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1 Howard Florey Institute,
2 Department of Biochemistry and Molecular Biology,
3 Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia
(Requests for offprints should be addressed to A L Gundlach; Email: a.gundlach{at}hfi.unimelb.edu.au)
(P Fu is now at Department of Medicine, Monash University, Alfred Hospital, Prahran, Victoria 3181, Australia)
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Introduction |
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A genetic lack of INSL3 causes cryptorchidism – a developmental disorder involving failure of testis descent associated with defective spermatogenesis and infertility, and a high risk of testicular malignancy (Hutson et al. 1997). INSL3 normally induces growth and differentiation of gubernacula during the initial trans-abdominal movement of the testes (Hutson et al. 1997, Adham et al. 2000). Male mice homozygous for a disruption of the Insl3 gene exhibit bilateral cryptorchidism (Nef & Parada 1999, Zimmermann et al. 1999), while female homozygotes display impaired fertility with abnormal oestrous cycles (Nef & Parada 1999). In contrast, transgenic overexpression of Insl3 causes ovaries to descend into the inguinal region due to an overdeveloped gubernaculum (Adham et al. 2002).
Recent data have demonstrated that the native receptor for INSL3 is the type C leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8) (Hsu et al. 2002, Kumagai et al. 2002) (known also as RXFP2; see Bathgate et al. 2005 for review). INSL3 bound with high affinity to LGR8 and receptor activation by INSL3 was associated with cAMP signalling. LGR8 mRNA was detected in human gubernaculum and treatment of gubernacular cells with INSL3 stimulated cAMP production and thymidine incorporation (Kumagai et al. 2002). Transgenic mice with a deletion of Lgr8 (also known as Great) exhibit cryptorchidism (Overbeek et al. 2001), a phenotype identical to Insl3-deficient mice (see Bogatcheva et al. 2003). Furthermore, analysis of human INSL3 and LGR8 mutations confirms cryptorchidism has a genetic aetiology related to these genes and INSL3–LGR8 signalling (Ferlin et al. 2003).
Importantly, LGR8 mRNA has been detected in a number of human tissues besides the gubernaculum, including ovary, testis, uterus, brain and kidney (Hsu et al. 2002). Until recently though, little has been published on the regional and cellular distribution of LGR8 expression or the functional role of INSL3 in these tissues of humans or other species (Kawamura et al. 2004). In an attempt to further elucidate the biology of the INSL3–LGR8 system in the rat, we cloned and sequenced rat Lgr8 cDNA and screened various tissues for Lgr8 mRNA expression using RT-PCR (Scott et al. 2005). Based on these initial findings, we employed in situ hybridization histochemistry to localize Lgr8 expression in adult rat kidney and subsequently examined the temporal and spatial expression profile during development, using in situ hybridization histochemistry and quantitative real-time PCR. Results revealed Lgr8 mRNA associated with mature glomeruli during development and in adulthood, and subsequent autoradiographic studies with 125I-labelled human INSL3 revealed specific binding sites associated with glomeruli in renal cortex. Therefore, using primary cultures of renal glomerular cells isolated from rat kidney (containing mesangial and other cell types), we identified Lgr8 mRNA expression in these cells by RT-PCR analysis and examined the effect of INSL3 treatment on their viability. (A preliminary account of some of these findings appeared in a monograph on the proceedings of the Relaxin 2004 Conference (Fu et al. 2005)).
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Materials and Methods |
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All procedures were approved by the Howard Florey Institute Animal Welfare Committee and were performed in strict accordance with guidelines of the National Health and Medical Research Council of Australia. Adult and postnatal Sprague–Dawley rats were deeply anaesthetized with the inhalation anaesthetic, isoflurane (Isoflo; Abbott Australia, Sydney, NSW, Australia) and then decapitated. Kidneys and other tissues were rapidly removed, frozen over liquid nitrogen and stored at –80 °C (see details below).
Database search, rat Lgr8 sequence amplification and alignment
As described briefly elsewhere, the protein sequence of human LGR8 (Accession No. NP_570718 [GenBank] ) was used to screen the National Center for Biotechnological Information (NCBI) high-throughput genome sequences (HTGSs) and fragmented hits were assembled through homology comparison with human LGR8 (Scott et al. 2005). Oligonucleotide primers based on the identified rat sequences were used to generate the full-length coding region via RT-PCR. Total RNA was extracted from dissected, snap-frozen rat tissues (testis, ovary, brain, kidney and embryonic day (E) 16 fetal gubernaculum) by homogenization in RNAwiz (Ambion Inc, Austin, TX, USA) and cDNA was synthesized from 2.5 µg of total RNA using an RT-kit (Promega) and random hexamer primers. Putative LGR8-coding regions were amplified from cDNA, using a Taq polymerase PCR kit (Eppendorf, Sydney, NSW, Australia), cloned into the pGEM-T vector (Promega) and confirmed using Big Dye Terminator sequencing (Applied Biosystems, Foster City, CA, USA). The full coding sequence was assembled from overlapping fragments and confirmed by RT-PCR, using primers from outside the coding region. Alignment of human, mouse and rat sequences was done using ClustalW (http://www.ebi.ac.uk/clustalw/) and Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html) (Scott et al. 2005).
