elevation of blood pressure (2). Mechanisms that may contribute to reduction of renal NO production include downregulation of renal nNOS, ecNOS, and iNOS expression (22, 26). Indeed, in a recent study, we demonstrated that 5/6 nephrectomy (5/6 Nx) in rats inhibits expression of nNOS mRNA and protein in the renal cortex and inner medulla (22).

Despite the discovery of an endogenous protein inhibitor of nNOS, little is known about its distribution and regulation in the kidney. Accordingly, the first objective of our study was to localize PIN expression in the kidney. Secondly, we determined whether PIN expression is altered in 5/6 Nx rats. Our data show that PIN is highly expressed in the endothelial cells of glomeruli, afferent arterioles, and vasa rectae, whereas in the inner medulla, labeling of the apical surface of IMCD cells was observed. In 5/6 Nx rats, inner medullary PIN expression is significantly upregulated, suggesting that PIN could contribute to a decrease in NO synthesis by this segment.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal surgery. Male Sprague-Dawley rats (200-250 g) were fed regular rat chow and had unlimited access to water. Rats were anesthetized with a mixture of oxygen (1 l/min) and halothane (gradually increased from 0.5% to 4.0%), and 5/6 Nx was performed as previously described (22). Briefly, the right kidney was exposed by midline laparotomy, and the right renal artery, vein, and ureter were ligated with silk, and the entire kidney was removed. The poles of the left kidney were removed using a Bovie. Sham rats underwent a similar procedure, except the kidneys were only touched with the instruments. Two weeks after 5/6 Nx, rats were killed, and the left kidney was immediately removed for dissection of cortex and inner medulla or used for immunohistochemistry. All experiments were approved by the Animal Care Committee at the University of Ottawa.

Reverse transcription-polymerase chain reaction. Total RNA was isolated from kidney cortex and inner medulla using a commercially available kit (RNeasy; Qiagen, Chatsworth, CA). Prior to RT-PCR, residual genomic DNA was digested by incubating RNA with amplification grade DNase I (GIBCO, Burlington, Ontario, Canada). RNA quality was determined by running samples on 1% agarose-formaldehyde gels stained with ethidium bromide, and RNA concentration and purity were determined by optical density measurement at 260 and 280 nm. RNA samples were uniformly of high quality by these standards.

RT-PCR was performed, essentially as described (22). One hundred nanograms of total RNA was reverse-transcribed and amplified in the presence of 1 µM of PIN-specific oligonucleotide primers. After initial denaturation at 94°C for 3 min, 40 cycles of amplification (94°C for 30 s, 60°C for 30 s, 72°C for 30 s) were performed, followed by final extension at 72°C for 10 min, in a Perkin-Elmer GeneAmp PCR system 2400 apparatus. The PCR products were separated on ethidium bromide-stained 2% agarose gels, then visualized and photographed under ultraviolet light.

The primer sequences for PIN were selected according to the published rat cDNA sequence (11). The upstream primer was 5'-GTAACCATGTGCGACCGGAA-3' (bases 91-110), and the downstream primer was 5'-TCCTCTTACCGGTGGACTGG-3' (bases 331-350), which were designed to generate a PCR product of 259 bp. The specificity of the primers was verified through GenBank.

The PCR fragment amplified with PIN-specific primers was subcloned into a pCR 4-TOPO plasmid (Invitrogen, Carlsbad, CA) and sequenced in the University of Ottawa DNA sequencing facility.

