Segmental expression of somatostatin receptor subtypes sst1 and sst2 in tubules and glomeruli of human kidney

Douglas A. Balster1, M. Sue O'Dorisio2, Monica A. Summers1, and Martin A. Turman1

1 Department of Pediatrics, The Ohio State University, and Children's Research Institute, Children's Hospital, Columbus, Ohio 43205; and 2 Department of Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa 52242


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Somatostatin is known to modulate mesangial and tubular cell function and growth, but the somatostatin receptor (sst) subtypes responsible for these effects have not been defined. There are at least five different sst receptor subtypes (sst1-sst5). We used RT-PCR to demonstrate that normal human kidney consistently expresses mRNA for sst1 and sst2 (9 of 9 donors). Some donors expressed sst4 or sst5 mRNA, but none expressed sst3 mRNA. Expression of sst1 and sst2 was further assessed by staining serial sections of normal human kidney with sst1 and sst2 antisera, Arachis hypogaea (AH) lectin (to define distal tubule/collecting duct cells), Phaseolus vulgaris lectin (proximal tubules), and Tamm-Horsfall protein (THP) antiserum (thick ascending limb of the loop of Henle). Specificity of antisera was demonstrated by transfection and absorption studies. Sst2, but not sst1, was expressed in glomeruli. Intense sst1 and sst2 staining localized exclusively to AH+ and THP+ tubules. Thus sst1 and sst2 subtype-selective analogs may be useful to beneficially modulate renal cell function in pathological conditions.

somatotropin release inhibitory factor; renal; mesangium; immunohistochemistry; reverse transcriptase-polymerase chain reaction


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SOMATOSTATIN IS WELL RECOGNIZED for its role as a neuropeptide, a hypothalamic inhibitor of growth hormone release, and a paracrine inhibitor of gut peptide secretion (21). Somatostatin also serves to modulate renal cell function and growth. Intravenous infusion of somatostatin decreases glomerular filtration rate, renal plasma flow, urine volume (40), and osmotic and free water clearance (34, 40). Furthermore, somatostatin increases the fractional excretion of sodium and phosphate while decreasing plasma renin activity and urinary excretion of PGE2, dopamine, epinephrine, and other vasoactive factors (34). Whereas some of the effects of systemic infusion of somatostatin are mediated by altering renal blood flow, several studies indicate that somatostatin also directly alters renal function at the cellular level. For example, somatostatin inhibits vasopressin-induced water permeability of microperfused rat papillae (22) and cAMP production in cultured rat renal collecting tubule cells (13). We have demonstrated that somatostatin inhibits epidermal growth factor-induced renal tubular cell proliferation (36). Somatostatin also inhibits serum-induced proliferation of rat mesangial cells (27) and ANG II-induced contraction of cultured human mesangial cells (3, 7, 8).

The ability of somatostatin to directly modulate renal cell function implies the presence of functional somatostatin receptors (sst) in the kidney. Hatzoglou et al. (10) demonstrated that opossum kidney (OK) tubular cells express somatostatin binding sites for octreotide by radioligand binding assays. High-affinity somatostatin binding sites were also detected in rabbit kidney extracts (26). There are at least five different somatostatin receptor subtypes (sst1-sst5). Mitsuma et al. reported that rat kidney expresses sst2 (16) and sst3 (15), but not sst1 (17) as assessed by immunohistochemistry. However, the kidney data were provided only in tabular form in these reports, so the segmental distribution of sst receptor expression in the rat kidney could not be ascertained.

In human kidney, Yamada et al. (41) detected sst1 and sst2 mRNA by Northern blot analysis. The majority of renal carcinomas express sites for somatostatin, as assessed by radioligand binding assays (24), and mRNA for sst2, as detected by RT-PCR (25, 39). Diez-Marques et al. (3) demonstrated specific binding of octreotide to cultured human mesangial cells corresponding to the ability of somatostatin to stimulate cGMP production and inhibit ANG II-induced mesangial cell contraction (8). Reubi et al. (23) detected specific binding of octreotide to human renal tubules, collecting ducts, and vasa recta by in situ radioligand binding assays.

The expression pattern of somatostatin receptor subtypes in different segments of the human kidney has not previously been described. This information is critical for developing strategies to utilize somatostatin subtype-selective analogs to beneficially modify renal transport functions or to treat renal diseases such as mesangial proliferative forms of glomerulonephritis. Herein, we report on somatostatin receptor subtype mRNA expression in normal human kidney for all five somatostatin receptor subtypes. We also demonstrate the segmental expression pattern of sst1 and sst2 by immunohistochemistry.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue procurement. Kidney tissue was provided by The Ohio State University and Children's Hospital Cooperative Human Tissue Network with approval from Children's Research Institute Human Subjects Internal Review Committee. Specimens were obtained from normal kidney adjacent to a renal tumor or from cadaveric kidneys unsuitable for renal transplanation.