Detection of Lgr8 mRNA in rat tissues and glomerular cells by RT-PCR
Rat tissues and samples of cultured rat glomerular cells were screened for Lgr8 mRNA expression using RT-PCR, as described (Kubota et al. 2002). Briefly, total RNA was extracted with RNAWiz reagent (Ambion Inc) according to the manufacturer’s instructions. Primers equivalent to nucleotides 1338–1358 and 2121–2141 of the final rat Lgr8 cDNA sequence were used for RT-PCR reactions. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control for the quality of the cDNA synthesis and was amplified using primers equivalent to nucleotides 789–808 and 1017–1036 of rat GAPDH cDNA (Accession No. NM_017008 [GenBank] ). All the primers were designed to cross intron–exon boundaries to control for genomic DNA contamination.
In situ hybridization histochemistry
Frozen kidney sections (14 µm) from both developing (E16, E18, postnatal day (P) 2, 5, 10, 20, 30 and 40, n 
3 for each age group) and adult rats were cut on a cryostat at –16 °C and thaw-mounted onto microscope slides coated with 0.2% poly-L-lysine (Mr 300 000). Slides were stored at –80 °C until use. Lgr8 mRNA was detected with three synthetic oligonucleotide probes corresponding to nucleotides 181–219, 1371–1409 and 2018–2056 of rat Lgr8 cDNA sequence. Probes were labelled with [
-35S]dATP ( 1200 Ci/mmol; Perkin-Elmer Life Sciences, Melbourne, VIC, Australia) using terminal deoxynucleotidyl transferase (Roche Diagnostics) to a specific activity of 1–2 x 109 d.p.m./µg. Sections were hybridized as described (Shen & Gundlach 1996, Wisden & Morris 2002) and then exposed to Kodak Biomax film (Integrated Sciences, Sydney, NSW, Australia) for 4–5 weeks, or coated with nuclear emulsion (Ilford K5, diluted 1:1 with dH2O; Ilford Imaging, Melbourne, VIC, Australia) and exposed for 20–25 weeks, prior to development, counterstaining with 0.01% thionin and analysis using bright- and dark-field microscopy. The specificity of the hybridization signal was assessed by confirming the similarity of hybridization patterns obtained with individual probes (data not shown) and hybridization in the presence of a 100-fold excess of unlabelled oligonucleotides.
Photography and image production
Digital images of film autoradiograms were produced using a Sony XC-77CE CCD video camera coupled to a Nikon 1:2.8, 55 mm lens and a light box, with MCID M2 software (Imaging Research Inc, St Catharines, OT, Canada). Digital micrographs of nuclear-emulsion-dipped, counterstained sections were produced under bright- and dark-field illumination on an Olympus IX70 microscope (Olympus Imaging, Melbourne, VIC, Australia) equipped with a SPOT real-time digital camera with Image-pro 4.5 software (Diagnostics Instruments, Sterling Heights, MI, USA). All micrographs were archived as high-resolution images in Adobe Photoshop (Version 5.0; Adobe Systems Inc, San Jose, CA, USA) and after any required cropping, adjustment for contrast or removal of obvious artefacts, plates were compiled and labelled in PowerPoint (Microsoft Corporation).
Real time-PCR analysis of Lgr8 mRNA in rat kidney
Kidney tissue from embryonic, developing and adult rats was finely diced under liquid nitrogen and immediately homogenized with RNAWiz reagent (Ambion Inc.). Total RNA was extracted and real-time PCR was used to quantify Lgr8 mRNA levels in each sample. Real-time PCR primers and probes were designed using Primer Express (Applied Biosystems). The primer sequences are located at nucleotides 1898–1917 and 1998–1977 of the Lgr8 cDNA respectively and span a 9.0 kb intron. Total RNA (1 µg) from each sample was used for the RT, in a 30 µl reaction (Siebel et al. 2002). The probe sequence, corresponding to nucleotides 1925–1961, was labelled at the 5'-end with 6-carboxy fluorescein (Qiagen) and included with the primers in each reaction tube. The relative CT standard curve method was adopted and ribosomal RNA (18S) was used as an internal, endogenous control, with primers and probe(s) as described (Siebel et al. 2002).
125I-INSL3 autoradiography
125I-Human INSL3 binding autoradiography was performed essentially as described in earlier studies with 33P-labelled human relaxin (Burazin et al. 2002). Slide-mounted cryostat sections (14 µm) of kidney were pre-incubated in 25 mM Hepes, 5 mM MgCl2, pH 7.2 for 30 min. Slides were then incubated in the same buffer plus 0.1% BSA (w/v), 0.015% Pefabloc (w/v) and 100 pM 125I-human INSL3 (Perkin-Elmer Life Sciences) at 21 °C for 1 h. Non-specific binding was determined by the addition of 100 nM unlabelled human INSL3 (Fu et al. 2004) to the incubation buffer. Following incubation, slides were washed twice for 5 min each in ice-cold incubation buffer and rinsed in ice-cold dH2O. Sections were allowed to air-dry overnight and were apposed to Kodak Biomax film for 5 days.