Immunohistochemistry. Animals were anesthetized with an intraperitoneal injection of 100 mg/kg pentobarbital sodium, and the kidneys were removed and immersed in Zamboni's fixative (2% paraformaldehyde and 15% picric acid in 0.1 M phosphate buffer, pH 7.3) at 4°C overnight. Tissue was then rinsed in 0.1 M PBS (pH 7.4) containing 10% sucrose and incubated overnight at 4°C. The kidneys were embedded in paraffin, and 5-µm sections were cut with a microtome (Jung model RM 2035) and mounted onto Superfrost microscope slides. The kidney slices were deparaffinized by incubation in xylene for 5 min at room temperature, then transferred into fresh xylene for another 5 min. The slices were rehydrated by sequential incubation in 100%, 95%, and 70% ethanol, for 5 min each at room temperature, followed by incubation in water for 2 min. The kidney slices were rinsed with PBS, and endogenous peroxidase activity was blocked by incubation in a solution of 0.75% H2O2 in methanol for 1 h at room temperature. The sections were then permeabilized with 0.3% Triton X-100 in PBS for 15 min at room temperature. Slides were then incubated in blocking buffer consisting of PBS supplemented with 5% normal donkey serum (Sigma-Aldrich, Oakville, Ontario, Canada) for 1 h at room temperature. A goat polyclonal PIN antibody (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in PBS supplemented with 2.5% donkey serum was added to the slides and incubated for 18 h at 4°C. To ensure specificity of staining with this antibody, additional slices were incubated in the presence of primary antibody that had been previously neutralized with the immunizing antigen (Santa Cruz Biotechnology) or in the presence of normal goat serum. In some experiments slides were incubated with 1:200 dilution of rabbit polyclonal antibody to nNOS (Santa Cruz Biotechnology) for 18 h at 4°C. The slides were then washed three times for 5 min each in PBS and incubated with donkey anti-goat biotinylated whole antibody (1:100 dilution; Amersham, Oakville, Ontario, Canada) for 1 h at room temperature. After three washes with PBS, the slides were incubated in 3% H2O2 for 20 min at room temperature followed by incubation with 1:50 dilution of streptavidin-horseradish peroxidase (Amersham) in PBS for 1 h at room temperature. The slides were washed and incubated with 3,3'-diaminobenzidine chromogen solution (DAB; BioGenex, San Ramon, CA) for 10 min. All slides were incubated in hematoxylin (Sigma), water, 95% and 100% ethanol, and xylene. The slides were covered with Permount (Fisher Scientific, Nepean, Ontario, Canada) and viewed with a Zeiss Axioplan microscope.

The specificity of the polyclonal PIN antibody utilized in immunohistochemistry studies was established by Western blotting (Fig. 1). The antibody detected the 10-kDa PIN in both rat IMCD lysates and in rat brain lysate (positive control) (Fig. 1A). In addition, in IMCD lysates the antibody detected three other proteins at approximately 18, 30, and 32 kDa. When the PIN antibody was preabsorbed with excess immunizing peptide, the signal for the 10-kDa PIN protein disappeared, but not the signals for the unidentified other three proteins (Fig. 1B).


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Fig. 1.   Western blot demonstrating specificity of anti-PIN antibody. A: Representative Western blot depicting PIN immunoreactivity with the polyclonal anti-PIN antibody utilized in immunohistochemical studies. Lane 1: 60 µg of inner medullary collecting duct (IMCD) lysate. Lane 2: rat brain lysate as positive control. Incubation with the anti-PIN antibody results in detection of 10-kDa PIN in lanes 1 and 2, and of three other proteins at 18, 30, and 32 kDa in lane 1. B: Preabsorption of anti-PIN antibody with immunizing peptide results in selective blockade of PIN protein immunoreactivity. PIN, protein inhibitor of nNOS; nNOS, neuronal nitric oxide synthase.