RT-PCR. RT-PCR was performed with previously described (2) oligonucleotide primers for all five sst receptor subtypes and c-abl, a tyrosine kinase protooncogene constitutively expressed in glomeruli and other cell types (2, 18, 30). Total RNA was isolated by using the TRIzol (GIBCO-BRL, Grand Island, NY) or RNAzol (CINNA/BIOTEX, Friendwood, TX) methods as described by the manufacturers. Total RNA (200 ng) was reverse transcribed with random hexamer primers followed by amplification of cDNA by PCR (GeneAmp kit; PerkinElmer Cetus, Norwalk, CT). For sst1-sst3, reaction mixtures were subjected to 33 cycles of 94°C for 1 min, 60°C for 2 min, and 72°C for 2 min For sst4, mixtures were subjected to 35 cycles of 94°C for 40 s, 62°C for 45 s, and 72°C for 1 min For sst5, PCR conditions included 1.5 mM MgCl2 and 1.5% deionized formamide; mixtures were subjected to 30 cycles of 94°C for 30 s, 64°C for 10 s, and 72°C for 1 min. After completion of PCR cycles, reactions were subjected to 72°C for 9 min. RT-PCR products were resolved by electrophoresis at 100 V through 1% agarose in 1× TAE (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0) and visualized with ethidium bromide.

Southern hybridization of sst receptor RT-PCR products. Southern analysis was used to confirm that sst1, sst2, and sst5 RT-PCR products contained the expected sst receptor subtype-specific sequences. As previously described (2), the Southern blot probes were complementary to regions of receptor cDNA nested between the binding sites for RT-PCR primers. The RT-PCR products were transferred to a nylon membrane and hybridized with the appropriate oligonucleotide probes that were 32P labeled with T4 polynucleotide kinase. Hybridization was performed in 50% formamide at 42°C overnight. The membranes were washed sequentially with 2× standard sodium citrate (SSC; 0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0) at room temperature, 2× SSC with 1% sodium dodecyl sulfate at 65°C, and then 0.1× SSC at room temperature. Bound 32P-labeled probe was detected by autoradiography. To control for nonspecific hybridization, c-abl RT-PCR products were also included on all blots.

Antisera preparation for immunohistochemistry of sst1 and sst2. Antisera against sst1 and sst2 were generated as previously described (2). Briefly, for generation of sst1 antiserum, an oligonucleotide coding for the NH2 terminal, 57 amino acids comprising the first extracellular domain of sst1 were cloned into pET-32a(+) vector (Novagen, Madison, WI) as a COOH-terminal fusion to thioredoxin by using T4 DNA ligase (TA cloning kit, Invitrogen, Carlsbad CA). Similarly, an oligonucleotide corresponding to the NH2 terminal, 45 amino acids of sst2 were cloned into pET-32a(+). After the truncated sst1 and sst2 peptides were expressed by AD494 bacteria (Novagen), peptides were purified by using the Pharmacia HisTrap column system (Amersham Pharmacia Biotech, Uppsala, Sweden). Rabbits were immunized with 500 µg of truncated sst1 or sst2 peptide in 0.5 ml 0.9% NaCl mixed with an equal volume of Freund's adjuvant. After four boosts (100 µg peptide each) at 3-wk intervals, antisera were collected and stored at -70°C.

Transient transfection. Expression vectors for sst1, sst2, and sst5 were generated as previously described (2). OK cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured as directed. Cells were periodically tested for mycoplasma contamination by using an RT-PCR mycoplasma detection kit (ATCC). OK cells (5,000 cells per well) were plated in 8-well chamber slides (Fisher Scientific, Pittsburgh, PA) and transfected by using Effectene transfection reagent as directed by the manufacturer (Qiagen, Valencia, CA). Briefly, 0.4 µg of plasmid DNA were mixed with Effectene reagent in a 1:20 ratio of DNA to Effectene. Cells were incubated with the transfection mixture overnight at 37°C. After incubation in regular growth medium at 37°C for an additional 24 h, the cells were washed once with PBS and then fixed with ice-cold 100% methanol for 10 min.

Immunofluorescence of transfected cells. For immunofluorescent staining, cells were rinsed with PBS, then blocked with "Power Block" (Biogenix, San Ramon, CA) for 30 min at room temperature. Cells were then incubated with 300 µl of antisera or preimmune serum. Anti-sst1 serum was diluted in Diluent buffer (Biomedia, Foster City, CA) 1:1,000 while anti-sst2 serum was diluted 1:2,000. For blocking studies, antisera were preincubated for 2 h at room temperature with 50 µg/ml of immunizing peptide. The mixtures were then subjected to centrifugation at 17,000 g at 4°C for 10 min, and the supernatant was used for staining. After incubation with antisera or preimmune serum at 37°C for 1 h, cells were washed three times with OptiMax wash buffer (Biogenix, San Ramon, CA). Cy3-conjugated affinity-purified anti-rabbit IgG antiserum (Jackson Laboratories, West Grove, PA) diluted 1:500 was then applied. After incubation for 1 h at 37°C in the dark, cells were washed three times with wash buffer. After being coverslipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA), cells were photographed with an Optronics Magnafire digital camera mounted on a Leica DMLB microscope.