Rat glomerular cell culture
Male Sprague–Dawley rats (n=12 per study) were anaesthetized using Isoflo and kidneys were rapidly removed and placed on ice. Renal cortical tissue was excised and minced with a razor blade into millimetre-sized pieces. Glomeruli were isolated by passing the minced cortex through a 150 µm steel sieve and collecting strained material on a 75 µm steel sieve. Intact glomeruli were removed from the sieve, rinsed twice in PBS and centrifuged at 400 g for 5 min. Glomeruli were resuspended and incubated with 0.1% collagenase (190 U/ml) at 37 °C for 30 min. After incubation, cells were centrifuged and the pellet was washed twice with PBS. Cells were then plated onto two 60 mm dishes in DMEM medium supplemented with 20% fetal calf serum, 2 µM glutamine, 5 µg/ml insulin, 100 U/ml penicillin, 100 µg/ml streptomycin (culture medium) in a 95% air–5% CO2 humidified atmosphere at 37 °C (Gruden et al. 1997).
Glomerular cell viability/proliferation assay
Glomerular cell samples, likely to be enriched in mesangial cells but also containing endothelial and epithelial cells, were seeded into 96-well plates at a density of 1 x 104 cells/well in culture medium in a 95% air–5% CO2 humidified atmosphere at 37 °C. After a 24 h incubation, the medium was replaced by serum-free medium containing 0.1% BSA and INSL3 or other peptide. Cells were incubated for a further 48 h and then cell viability/proliferation was assessed by an MTT assay (Hansen et al. 1989, Loveland et al. 1992). Briefly, MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; final concentration 0.5 µg/ml) was added, the cells were incubated for 3 h and then the insoluble reaction product was dissolved by the addition of 100 µl of cell lysis buffer (20% w/v SDS, 50% N,N'-dimethyl-formamide). In several wells, cells were lysed by the addition of cell-lysis buffer immediately prior the addition of MTT, to control for background absorbance. Absorbance of each well was measured at 595 nm using a Microplate Reader Model 3550-UV Spectrophotometer (Bio-Rad Laboratories). Data are expressed as a percentage of the maximum control response ± S.D. from three to five independent experiments. Statistical differences were assessed using one-way ANOVA.
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Results |
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In preliminary studies, rat Lgr8 gene was cloned and sequenced (see Scott et al. 2005) and subsequently its distribution in different rat tissues was explored by RT-PCR. Briefly, sequences homologous to regions of the human LGR8 cDNA were identified in bacterial artificial chromosome clones in the NCBI HTGS database. Using derived, specific RT-PCR primers the full rat Lgr8 cDNA was assembled by sequencing multiple, independent overlapping clones from E16 fetal rat gubernaculum (Genebank Accession No. AY906861 [GenBank] ). Cells transfected with full-length rat LGR8 demonstrate high-affinity binding for 125I-INSL3 and respond to ligand stimulation with cAMP accumulation (Scott et al. 2005). RT-PCR analysis of rat tissues revealed strong Lgr8 expression in gubernaculum, testis and ovary (data not shown; Kawamura et al. 2004). Notably, Lgr8 mRNA was also found in rat brain (Shen et al. 2005) and kidney (Fu et al. 2005; this study).
Detection of regional and cellular Lgr8 mRNA expression in adult and developing kidney
In an attempt to discover the regional and cellular distribution of these receptors in rat kidney, frozen longitudinal sections from adult (8- to 10-week-old) male rats were subjected to in situ hybridization histochemistry for Lgr8 mRNA using 35S-labelled oligonucleotides. Relatively low-resolution film autoradiograms revealed the expression of Lgr8 mRNA restricted to the renal cortex, in a pattern consistent with labelling of cells within the glomeruli (Fig. 1A
). The specificity of the Lgr8 mRNA signal detected was confirmed by several methods. In competition experiments, the signal was abolished when a 100-fold excess of unlabelled oligonucleotides was added to the incubation (Fig. 1A'). Secondly, sections hybridized with the three individual oligonucleotide probes displayed the same distribution of labelling (data not shown), and finally the oligonucleotide probes used produced strong, specific labelling of sections of gubernaculum from a 17-day rat embryo and adult testis (data not shown; Scott et al. 2005). Microscopic analysis of high-resolution nuclear emulsion autoradiograms from counterstained kidney sections indicated that Lgr8 mRNA expression was restricted to putative mesangial cells within the adult glomeruli (Fig. 1B and C), with no obvious specific hybridization observed in glomerular podocyte or epithelial cells (Hoffmann et al. 2004). Notably, parallel in situ hybridization studies with Lgr7-directed oligonucleotides (Burazin et al. 2005) failed to detect Lgr7 mRNA-positive cells in rat kidney (data not shown).