Western blotting. Cortexes and inner medullas from sham and 5/6 Nx rats were homogenized with a cell disrupter in boiling lysis buffer (1% SDS and 10 mM Tris · HCl, pH 7.4). Western blot analysis was performed as previously described (13). Briefly, the lysate was boiled for 10 min, followed by centrifugation at 12,000 g for 2 min to remove insoluble debris. Protein concentrations in the supernatant were determined by the Bradford method (Bio-Rad, Montreal, Quebec, Canada) using BSA (Sigma) as standard. Tissue lysates (20 µg for inner medulla and 40 µg for cortex) were separated on 7.5% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% skimmed milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 (TBS-T) and 0.01% sodium azide, for 1 h at room temperature. The membranes were then incubated for 18 h at 4°C with 1:250 dilution of mouse monoclonal antibody to PIN (Transduction Laboratories, Lexington, KY), followed by incubation with 1:2,000 dilution of anti-mouse secondary antibody conjugated to horseradish peroxidase (Amersham). A monoclonal antibody to PIN was used for Western blotting, since the polyclonal antibody used in the immunohistochemical experiments had lower sensitivity under the experimental conditions. Primary antibodies were diluted in TBS-T supplemented with 5% skimmed milk and 0.01% sodium azide, whereas the secondary antibodies were diluted in TBS-T supplemented with 2% milk. Proteins were detected by enhanced chemiluminescence (ECL, Amersham) on Hyperfilm (Amersham), according to the manufacturer's instructions. Prestained standards were used as molecular weight markers (Bio-Rad), and rat brain protein (4 µg, Transduction Laboratories) was used as a positive control for PIN. To control for protein loading, all membranes were stripped and probed with a monoclonal anti-beta -actin antibody (mouse ascites fluid, Sigma), which recognized the beta -actin protein at ~45 kDa. Signals on Western blots were quantified by densitometry and corrected for the beta -actin signal, using an image analysis software program (NIH Image 1.47).

Statistics. Results are means ± SE. Data were analyzed by the unpaired Student t-test. P < 0.05 was considered as significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Localization of PIN. Using RT-PCR, mRNA expression for PIN was readily detected in both cortex and inner medulla of the rat kidney (Fig. 2). By DNA sequencing, the nucleotide sequence of the PIN PCR product was identical to the published sequence for rat PIN (bases 91-350) (11). To localize PIN expression in the kidney, we used immunohistochemistry. In the cortex, staining for PIN was observed in endothelial cells of the glomeruli and the afferent arterioles (Fig. 3A), with no staining in macula densa cells. In the outer medulla, endothelial cells of the vasa recta stained for the presence of PIN (Fig. 3C), whereas in the inner medulla, staining for PIN was observed on the apical surface of cells of the IMCD (Fig. 3E). IMCD cells also demonstrated diffuse cytoplasmic staining for nNOS by immunohistochemistry (Fig. 3G), consistent with our previous studies (22, 23) and with recent work demonstrating relatively high levels of NOS activity in this segment (27). The specificity of PIN staining was verified by incubating kidney slices with PIN antibody that had been previously neutralized with immunizing antigen, which resulted in complete loss of labeling in cortex, outer medulla, and inner medulla (Fig. 3, B, D, and F). In addition, incubation with an identical dilution of nonimmune serum from goat resulted in absence of PIN staining of kidney slices, further demonstrating specificity of staining with the PIN antibody.


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Fig. 2.   RT-PCR for PIN in the kidney. Representative ethidium bromide-stained agarose gel of cortical (lane 1) and inner medullary (lane 2) cDNA product (259 bp). Lane 3 depicts negative control obtained by omitting reverse transcriptase. Left lane, molecular standard.



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Fig. 3.   Immunolocalization of PIN. Immunohistochemical micrographs of cortex (A), outer medulla (C), and inner medulla (E) of rat kidney labeled with polyclonal PIN antibody (arrows). In A, top arrow points to endothelial cells within a glomerulus; bottom arrow indicates endothelial cells of afferent arteriole. Incubation of kidney sections with anti-PIN antibody preabsorbed with immunizing antigen demonstrates lack of staining in cortex, outer medulla, and inner medulla (B, D, and F, respectively). Micrograph of inner medulla labeled with a polyclonal antibody to nNOS is shown in G, and depicts diffuse cytoplasmic staining (brown) of IMCD cells. To demonstrate antibody specificity, incubation of kidney section with anti-nNOS antibody preabsorbed with immunizing antigen demonstrates lack of inner medullary staining (H).