Immunohistochemical staining of normal human kidney. For immunohistochemical analysis of sst1 and sst2 expression, 4-µm sections of paraffin-embedded normal human kidney were placed on charged slides and deparaffinized. To block endogenous peroxidase activity, slides were incubated with PeroxiDaze 1 (Biocare Medical, Walnut Creek, CA) for 15 min at room temperature. Endogenous biotin was blocked by incubating sections in avidin-blocking solution for 15 min followed by biotin-blocking solution for 15 min (Vector Laboratories). Sections were then incubated in Power Block for 10 min. Antisera were diluted and absorbed with immunizing peptide as described above. Slides were incubated with preimmune serum or antisera at 4°C overnight. Sections were then processed with the "super sensitive multilink" staining system as described by the manufacturer (Biogenix). Briefly, after slides were washed three times, biotinylated secondary antibody was applied for 20 min at room temperature. Slides were then washed three times in wash buffer for 20 min each wash, followed by incubation in peroxidase-conjugated streptavidin for 20 min. After washing of slides three times for 20 min each, 3-amino-9-ethyl-carbazole chromogen was applied for 3 to 10 min depending on development of slides. Staining was stopped by immersion in distilled water. Slides were counterstained with hematoxylin for 10 s, rinsed in water, immersed in ammonia water for 15 s, and then cemented with Crystal Mount (Biomedia), placed on a 60°C surface for 15 min, coverslipped with Permamount (Biogenix), and photographed as described above.

To determine which tubular segments express sst1 and sst2, serial sections were also stained with Arachis hypogaea (AH) lectin (to define distal tubule/collecting duct cells), Phaseolus vulgaris (PHA-E) lectin (to define proximal tubules), and Tamm-Horsfall protein (THP) antiserum (to define the thick ascending limb of the loop of Henle) (19). Lectins were obtained from Sigma (St. Louis, MO). For THP staining, a goat anti-THP (anti-human uromucoid) antiserum (Cappel/ICN, Costa Mesa, CA) was used at a 1:100 dilution. THP staining was performed as for anti-sst receptor staining except that a 1:100 dilution of biotinylated anti-sheep IgG (Vector) was used instead of the BioGenix multilink secondary antibody. For lectin staining, sections were prepared and blocked as for immunostaining. Sections were then incubated with biotinylated AH lectin or PHA-E lectin. Biotinylated AH and PHA-E were diluted to 0.01 mg/ml and 0.002 mg/ml, respectively. After incubation at room temperature for 1 h, slides were washed three times in wash buffer for 20 min each wash, followed by incubation in peroxidase-conjugated streptavidin for 20 min. Slides were then processed as for immunohistochemistry.

To determine the fraction of tubules coexpressing somatostatin receptor and THP, AH lectin, or PHA-E lectin, the same region of serial sections was photographed for each stain. Four separate regions were photographed, magnified, and then compared. All stained tubules in one serial section that could be clearly identified in a corresponding serial section by local landmarks were evaluated.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Human kidneys express mRNA for several somatostatin receptor subtypes. For RT-PCR analysis, kidney tissue from nine different donors was tested. These donors ranged in age from childhood to 69 yr of age and were evenly distributed between male and female donors (Table 1). Unfortunately, racial information on the donors was not provided with the procurement information.

                              
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Table 1.   Description of donors and summary of RT-PCR results

In preliminary experiments, adequacy of primers was confirmed and conditions optimized by amplifying all five sst receptor subtypes from genomic DNA. For RT-PCR, the presence of adequate amounts of RNA was confirmed by performing RT-PCR with primers specific for the protooncogene, c-abl. The sst receptor genes are intronless. Therefore, products obtained from contaminating genomic DNA are the same size as from mRNA. However, the primers for c-abl amplify across an intron/exon splice site. Reactions for c-abl did not demonstrate the presence of the genomic c-abl product (Fig. 1) indicating the absence of genomic DNA. In addition, parallel RT-PCR mixtures without RT were included in each experiment (Fig. 1), and no contaminating DNA was detected.


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Fig. 1.   Detection of human kidney somatostatin receptor (sst) mRNA expression by RT-PCR. Total RNA isolated from normal human kidney was subjected to RT-PCR with oligonucleotides specific for each sst receptor subtype or the protooncogene, c-abl. To control for contaminating genomic DNA, a c-abl sample was processed in an identical manner except RT was omitted (-RT). Products were visualized by agarose gel electrophoresis and ethidium bromide staining. Left lane: DNA molecular weight (MW) markers (100-bp ladder). This is a representative result from a single donor (donor 6). The parallel -RT controls for each sst receptor subtype, which were included in all experiments, are not shown.

RT-PCR of normal human kidney RNA with sst receptor subtype-specific primers resulted in single products of the appropriate size for sst1 and sst2 for all nine donors (Fig. 1 and Table 1). A single donor of seven expressed sst4 and four of nine donors expressed sst5. sst3 mRNA was not detected in any of seven donors tested. These results indicate that mRNA for sst1 and sst2 is consistently expressed in normal human kidney.