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Subsequent to the identification of cell-specific expression in adult kidney, we assessed the possible renal expression of Lgr8 mRNA in samples of developing kidney to provide further possible insights into the functional involvement of LGR8. In these studies the profile of Lgr8 expression during fetal and postnatal kidney development was examined by in situ hybridization and quantitative real-time RT-PCR. Although expression of Lgr8 was detectable in frozen sections from E16 through to adulthood, highest relative levels were observed in the embryonic stages, around E18 (Fig. 2). Shortly after birth at P2–5, a sharp decrease in Lgr8 mRNA levels was observed and the reduction continued until Lgr8 transcripts reached their lowest level in adult kidney (Fig. 2). Kidney sections from P2 rats, in which glomerulogenesis is still occurring, displayed strong Lgr8 expression in mature glomeruli, whereas little or no distinct signal was observed in immature glomeruli or the nephrogenic zone, located at the edge of the renal cortex (Fig. 3A–A''). In P10 kidney, the nephrogenic zone is absent as glomerulogenesis has been completed by this age and Lgr8 expression is abundant in mature glomeruli (Fig. 3B–B'). At all stages of nephron development studied, Lgr8 expression appeared to be restricted to cells of mature glomeruli and was not detected in any other tissue regions or cell types.
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). There was no significant change in Lgr8 mRNA levels between P5 and 10, but at P20 the levels were 12-fold lower than at P2. By adulthood, there was a further reduction in Lgr8 mRNA levels representing a 48-fold decrease in Lgr8 mRNA expression from P2. Lgr8 transcript levels measured at P20 and adulthood were also significantly lower than those measured at P5 and 10 (Fig. 4A). Notably, parallel RT-PCR studies failed to detect Insl3 mRNA in embryonic, postnatal or adult kidney, despite effective detection of high levels of transcripts in adult testis, as a positive control for the primers and protocol used (data not shown).
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INSL3 has to date been largely considered as a male hormone and investigated for its capacity to stimulate gubernaculum growth and differentiation during the process of testicular descent. The serum concentration of INSL3 is lower in adult females that males in various species (Büllesbach et al. 1999, Boockfor et al. 2001), but INSL3 is also produced by tissues such as the ovary (Büllesbach et al. 1999). Comparison of levels of Lgr8 mRNA in adult male and female kidney by quantitative real-time RT-PCR analysis revealed that Lgr8 mRNA levels were some 6-fold higher in the female than male (Fig. 4B
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Detection of 125I-INSL3-binding sites in developing and adult kidney
In studies aimed at revealing the presence and distribution of functional LGR8 in rat kidney, frozen longitudinal sections from adult and developing rats were incubated with 125I-human INSL3. Specific 125I-INSL3-binding sites were detected by X-ray film autoradiography in the renal cortex, in a pattern consistent with labelling of cells within the glomeruli (Fig. 5A). The specificity of the binding detected was confirmed by its inhibition in the presence of 100 nM unlabelled human INSL3 (Fig. 5B). In addition, specific binding was also detected in renal cortex (glomeruli) of kidney from postnatal rats (Fig. 5C–H), indicating the presence of functional LGR8 throughout this period. In kidney sections from P2–5 rats, in which very strong Lgr8 mRNA expression was detected, 125I-INSL3-binding densities associated with glomeruli were lower than those seen in more mature rats (Fig. 5C–H).
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In studies aimed at examining the presence of functional renal LGR8, adult rat glomerular cells were isolated and cultured under standard conditions (e.g. Gruden et al. 1997; see Methods). Cells were propagated in culture and passaged a total of ten times before total RNA from these cells was isolated and subjected to RT-PCR analysis for Lgr8 mRNA. The predicted 101 bp band specific for Lgr8 was detected, while under the same conditions no Lgr7 mRNA expression was detected in these cells (Fig. 6). The housekeeping gene encoding GAPDH RNA was used as a control for sample RNA content/lane loading. Total RNAs isolated from kidney, E17 gubernaculum and cervix were used as positive controls for Lgr8 and Lgr7 expression. In contrast to in situ hybridization studies, weak expression of Lgr7 mRNA was detected in whole kidney by RT-PCR (Fig. 6), possibly associated with expression by the renal vasculature (Danielson et al. 1999).