Kidney slices obtained from rats that underwent 5/6 nephrectomy were also examined for PIN distribution. The localization of PIN was unchanged compared with sham rats, with apical staining of IMCD, and staining of endothelial cells of the vasa rectae, afferent arterioles, and glomeruli.

Effect of 5/6 Nx on expression of PIN in inner medulla. We have previously demonstrated that nNOS mRNA and protein expression is significantly downregulated in the cortex and inner medulla of 5/6 Nx rats (22). Accordingly, we studied the effect of 5/6 Nx on the expression of PIN, by Western blotting. Fourteen days after 5/6 Nx, PIN expression was significantly upregulated in the inner medulla (sham, 0.18 ± 0.07 vs. 5/6 Nx, 0.58 ± 0.13 arbitrary units; P < 0.02, n = 6) (Fig. 4). Although by immunohistochemistry we were able to detect expression of PIN in glomerular and afferent arteriolar endothelial cells, Western blotting on proteins (40 µg) extracted from the cortex revealed only a very faint band that did not permit adequate quantification by densitometry.



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Fig. 4.   Effect of 5/6 nephrectomy (5/6 Nx) on inner medullary expression of PIN. A: representative Western blot of PIN expression in inner medulla of sham and 5/6 Nx rats. Lane 1: rat brain lysate (positive control). Lane 2: 20 µg total protein from sham rat. Lane 3: 20 µg total protein from 5/6 Nx rat. Corresponding bands for beta -actin are depicted, demonstrating equal loading of proteins. B: bar graph depicting densitometric quantification of Western blot signals for PIN in inner medulla. Results are means ± SE (n = 6). *P < 0.02.

In all experiments, to control for possible variations in protein loading, membranes were stripped and probed for beta -actin. There were no differences in beta -actin protein abundance between samples derived from either sham or 5/6 Nx rats.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A gene encoding PIN has recently been cloned from rabbit and consists of three exons and two introns, spanning ~2.3 kb of genomic DNA (12). Northern and Western blot analyses revealed that PIN is expressed in the testes, brain, and respiratory muscles of humans, rats, and mice (7, 8). PIN mRNA has also been detected by Northern blot analysis of whole human kidney RNA (5), but no studies have localized PIN within the kidney. In the present study, we detected PIN immunoreactivity in endothelial cells of the glomeruli, afferent arterioles, and vasa rectae. In the inner medulla, we observed intense staining for PIN on the apical surface of IMCD cells, a major site of nNOS mRNA and protein expression in the kidney (22, 23) and the intrarenal site with the highest enzymatic activity for NO production (27). The expression of both PIN and nNOS in the IMCD suggests that nNOS activity, and hence NO production, may be tightly regulated by PIN. Although the macula densa expresses nNOS protein (3), it lacked immunoreactivity for PIN, suggesting that nNOS activity in the macula densa may be regulated in a PIN-independent fashion. Although nNOS mRNA has been detected in the vasa recta (24), the afferent arteriole does not express significant levels of nNOS protein (3). Similarly, although nNOS has been detected in glomerular epithelial cells (3), it has not been detected in glomerular endothelial cells (3). The significance of the disparity in the distribution of nNOS and PIN in the kidney is unclear but suggests that PIN may have other functions in addition to regulation of nNOS activity.