The RT-PCR products for sst1, sst2, and sst5 were analyzed further by Southern blot analysis to confirm that they contained the expected sst receptor subtype-specific nucleotide sequences. RT-PCR products were hybridized with a 32P-labeled oligonucleotide complementary to sst receptor subtype-specific cDNA sequences nested between binding sites for the RT-PCR primers. To control for nonspecific hybridization, c-abl RT-PCR products were also included on all blots. RT-PCR products from two donors (donors 1 and 9) were tested for sst1, sst2, and sst5. The 32P-labeled sst receptor probes hybridized specifically to the appropriate sst receptor RT-PCR products (Fig. 2), but not to c-abl RT-PCR products (not shown). Also, no hybridization occurred when RT was omitted from the RT reaction (Fig. 2), confirming the absence of contaminating genomic DNA. The ability of sst receptor subtype-specific probes to hybridize exclusively to the expected RT-PCR products confirms that the sst receptor subtype RT-PCR products are not amplification artifacts.


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Fig. 2.   Southern blot analysis of sst receptor RT-PCR products. sst1, sst2, and sst5 RT-PCR products from donor 1 were transferred to a nylon membrane and incubated with 32P-labeled sst receptor subtype-specific synthetic oligonucleotide probes. Bound probe was visualized by autoradiography. To control for contaminating genomic DNA, samples were processed in an identical manner except RT was omitted. A 123-bp DNA MW ladder from one of the corresponding ethidium bromide-stained agarose gels is shown (left lane). This is a representative result from RT-PCR products obtained from donor 1. Similar results were obtained with RT-PCR products from donor 9 (not shown).

Immunohistochemical assessment of sst1 and sst2 expression in normal human kidney. We next tested for expression of sst1 and sst2 by immunohistochemical staining. To document that the anti-sst1 and anti-sst2 antisera bind specifically to the appropriate sst receptor subtype in kidney cells, opossum kidney (OK) cells were transfected with sst1, sst2, or sst5 expression vectors and then assessed for binding by anti-sst1 or anti-sst2 antisera by using immunofluorescence (Fig. 3). The antisera reacted only with cells transfected with the corresponding sst receptor expression vector. This result indicates that the antisera do not cross-react with other somatostatin subtypes. In addition, the specificity of the antisera was demonstrated by the ability of the immunizing peptide to block binding of the corresponding antisera to transfected cells (Fig. 3).


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Fig. 3.   Immunofluorescent staining of opossum kidney (OK) cells transfected (Tx) with sst1, sst2, or sst5 expression vectors. OK cells were transfected with either sst1 (A-D), sst2 (E-H), or sst5 (I-K) transfection vectors and then stained with anti-sst1 antiserum (A, E, I), anti-sst2 antiserum (B, F, J), or preimmune serum (C, G, K). In addition, sst1 and sst2 antisera were preabsorbed with sst1 immunizing peptide (D) or sst2 immunizing peptide (H), respectively, before staining the corresponding transfected cells.

We next assessed sst1 and sst2 expression in paraffin-embedded sections of normal human kidney (Fig. 4). Antibody specificity was demonstrated by a lack of staining with preimmune serum (Fig. 4A) and by blocking of binding when antiserum was preincubated with the corresponding immunizing peptide (Fig. 4, D and H).


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Fig. 4.   Immunohistochemical staining of normal human kidney with sst1 and sst2 antisera. Paraffin sections of normal human kidney were stained as described in METHODS with preimmune serum (A), sst1 antiserum (B-D), or sst2 antiserum (E-H). For D and H, respectively, sst1 and sst2 antisera were preabsorbed with the corresponding immunizing peptide before staining. A, C, D, G, and H are ×100 magnification. B and F are ×200 magnification and E is ×400 magnification. In C, the arrowheads labeled "G" point to unstained glomeruli.

Staining patterns from kidney tissue from four different donors were assessed (2 male and 2 female donors, age ranging from 4 to 64 yr). sst1 was expressed exclusively in tubular cells (Fig. 4, B and C), whereas sst2 was expressed in both tubular and glomerular cells (Fig. 4, E-G). Expression of sst2 in glomeruli was most consistent with a mesangial staining pattern (Fig. 4E), but the specific glomerular cell type(s) expressing sst2 could not be ascertained. In addition, the intensity of glomerular staining was variable among donors, ranging from light and focal staining to intense and more diffuse staining. However, glomeruli from all four donors were clearly positive for sst2, but not sst1.

In most tubules, staining for both sst1 and sst2 was diffuse throughout the cytoplasm. However, in some tubules, expression was predominately on the apical surface without diffuse cytoplasmic staining (not shown). Comparison of serial sections demonstrated that of all tubules expressing sst1 or sst2, 40% (89/221) expressed both sst1 and sst2, 54% (119/221) expressed sst2+ but not sst1, and only 6% (13/221) expressed sst1 but not sst2 (Figs. 4 and 5). These results indicate that sst2 is expressed more widely than sst1, but both sst1 and sst2 are expressed in renal tubules in vivo.