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Glomerular cells were incubated in serum-free medium containing 0.1% BSA to assay cell viability and proliferation and treatment with 10 nM INSL3 for 48 h resulted in a 25% decrease in cell proliferation (P<0.01; Fig. 7A). In contrast, treatment of these cells with another insulin/relaxin family peptide, INSL4 (10 nM), had no significant effect on cell numbers. Human relaxin, which has been shown to bind to LGR8 transfected into human fetal kidney fibroblast 293T cells and induce cellular cAMP accumulation, had no effect on cell proliferation at 10 nM, but a 100 nM concentration produced an ~15% decrease (P<0.05; Fig. 7A), consistent with a lower-affinity interaction with LGR8 than INSL3. The effect of INSL3 was also shown to be dose-dependent (Fig. 7B), with an approximate IC50 between 1 and 10 nM. There is a possibility that INSL3 and the other peptides added to the incubation may have had differential or separate effects on the different cell types present in the mixed culture that may have biased the results observed and further specific tests are required to determine whether INSL3 promotes apoptosis in this system, but otherwise these data indicate that INSL3 is a specific and potent inhibitor of renal cell proliferation.
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Discussion |
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In situ hybridization studies with 35S-labelled oligonucleotides consistently and exclusively detected Lgr8 mRNA in mature glomeruli (possibly in mesangial cells) during development and in adulthood. Levels of renal Lgr8 mRNA expression were also quantified at various stages of kidney development by real-time PCR, with highest levels detected at E18 and expression observed at all times studied. Radioligand binding studies with 125I-INSL3 confirmed the presence of somewhat differing levels of functional LGR8 binding sites in renal glomeruli during development and in adulthood. These anatomical findings were extended by in vitro studies that revealed that cultured glomerular cells isolated from adult kidney expressed Lgr8 transcripts and that treatment of these cells with INSL3 reduced their number by inhibiting proliferation (or promoting apoptosis?). These data indicate that functional LGR8 (receptors) might be present on mesangial cells and/or on endothelial and epithelial cells, and is consistent with the abundant, specific expression of LGR8 by cells in mature glomeruli in embryonic, early postnatal and adult kidney. It suggests a potentially important role for INSL3–LGR8 signalling in regulating the development, maturation and/or normal function of these renal cells in rat and possibly other species, including mouse and human.
Glomerulogenesis involves four major cellular processes – migration, proliferation, differentiation and apoptosis. Active migration, proliferation and differentiation of mesangial cells occur largely in immature glomeruli. Glomerular mesangial cells are, however, known to have a limited capacity for proliferation and cease to proliferate when mature glomeruli are formed. In normal adult kidney, the turnover rate of mesangial cells is 1% per day (Pabst & Sterzel 1983). The Lgr8/LGR8 expression pattern observed is very similar to that for the angiotensin II receptor-1 (Harrison-Bernard et al. 1997, Norwood et al. 1997, Ma et al. 2004), with both mRNAs/proteins highly expressed in mature glomeruli (possibly by mesangial cells), but not in more immature comma- or S-shaped bodies – implying that like angiotensin II, INSL3 is more likely to be involved in the inhibition of mesangial cell proliferation and/or apoptosis during glomerulogenesis, than in differentiation and/or migration (Ma et al. 2004). This speculation is supported by the current in vitro experiments in which treatment of cultured glomerular cells (enriched in mesangial cells) with synthetic INSL3 resulted in apparent inhibition of proliferation, suggesting that like transforming growth factor-ß (TGF-ß) (Jaffer et al. 1989), INSL3 (circulating) is a growth-inhibitory factor for these renal cells.
Glomerular mesangial cells play an important role in maintaining the structure/function of the capillary ultra-filtration apparatus. Maintaining the correct number of mesangial cells is important for renal physiology and abnormalities are associated with renal pathology. Thus, uncontrolled mesangial cell proliferation is a primary event leading to the pathogenesis of progressive renal diseases including diabetic nephropathy, membrane-proliferative glomerulonephritis and lupus nephritis (Haas et al. 1999). Increased mesangial cell proliferation may also be essential for glomerular repair. Several growth factors and mediators can regulate mesangial cell proliferation, including platelet-derived growth factor (Blom et al. 2001), basic fibroblast growth factor (Floege et al. 1993), insulin-like growth factors (Feld et al. 1995) and TGF-ß (Jaffer et al. 1989). Among these, TGF-ß is the only peptide with anti-mitogenic and anti-inflammatory actions (Schoecklmann et al. 1997, 1999), but TGF-ß also plays an important role in the pathogenesis of glomerular disease and mediates extracellular matrix deposition leading to glomerulosclerosis (Shankland & Johnson 1998). Nonetheless, the possibility that INSL3 inhibits mesangial cell proliferation (and/or promotes apoptosis) predicts a potentially beneficial application of the INSL3–LGR8 system in glomerular diseases, especially those involving excessive mesangial cell proliferation.
A characteristic action of INSL3 is to stimulate gubernacular cell proliferation and differentiation during testicular descent (see Introduction). LGR8 transcripts are present in human gubernaculum and INSL3 stimulates cAMP production and thymidine incorporation in cultured E17 gubernaculum (Kubota et al. 2002, Kumagai et al. 2002). In rat testis, however, INSL3 inhibits germ cell apoptosis (Kawamura et al. 2004). Thus, in rat kidney INSL3 may have a different function to that in gubernacular or germ cells – inhibiting cell proliferation. Such differential, cell-type specific effects have been observed for other peptide ligand–receptor systems, including members of the insulin/relaxin superfamily (Saile et al. 2004).