The cDNA for PIN, otherwise termed light-chain dynein, was simultaneously cloned by two separate groups (5, 11). One group identified this novel protein as an inhibitor of nNOS (11), whereas the other group identified it as a light-chain component of the dynein complex (5). Dyneins are highly complex molecular microtubule-based motors responsible for translocation of membranous vesicles, nuclear migration, and movement of organelles (10, 21). Cytoplasmic dynein is a multimeric protein consisting of two heavy chains, two or three intermediate chains, and varying numbers of light chains (9). The two heavy chains contain regions involved in ATP hydrolysis, whereas the intermediate chains are thought to target dynein within the cell and may link the enzyme to the surface of membranous organelles, kinetochores, and vesicles (9). The role of PIN or of any other light chain dynein in mammalian cells is unknown, but in Drosophila, partial loss-of-function mutations in light chain dynein are associated with morphogenic defects in bristle and wing development (5). In addition, diverse proteins have been found to interact and associate with PIN, including flagellar dynein (14), cytosolic dynein (13), the signaling molecule Ikappa Balpha (4), and nNOS (6, 11). For example, in skeletal muscle PIN is found associated with the dynein complex as well as in the cytoplasm, suggesting it performs different functions (8). In Madin-Darby canine kidney (MDCK) cells, dynein is one of the microtubule motors involved in the apical transport of vesicles, suggesting its involvement in the maintenance of a polarized epithelium (17). Dynein may also be involved in the transport of apical water and ion channels in renal epithelia. Indeed, in IMCD cells, the intermediate dynein chain is associated with aquaporin-2-containing vesicles (18). The role of PIN in these processes is unknown, and to our knowledge, the present study is the first to determine its localization in the kidney. Our data, demonstrating localization of PIN in IMCD cells, suggest as one possibility that PIN may be involved in regulation of vesicular transport and in regulation of nNOS activity in this segment.

In rats with renal mass reduction, both renal and systemic NO synthesis are reduced (1). Administration of L-arginine to 5/6 Nx rats reduced proteinuria, normalized creatinine clearance, and increased fractional excretion of sodium, suggesting that NO is renoprotective in this model (2). In a recent study, we (22) showed that nNOS mRNA and protein expression is significantly downregulated in the cortex and inner medulla of 5/6 Nx rats. Furthermore, Vaziri et al. (26) demonstrated that expression of intrarenal ecNOS and iNOS is downregulated in this model of CRF. The decrease in NO production in CRF may also be due to an increase in the abundance of circulating endogenous inhibitors of NOS. The synthesis of NO can be inhibited by analogs of arginine, some of which are present in human plasma and are excreted in the urine (25). Indeed, Vallance et al. (25) showed that in patients with end-stage renal disease, the circulating concentration of the endogenous NOS inhibitor NG,NG-dimethylarginine (ADMA) is significantly increased, to levels that can inhibit NOS activity. In addition, urinary ADMA excretion significantly correlates with mean arterial pressure in Dahl salt-sensitive rats fed a high-salt diet, suggesting that ADMA may contribute to inhibition of NO synthesis and development of high blood pressure in this rat model of hypertension (19). Our results indicate that PIN expression in the inner medulla was significantly upregulated in 5/6 Nx rats, suggesting that PIN could contribute to a decrease in NO synthesis in the inner medulla, accompanying CRF. Furthermore, it is possible that decreased NO production could contribute to long-term elevation of blood pressure, since inhibition of inner medullary nNOS expression has been shown to induce salt-sensitive hypertension in rats (20).

In summary, this study shows that the endogenous inhibitor of nNOS, PIN, is expressed in the endothelial cells of the glomeruli, afferent arterioles, and vasa rectae. In the inner medulla, PIN is expressed in the IMCD, a site of high nNOS expression in the kidney. In 5/6 Nx rats, upregulation of PIN expression may contribute to salt retention and increased blood pressure, via inhibition of local NO synthesis.


    ACKNOWLEDGEMENTS

We thank Dr. S. Robertson (Dept. of Pathology, Ottawa Hospital) for assistance with the immunolocalization studies and Joseph Zimpelmann for excellent technical assistance.


    FOOTNOTES

This work was supported by Medical Research Council of Canada Grant MT-11560 (to K. D. Burns).

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 and other correspondence: K. D. Burns, Division of Nephrology, 501 Smyth Rd., Rm LM-18, Ottawa, Ontario, Canada K1H 8L6 (E-mail: kburns{at}ogh.on.ca).

Received 1 July 1999; accepted in final form 9 December 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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