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Fig. 5.   Staining of serial sections of normal human kidney with sst1 and sst2 antisera and segment-specific markers. Paraffin sections of normal human kidney were stained as described in METHODS with antiserum against sst1 (A), sst2 (B), Tamm-Horsfall protein (THP; C), or with biotinylated Arachis hypogaea (AH) lectin (D) or Phaseolus vulgaris (PHA-E) lectin (E). Original magnification ×100. The pictures are focused on one portion of the field for clarity. Single-lined arrowhead denotes a tubular segment positive for sst1, sst2, and AH, but negative for THP and PHA-E. The double-lined arrowhead denotes a tubular segment positive for sst2, AH, and THP, but negative for sst1 and PHA-E.

To define which tubular segments express sst1 and/or sst2, serial sections were stained with AH lectin (to define distal tubule/collecting duct cells), PHA-E lectin (to define proximal tubules), and THP antiserum (to define the thick ascending limb of the loop of Henle) (19). THP and AH staining overlapped considerably; of all tubules positive for either AH or THP, 43% (97 of 226 tubules) were positive for both AH and THP, 23% (52 of 226) for AH only, and 34% (77 of 226) for THP only (Figs. 5 and 6). There was no overlap between PHA-E+ and AH+ or THP+ tubules, demonstrating the specificity of these lectins and antiserum for specific tubular segments.


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Fig. 6.   Summary of results of tubular segment staining. Serial sections of human kidney were stained for sst1, sst2, THP, AH lectin, and PHA-E lectin. Tubules that were strongly positive were correlated with staining in other serial sections. The percentage of tubules that costained was then calculated for each combination of antiserum and lectin. A: percentage of AH+ and/or THP+ tubules that were positive for sst1+ or sst2+. B: percentage of sst1+ or sst2+ tubules that were AH+ and/or THP+ tubules. Calculations are based on all identifiable tubules in 4 different ×100 microscopic fields from 2 different donors.

Tubules that could be clearly identified in corresponding serial sections were assessed for expression of sst1, sst2, THP, AH lectin, and PHA-E lectin (Figs. 5 and 6). For all tubules expressing AH, but not THP, 65 and 73% expressed sst1 and sst2, respectively (Fig. 6A). For tubules expressing both AH and THP, 51 and 84% expressed sst1 and sst2, respectively (Fig. 6A). For tubules expressing THP, but not AH, only 14% expressed sst1, whereas 86% expressed sst2 (Fig. 6A). Conversely, when all distal tubules or collecting ducts that expressed sst1 were evaluated, 50% were AH+/THP-, 39% were AH+/THP+, and 11% were THP+/AH- (Fig. 6B). Of all distal tubules or collecting ducts that expressed sst2, 30% were AH+/THP-, 35% were AH+/THP+, and 35% were THP+/AH- (Fig. 6B). These results indicate that sst1 and sst2 are expressed in overlapping, but distinct, patterns in human renal cells in vivo. sst1 is expressed predominately in AH+ collecting duct cells or distal tubular cells and less commonly in THP+ tubules. In contrast, sst2 is more widely expressed than sst1 and is expressed in the majority of AH+ and THP+ tubules.

PHA-E+ tubules never stained intensely positive for either sst1 or sst2 (Fig. 5). However, many proximal tubules were lightly stained with sst2 above the background staining obtained with preimmune serum. Such sst2 staining is especially evident in Fig. 5B. Occasional proximal tubules were also weakly positive for sst1. Proximal tubular staining was blocked by preincubation with immunizing peptide. These results suggest that proximal tubules also express sst2 and sst1 but at a much lower level than in AH+ and/or THP+ tubules.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we discovered that somatostatin receptor subtypes 1 and 2 are expressed widely throughout the distal nephron and collecting duct in normal human kidney. The physiological source of somatostatin that might bind to these receptors is not known. We previously reported that freshly isolated human renal cortex and cultured human mesangial and tubular epithelial cells express somatostatin mRNA and that mesangial and tubular cells secrete somatostatin peptide (35, 37). The finding that renal cells produce somatostatin and express somatostatin receptors implies that somatostatin may modulate renal function in an autocrine/paracrine manner. In addition, circulating somatostatin may modulate renal function. Fasting plasma somatostatin concentrations are less than 0.02 nM in normal plasma samples (20), which is below the 0.5 nM equilibrium dissociation constant (Kd) calculated for somatostatin receptors in intact human kidney (23). However, somatostatin filtered from the circulation or secreted into the urinary space could modulate downstream tubular cell function.

The finding that sst1 and sst2 are expressed predominately in the distal nephron and collecting duct implies that these receptors play an important role in renal function, perhaps for tubular transport or regulation of cell proliferation and differentiation. Somatostatin inhibits contraction of human mesangial cells (7, 8), vasopressin-induced water permeability of microperfused rat papillae (22), cAMP production in cultured rat renal collecting tubule cells (13), and proliferation of OK proximal tubular cells (10), human renal tubular cells (36), and rat mesangial cells (27).