Expression of Lgr8 mRNA was 6-fold higher in female than male rat adult kidney. The serum INSL3 concentration is reportedly higher in adult men than in post-pubertal women (Büllesbach et al. 1999) and higher in adult male rats than females (Boockfor et al. 2001). Whether the higher level of Lgr8 in female rat kidney reflects a biological compensation for the low level of INSL3 in female serum and is reflected by a correspondingly higher expression of LGR8 protein in female kidney requires further investigation, along with studies of the precise relationship between gene and protein expression in renal glomeruli during development (in males and females). In this respect it is possible that levels of 125I-INSL3 binding observed accurately reflect the densities of LGR8 protein present in developing kidney or that binding levels are influenced by the level of endogenous ligand present in the tissue prior to its collection. The ratio of transcript to protein and the exact nature of the glomerular cells expressing LGR8 (mesangial or other?) might be better understood following the eventual development and application of specific antisera to rat LGR8. Nevertheless our current findings, along with the apparent absence of endogenous renal Insl3 expression, suggest that actions of circulating INSL3 at LGR8 may play a role in regulating both male and female kidney function.
Insl3- and Lgr8-deficient mice have been generated by several independent groups (Nef & Parada 1999, Zimmermann et al. 1999, Overbeek et al. 2001, Kamat et al. 2004), but their investigations have not reported any phenotypic kidney abnormalities. This suggests that INSL3 may not be essential for mouse nephrogenesis, but at this stage some role for INSL3 in embryonic kidney development cannot be precluded. It is perhaps likely that other mediators can compensate for the lack of INSL3 and/or LGR8 activity, but alternatively there may be some minor phenotypic changes in these knockout mice which have not been discovered. Interestingly, Nef & Parada (1999) reported that mice lacking Insl3 produced a somewhat smaller average litter size, but it remains to be determined whether this is due to some germ cell abnormalities (Kawamura et al. 2004). Future studies in these insl3–/– mice should allow clarification of the physiological role of the INSL3–LGR8 system in renal development and normal renal function. In this context, the distribution of LGR8 should also be assessed in wild-type mouse kidney and compared with that in the rat.
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References |
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Adham IM, Steding G, Thamm T, Bullesbach EE, Schwabe C, Paprotta I & Engel W 2002 The overexpression of the Insl3 gene in female mice causes descent of the ovaries. Molecular Endocrinology 16 244–252.
Balvers M, Spiess AN, Domagalski R, Hunt N, Kilic E, Mukhopadhyay AK, Hanks E, Charlton HM & Ivell R 1998 Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary. Endocrinology 136 2960–2970.
Bathgate RAD, Samuel CS, Burazin TCD, Gundlach AL & Tregear GW 2003 Relaxin: new peptides, receptors and novel actions. Trends in Endocrinology and Metabolism 14 207–213.[CrossRef][ISI][Medline]
Bathgate RAD, Ivell R, Sanborn BM, Sherwood OD & Summers RJ 2005 Receptors for relaxin family peptides. Annals of the New York Academy of Sciences 1041 61–76.
Blom IE, van Dijk AJ, Wieten L, Duran K, Ito Y, Kleij L, deNichilo M, Rabelink TJ, Weening JJ, Aten J et al. 2001 In vitro evidence for differential involvement of CTGF, TGFß, and PDGF-BB in mesangial response to injury. Nephrology, Dialysis, Transplantation 16 1139–1148.
Bogatcheva NV & Agoulnik AI 2005 INSL3/LGR8 role in testicular descent and cryptorchidism. Reproductive Biomedicine Online 10 49–54.[ISI][Medline]
Bogatcheva NV, Truong A, Feng S, Engel W, Adham IM & Agoulnik AI 2003 GREAT/LGR8 is the only receptor for insulin-like 3 peptide. Molecular Endocrinology 17 2639–2646.
Boockfor FR, Fullbright G, Bullesbach EE & Schwabe C 2001 Relaxin-like factor (RLF) serum concentrations and gubernaculum RLF receptor display in relation to pre- and neonatal development of rats. Reproduction 122 899–906.[Abstract]
Büllesbach EE & Schwabe C 2005 LGR8 signal activation by the relaxin-like factor. Journal of Biological Chemistry 280 14586–14590.
Büllesbach EE, Rhodes R, Rembiesa B & Schwabe C 1999 The relaxin-like factor is a hormone. Endocrine 10 167–169.[CrossRef][ISI][Medline]
Burazin TCD, Bathgate RAD, Macris M, Layfield S, Gundlach AL & Tregear GW 2002 Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain. Journal of Neurochemistry 82 1553–1557.[CrossRef][ISI][Medline]
Burazin TCD, Johnson KJ, Ma S, Bathgate RAD, Tregear GW & Gundlach AL 2005 Localization of LGR7 (relaxin receptor) mRNA and protein in rat forebrain: Correlation with relaxin binding site distribution. Annals of the New York Academy of Sciences 1041 205–210.