In addition to these effects, somatostatin may have other direct effects on renal cell function and gene expression because somatostatin receptors are linked to a multitude of intracellular signaling pathways. For example, depending on which sst receptor subtype and G proteins are expressed in various cells, somatostatin can lead to inhibition of adenylate cyclase, to stimulation of tyrosine or serine/threonine phosphatase activity, activation of guanylate cyclase, or to modulation of calcium or potassium fluxes (8, 21, 41). Somatostatin also directly inhibits insulin-like growth factor-1 (IGF-1) synthesis (29) and modulates expression of IGF-1 binding protein-1 (6). Furthermore, somatostatin leads to inhibition of c-fos transcription and AP-1 DNA binding (31-33). The widespread expression of somatostatin receptors in the kidney implies that somatostatin plays an important role in regulating renal cell function. Therefore, future studies aimed at understanding how somatostatin modulates kidney function, growth, and development in normal and pathological states are highly warranted.

The availability of somatostatin receptor subtype-selective analogs may allow precise manipulation of the response of renal cells to somatostatin. In recent years somatostatin analogs have been used in the treatment of a wide variety of diseases (21). These analogs have been found to be exceptionally safe and cause relatively few side effects. We propose that somatostatin analogs may be useful to treat several renal disorders. The capacity of somatostatin analogs to inhibit proliferation of tumor cells, which correlates with sst receptor expression, is presently being utilized clinically to treat sst receptor-positive tumors (11). Our observation that somatostatin inhibits renal tubular cell proliferation (36) and the fact that sst receptor binding sites are present on 72% of renal cell carcinomas (24) indicates that somatostatin analogs may be useful for diagnosis and treatment of this malignancy (4). Somatostatin not only inhibits proliferation of cultured rat mesangial cells (27), it also decreases mesangial cell proliferation in vivo after subtotal nephrectomy in rats (38). Because many forms of acute glomerulonephritis, including IgA nephropathy, membranoproliferative glomerulonephritis, and proliferative lupus nephritis, are characterized by excessive mesangial cell proliferation (1), somatostatin analogs may be useful for inhibiting excessive mesangial cell proliferation in such diseases.

Systemic administration of the somatostatin analog octreotide (100 µg twice per day) for 6 mo after induction of diabetes with streptozotocin in rats decreases renal hypertrophy and urinary albumin excretion (5). Similarly, Igarashi et al. (12) reported that octreotide treatment decreased albuminuria in diabetic rats. Furthermore, Gronbaek et al. (9) demonstrated that after three mo of untreated streptozotocin-induced diabetes, the combination of octreotide and captopril (an angiotensin converting enzyme inhibitor) was superior to captopril alone in reducing urinary albumin excretion and renal growth. Although such animal studies of diabetic nephropathy are promising, results in small clinical trials have been variable (14, 28). However, as more knowledge is gained about the renal effects of somatostatin and somatostatin receptor expression, use of somatostatin analogs may be optimized to achieve results in humans that are similar to the results obtained in animal models of diabetic nephropathy.

We speculate that the binding of somatostatin to sst1 compared with sst2 will have distinct effects. The lack of staining for sst1 in the glomerulus suggests that only sst2-selective analogs will influence mesangial cell function. The regulation of sst receptor expression in the kidney is not known. Thus the pattern of sst receptor subtype expression may be altered under pathological conditions. Because somatostatin influences a multitude of signaling pathways, it is clear that as more is elucidated about how somatostatin impacts on renal signaling pathways, and how renal somatostatin receptor expression is regulated, novel methods to use sst receptor subtype-selective somatostatin analogs to modulate renal cell function in pathological conditions will be developed.


    ACKNOWLEDGEMENTS

We thank Courtney A. Apple for superb technical help and Carl M. Bates for helpful review of the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-51707 (M. A. Turman) and The Children's Research Institute, Children's Hospital, Columbus, Ohio (M. A. Turman).

Address for reprint requests and other correspondence: M. A. Turman , Section of Nephrology, Children's Hospital, 700 Children's Drive, Columbus, OH 43205 (E-mail: mturman{at}chi.osu.edu).

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. Section 1734 solely to indicate this fact.

Received 2 August 2000; accepted in final form 1 November 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abboud, HE. Growth factors and the mesangium. J Am Soc Nephrol 2: S185-S189, 1992[Abstract].

2.   Albers, AR, O'Dorisio MS, Balster DA, Caprara M, Gosh P, Chen F, Hoeger C, Rivier J, Wenger GD, O'Dorisio TM, and Qualman SJ. Somatostatin receptor gene expression in neuroblastoma. Regul Pept 88: 61-73, 2000[ISI][Medline].

3.   Díez-Marqués, ML, García-Escribano C, Medina J, Boyano-Adanez MC, Arilla E, Torrecilla G, Rodríguez-Puyol D, and Rodríguez-Puyol M. Effects of somatostatin on cultured human mesangial cells. Endocrinology 136: 3444-3451, 1995[Abstract].