Danielson LA, Sherwood OD & Conrad KP 1999 Relaxin is a potent renal vasodilator in conscious rats. Journal of Clinical Investigation 103 525–533.[ISI][Medline]
Feld SM, Hirschberg R, Artishevsky A, Nast C & Adler SG 1995 Insulin-like growth factor I induces mesangial proliferation and increases mRNA and secretion of collagen. Kidney International 48 45–51.[ISI][Medline]
Ferlin A, Simonato M, Bartoloni L, Rizzo G, Bettella A, Dottorini T, Dallapiccola B & Foresta C 2003 The INSL3–LGR8/GREAT ligand-receptor pair in human cryptorchidism. Journal of Clinical Endocrinology and Metabolism 88 4273–4279.
Floege J, Eng E, Young BA, Alpers CE, Barrett TB, Bowen-Pope DF & Johnson RJ 1993 Infusion of platelet-derived growth factor or basic fibroblast growth factor induces selective glomerular mesangial cell proliferation and matrix accumulation in rats. Journal of Clinical Investigation 92 2952–2962.[ISI][Medline]
Fu P, Layfield S, Ferraro T, Tomiyama H, Hutson J, Otvos LJ, Tregear GW, Bathgate RAD & Wade JD 2004 Synthesis, conformation, receptor binding and biological activities of monobiotinylated human insulin-like peptide 3. Journal of Peptide Research 63 91–98.[CrossRef][ISI][Medline]
Fu P, Shen P-J, Zhao C-X, Scott DJ, Samuel CS, Wade JD, Tregear GW, Bathgate RAD & Gundlach AL 2005 Detection, localization and action of the INSL3 receptor – LGR8 – in rat kidney. Annals of the New York Academy of Sciences 1041 516–519.
Gruden G, Thomas S, Burt D, Lane S, Chusney G, Sacks S & Viberti G 1997 Mechanical stretch induces vascular permeability factor in human mesangial cells: mechanisms of signal transduction. PNAS 94 12112–12116.
Haas CS, Schocklmann HO, Lang S, Kralewski M & Sterzel RB 1999 Regulatory mechanism in glomerular mesangial cell proliferation. Journal of Nephrology 12 405–415.[ISI][Medline]
Hansen MB, Nielsen SE & Berg K 1989 Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. Journal of Immunological Methods 119 203–210.[CrossRef][ISI][Medline]
Harrison-Bernard LM, Navar LG, Ho MM, Vinson GP & el-Dahr SS 1997 Immunohistochemical localization of ANG II AT1 receptor in adult rat kidney using a monoclonal antibody. American Journal of Physiology 273 F170–F177.[ISI][Medline]
Hoffmann S, Podlich D, Hahnel B, Kriz W & Gretz N 2004 Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats. Journal of the American Society of Nephrology 15 1475–1487.
Hsu SYT 2003 New insights into the evolution of the relaxin-LGR signaling system. Trends in Endocrinology and Metabolism 14 303–309.[CrossRef][ISI][Medline]
Hsu SYT, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD & Hsueh AJW 2002 Activation of orphan receptors by the hormone relaxin. Science 295 671–674.
Hutson JM, Hasthorpe S & Heyns CF 1997 Anatomical and functional aspects of testicular descent and cryptorchidism. Endocrine Reviews 18 259–280.
Ivell R 1997 Biology of the relaxin-like factor (RLF). Reviews of Reproduction 2 133–138.[Abstract]
Jaffer F, Saunders C, Shultz P, Throckmorton D, Weinshell E & Abboud HE 1989 Regulation of mesangial cell growth by polypeptide mitogens. Inhibitory role of transforming growth factor beta. American Journal of Pathology 135 261–269.[Abstract]
Kamat AA, Feng S, Bogatcheva NV, Truong A, Bishop CE & Agoulnik AI 2004 Genetic targeting of relaxin and insulin-like factor 3 receptors in mice. Endocrinology 145 4712–4720.
Kawamura K, Kumagai J, Sudo S, Chun S-Y, Pisarska M, Morita H, Toppari J, Fu P, Wade JD, Bathgate RAD et al. 2004 Paracrine regulation of oocyte maturation and male germ cell survival. PNAS 101 7323–7328.
Kubota Y, Temelcos C, Bathgate RAD, Smith KJ, Scott DJ, Zhao C & Hutson JM 2002 The role of insulin 3, testosterone, Mullerian inhibiting substance and relaxin in rat gubernacular growth. Molecular Human Reproduction 8 900–905.
Kumagai J, Hsu SY, Matsumi H, Roh JS, Fu P, Wade JD, Bathgate RAD & Hsueh AJW 2002 INSL3/Leydig insulin-like peptide activates the LGR8 receptor important in testis descent. Journal of Biological Chemistry 277 31283–31286.
Loveland BE, Johns TG, Mackay IR, Vaillant F, Wang ZX & Hertzog PJ 1992 Validation of the MTT dye assay for enumeration of cells in proliferative and antiproliferative assays. Biochemistry International 27 501–510.[ISI][Medline]
Ma J, Matsusaka T, Yang H, Kawachi H, Shimizu F, Isaka Y, Imai E, Kon V & Ichikawa I 2004 Local actions of endogenous angiotensin II in injured glomeruli. Journal of the American Society of Nephrology 15 1268–1276.
Nef S & Parada LF 1999 Cryptorchidism in mice mutant for Insl3. Nature Genetics 22 295–299.[CrossRef][ISI][Medline]
Norwood VF, Craig MR, Harris JM & Gomez RA 1997 Differential expression of angiotensin II receptors during early renal morphogenesis. American Journal of Physiology 272 R662–R668.[ISI][Medline]
Overbeek PA, Gorlov IP, Sutherland RW, Houston JB, Harrison WR, Boettger-Tong HL, Bishop CE & Agoulnik A 2001 A transgenic insertion causing cryptorchidism in mice. Genesis 30 26–35.[CrossRef][ISI][Medline]
Pabst R & Sterzel RB 1983 Cell renewal of glomerular cell types in normal rats. An autoradiographic analysis. Kidney International 24 626–631.[ISI][Medline]
Saile B, DiRocco P, Dudas J, El-Armouche H, Sebb H, Eisenbach C, Neubauer K & Ramadori G 2004 IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts (rMF). Laboratory Investigation 84 1037–1049.[CrossRef][ISI]
Schoecklmann HO, Rupprecht HD, Zauner I & Sterzel RB 1997 TGF-beta1-induced cell cycle arrest in renal mesangial cells involves inhibition of cyclin E-cdk 2 activation and retinoblastoma protein phosphorylation. Kidney International 51 1228–1236.[ISI][Medline]
Schoecklmann HO, Lang S & Sterzel RB 1999 Regulation of mesangial cell proliferation. Kidney International 56 1199–1207.[CrossRef][ISI][Medline]
Scott DJ, Layfield S, Riesewijk A, Morita H, Tregear GW & Bathgate RAD 2004 Identification and characterization of the mouse and rat relaxin receptors as the novel orthologs of human LGR7. Clinical and Experimental Pharmacology and Physiology 31 828–832.[CrossRef][ISI][Medline]
Scott DJ, Fu P, Shen P-J, Gundlach AL, Layfield S, Riesewijk A, Tomiyama H, Hutson JM, Tregear GW & Bathgate RAD 2005 Characterization of the rat INSL3 receptor. Annals of the New York Academy of Sciences 1041 13–16.
Shankland SJ & Johnson RJ 1998 TGF-ß in glomerular disease. Mineral and Electrolyte Metabolism 24 168–173.[CrossRef][ISI][Medline]
Shen P-J & Gundlach AL 1996 Chromogranin mRNA levels in the brain as a marker for acute and chronic changes in neuronal activity: Effect of treatments including seizures, osmotic stimulation and axotomy in the rat. European Journal of Neuroscience 8 988–1000.[CrossRef][Medline]
Shen P-J, Fu P, Phelan KD, Scott DJ, Layfield S, Tregear GW, Bathgate RAD & Gundlach AL 2005 Restricted expression of LGR8 in intralaminar thalamic nuclei of rat brain suggests a role in sensorimotor systems. Annals of the New York Academy of Sciences 1041 510–515.
Siebel AL, Gehring HM, Nave CD, Bathgate RAD, Borchers CE & Parry LJ 2002 The upregulation of mesotocin receptors in the tammar wallaby myometrium is pregnancy-specific and independent of estrogen. Biology of Reproduction 66 1237–1243.
Spiess AN, Balvers M, Tena-Sempere M, Huhtaniemi I, Parry L & Ivell R 1999 Structure and expression of the rat relaxin-like factor (RLF) gene. Molecular Reproduction and Development 54 319–325.[CrossRef][ISI][Medline]
Wisden W & Morris BJ 2002 In situ hybridization with oligonucleotide probes. International Review of Neurobiology 47 3–59.[ISI][Medline]
Zimmermann S, Steding G, Emmen JM, Brinkmann AO, Nayernia K, Holstein AF, Engel W & Adham IM 1999 Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Molecular Endocrinology 13 681–691.
Received in final form 19 January 2006
Accepted 6 February 2006
Made available online as an Accepted Preprint 13 March 2006
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0.05 compared with E18; 
P
P<0.05 compared with P5 (and P10) (one-way ANOVA). (B) Comparison of Lgr8 mRNA levels in adult male and female kidneys, with mRNA levels in the male group represented as 1.0. Values represent means ± S.E.M. (n = 5 rats). *P<0.05 (one-way ANOVA).