4.   Flamen, P, Bossuyt A, De Greve J, Pipeleers-Marichal M, Keuppens F, and Somers G. Imaging of renal cell cancer with radiolabelled octreotide. Nucl Med Commun 14: 873-877, 1993[ISI][Medline].

5.   Flyvbjerg, A, Marshall SM, Frystyk J, Hansen KW, Harris AG, and Orskov H. Octreotide administration in diabetic rats: effects on renal hypertrophy and urinary albumin excretion. Kidney Int 41: 805-812, 1992[ISI][Medline].

6.   Flyvbjerg, A, Schuller AG, van Neck JW, Groffen C, Orskov H, and Drop SL. Stimulation of hepatic insulin-like growth factor-binding protein-1 and -3 gene expression by octreotide in rats. J Endocrinol 147: 545-551, 1995[Abstract].

7.   Garcia-Escribano, C, Diez-Marques ML, Gonzalez-Rubio M, Rodriguez-Puyol M, and Rodriguez-Puyol D. Somatostatin antagonizes angiotensin II effects on mesangial cell contraction and glomerular filtration. Kidney Int 43: 324-333, 1993[ISI][Medline].

8.   Garcia-Escribano, C, Diez-Marques ML, Medina-Alonso J, Rodriguez-Puyol M, and Rodriguez-Puyol D. Somatostatin activates particulate guanylate cyclase in cultured rat mesangial cells. Kidney Int 46: 1611-1615, 1994[ISI][Medline].

9.   Gronbaek, H, Vogel I, Osterby R, Lancranjan I, Flyvbjerg A, and Orskov H. Effect of octreotide, captopril or insulin on renal changes and UAE in long-term experimental diabetes. Kidney Int 53: 173-180, 1998[ISI][Medline].

10.   Hatzoglou, A, Bakogeorgou E, Papakonstanti E, Stournaras C, Emmanouel DS, and Castanas E. Identification and characterization of opioid and somatostatin binding sites in the opossum kidney (OK) cell line and their effect on growth. J Cell Biochem 63: 410-421, 1996[ISI][Medline].

11.   Hofland, LJ, Visser-Wisselaar HA, and Lamberts SWJ Somatostatin analogs: clinical application in relation to human somatostatin receptor subtypes. Biochem Pharmacol 50: 287-297, 1995[ISI][Medline].

12.   Igarashi, K, Nakazawa A, Tani N, Yamazaki M, Ito S, and Shibata A. Effect of a somatostatin analogue (SMS 201-995) on renal function and urinary protein excretion in diabetic rats. J Diabetes Complications 5: 181-183, 1991.

13.   Ishikawa, S, Saito T, and Kuzuya T. Reversal of somatostatin inhibition of AVP-induced cAMP by pertussis toxin. Kidney Int 33: 536-542, 1988[ISI][Medline].

14.   Jacobs, ML, Derkx FHM, Stijnen T, Lamberts SWJ, and Weber RFA Effect of long-acting somatostatin analog (Somatulin) on renal hyperfiltration in patients with IDDM. Diabetes Care 20: 632-636, 1997[Abstract].

15.   Mitsuma, T, Rhue N, Hirooka Y, Kayama M, Mori Y, Wago T, Takagi J, Ping J, Nogimori T, and Sakai J. Distribution of somatostatin receptor type 3 in the rat: immunohistochemical study. Endocr Regul 31: 187-192, 1997[Medline].

16.   Mitsuma, T, Rhue N, Kayama M, Izumi M, Adachi K, Ohtake M, Yuichi M, Hirooka Y, Nogimori T, and Sakai J. Distribution of somatostatin receptor type 2 in the rat: immunohistochemical study. Endocr Regul 30: 67-72, 1996[Medline].

17.   Mitsuma, T, Rhue N, Kayama M, Yokoi Y, Izumi M, Adachi K, Hirooka Y, Nogimori T, Sakai J, and Sugei I. Distribution of somatostatin receptor type I in the rat. An immunohistochemical study. Endocr Regul 29: 189-193, 1995[Medline].

18.   Murad, AO, de Cock J, Brown D, and Smerdon MJ. Variations in transcription-repair coupling in mouse cells. J Biol Chem 270: 3949-3957, 1995[Abstract/Free Full Text].

19.   Neufeld, TK, Douglass D, Grant M, Ye M, Silva F, Nadasdy T, and Grantham JJ. In vitro formation and expansion of cysts-derived from human renal cortex epithelial cells. Kidney Int 41: 1222-1236, 1992[ISI][Medline].

20.   O'Dorisio, TM, Mekhjian HS, Ellison EC, O'Dorisio MS, Gaginella TS, and Woltering EA. Role of peptide radioimmunoassay in understanding peptide-peptide interactions and clinical expression of gastroenteropancreatic endocrine tumors. Am J Med 82: 60-67, 1987[ISI][Medline].

21.   Patel, YC. Somatostatin and its receptor family. Front Neuroendocrinol 20: 157-198, 1999[ISI][Medline].

22.   Ray, C, Carney S, Morgan T, and Gillies A. Somatostatin as a modulator of distal nephron water permeability. Clin Sci (Lond) 84: 455-460, 1993[ISI][Medline].

23.   Reubi, JC, Horisberger U, Studer UE, Waser B, and Laissue JA. Human kidney as target for somatostatin: high affinity receptors in tubules and vasa recta. J Clin Endocrinol Metab 77: 1323-1328, 1993[Abstract].

24.   Reubi, JC, and Kvols L. Somatostatin receptors in human renal cell carcinomas. Cancer Res 52: 6074-6078, 1992[Abstract].

25.   Reubi, JC, Schaer JC, Waser B, and Mengod G. Expression and localization of somatostatin receptor sstR1, sstR2, and sstR3 messenger RNAs in primary human tumors using in situ hybridization. Cancer Res 54: 3455-3459, 1994[Abstract].

26.   Roca, B, Arilla E, and Prieto JC. Evidence for somatostatin binding sites in rabbit kidney. Regul Pept 13: 273-281, 1986[ISI][Medline].

27.   Ruiz-Torres, P, Lucio FJ, Gonzalez-Rubio M, Rodriguez-Puyol M, and Rodriguez-Puyol D. A dual effect of somatostatin on the proliferation of cultured rat mesangial cells. Biochem Biophys Res Commun 195: 1057-1062, 1993[ISI][Medline].

28.   Serri, O, Beauregard H, Brazeau P, Abribat T, Lambert J, Harris A, and Vachon L. Somatostatin analogue, octreotide, reduces increased glomerular filtration rate and kidney size in insulin-dependent diabetes. JAMA 265: 888-892, 1991[Abstract].

29.   Serri, O, Brazeau P, Kachra Z, and Posner B. Octreotide inhibits insulin-like growth factor-I hepatic gene expression in the hypophysectomized rat: evidence for a direct and indirect mechanism of action. Endocrinology 130: 1816-1821, 1992[Abstract].

30.   Takahashi, T, Shirasawa T, Miyake K, Yahagi Y, Maruyama N, Kasahara N, Kawamura T, Matsumura O, Mitarai T, and Sakai O. Protein tyrosine kinases expressed in glomeruli and cultured glomerular cells: Flt-1 and VEGF expression in renal mesangial cells. Biochem Biophys Res Commun 209: 218-226, 1995[ISI][Medline].

31.   Todisco, A, Campbell V, Dickinson CJ, DelValle J, and Yamada T. Molecular basis for somatostatin action: inhibition of c-fos expression and AP-1 binding. Am J Physiol Gastrointest Liver Physiol 267: G245-G253, 1994[Abstract/Free Full Text].

32.   Todisco, A, Seva C, Takeuchi Y, Dickinson CJ, and Yamada T. Somatostatin inhibits AP-1 function via multiple protein phosphatases. Am J Physiol Gastrointest Liver Physiol 269: G160-G166, 1995[Abstract/Free Full Text].

33.   Todisco, A, Takeuchi Y, Yamada J, Sadoshima JI, and Yamada T. Molecular mechanisms for somatostatin inhibition of c-fos gene expression. Am J Physiol Gastrointest Liver Physiol 272: G721-G726, 1997[Abstract/Free Full Text].

34.   Tulassay, T, Tulassay Z, Rascher W, Szucs L, Seyberth HW, and Nagy I. Effect of somatostatin on kidney function and vasoactive hormone systems in healthy subjects. Klin Wochenschr 69: 486-490, 1991[ISI][Medline].

35.   Turman, MA, and Apple CA. Human proximal tubular epithelial cells express somatostatin: regulation by growth factors and cAMP. Am J Physiol Renal Physiol 274: F1095-F1101, 1998[Abstract/Free Full Text].

36.   Turman, MA, and Kaisser TE. Somatostatin inhibits epidermal growth factor-induced mitogenesis in human proximal tubular cells. Pediatr Nephrol 12: C94-C94, 1998.

37.   Turman, MA, O'Dorisio MS, O'Dorisio TM, Apple CA, and Albers AR. Somatostatin expression in human renal cortex and mesangial cells. Regul Pept 68: 15-21, 1997[ISI][Medline].

38.   Uemasu, J, Tokumoto A, Godai K, Kawasaki H, and Hirayama C. Effects of chronic administration of somatostatin analogue SMS 201-995 on the progression of chronic renal failure in subtotal nephrectomized rats. Exp Clin Endocrinol 96: 97-104, 1990[ISI][Medline].

39.   Vikic-Topic, S, Raisch KP, Kvols LK, and Vuk-Pavlovic S. Expression of somatostatin receptor subtypes in breast carcinoma, carcinoid tumor, and renal cell carcinoma. J Clin Endocrinol Metab 80: 2974-2979, 1995[Abstract].

40.   Vora, JP, Owens DR, Ryder R, Atiea J, Luzio S, and Hayes TM. Effect of somatostatin on renal function. Br Med J 292: 1701-1702, 1986[ISI][Medline].

41.   Yamada, Y, Post SR, Wang K, Tager HS, Bell GI, and Seino S. Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney. Proc Natl Acad Sci USA 89: 251-255, 1992[Abstract].


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