Calcitonin receptor antibody validation and expression in the rodent brain.

BACKGROUND AND AIM: Therapeutics that reduce calcitonin gene-related peptide activity are effective migraine treatments. However, gaps remain in our understanding of the molecular mechanisms that link calcitonin gene-related peptide to migraine. The amylin 1 receptor responds potently to calcitonin gene-related peptide, and to the related peptide amylin, but its role in relation to either peptide or to migraine is unclear. We sought to better understand the expression of the amylin 1 receptor protein subunit, the calcitonin receptor, in the rodent brain. METHODS: We profiled three antibodies for immunodetection of calcitonin receptor, using immunocytochemistry, western blotting, and calcitonin receptor conditional knockout mouse tissue. Selected migraine-relevant rat brain regions were then examined for calcitonin receptor-like immunoreactivity. RESULTS: All three antibodies detected calcitonin receptor protein but only one (188/10) produced robust immunostaining in rodent brain, under the conditions used. Calcitonin receptor-like immunoreactivity was apparent in the rat brainstem and midbrain including the locus coeruleus, periaqueductal grey and spinal trigeminal nucleus. CONCLUSIONS: Anti-calcitonin receptor antibodies require comprehensive profiling to ensure confidence in the detection of calcitonin receptor. Using a validated antibody, calcitonin receptor-like immunoreactivity was detected in several brain regions relevant to migraine. Further research is needed to understand the functional consequences of calcitonin receptor expression for calcitonin gene-related peptide or amylin physiology and pathophysiology.

Strategic Development of an Immunotoxin for the Treatment of Glioblastoma and Other Tumours Expressing the Calcitonin Receptor.

New strategies aimed at treatment of glioblastoma are frequently proposed to overcome poor prognosis. Recently, research has focused on glioma stem cells (GSCs), some quiescent, which drive expansion of glioblastoma and provide the complexity and heterogeneity of the tumour hierarchy. Targeting quiescent GSCs is beyond the capability of conventional drugs such as temozolomide. Here, we discuss the proposal that the calcitonin receptor (CT Receptor), expressed in 76-86% of patient biopsies, is expressed by both malignant glioma cells and GSCs. Forty-two percent (42%) of high-grade glioma (HGG; representative of GSCs) cell lines available from one source express CT Receptor protein in cell culture. The pharmacological calcitonin (CT)-response profiles of four of the HGG cell lines were reported, suggesting mutational/splicing inactivation. Alternative splicing, commonly associated with cancer cells, could result in the predominant expression of the insert-positive isoform and explain the atypical pharmacology exhibited by CT non-responders. A role for the CT Receptor as a putative tumour suppressor and/or oncoprotein is discussed. Both CT responders and non-responders were sensitive to immunotoxins based on an anti-CT Receptor antibody conjugated to ribosomal-inactivating proteins. Sensitivity was increased by several logs with the triterpene glycoside SO1861, an endosomal escape enhancer. Under these conditions, the immunotoxins were 250-300 times more potent than an equivalent antibody conjugated with monomethyl auristatin E. Further refinements for improving the penetration of solid tumours are discussed. With this knowledge, a potential strategy for effective targeting of CSCs expressing this receptor is proposed for the treatment of GBM.

Building the case for the calcitonin receptor as a viable target for the treatment of glioblastoma.

Researchers are actively seeking novel targeted therapies for the brain tumour glioblastoma (GBM) as the mean survival is less than 15 months. Here we discuss the proposal that the calcitonin receptor (CT Receptor), expressed in 76-86% of patient biopsies, is expressed by both malignant glioma cells and putative glioma stem cells (GSCs), and therefore represents a potential therapeutic target. Forty-two per cent (42%) of high-grade glioma (HGG; representative of GSCs) cell lines express CT Receptor protein. CT Receptors are widely expressed throughout the life cycle of organisms and in some instances promote apoptosis. Which of the common isoforms of the CT Receptor are predominantly expressed is currently unknown, but a functional response to cell stress of the insert-positive isoform is hypothesised. A model for resistant malignancies is one in which chemotherapy plays a direct role in activating quiescent stem cells for replacement of the tumour tissue hierarchy. The putative role that the CT Receptor plays in maintenance of quiescent cancer stem cells is discussed in view of the activation of the Notch-CT Receptor-collagen V axis in quiescent muscle (satellite) stem cells. The pharmacological CT response profiles of four of the HGG cell lines were reported. Both CT responders and non-responders were sensitive to an immunotoxin based on an anti-CT Receptor antibody. The CALCR mRNA exhibits alternative splicing commonly associated with cancer cells, which could result in the atypical pharmacology exhibited by CT non-responders and an explanation of tumour suppression. Due to the inherent instability of CALCR mRNA, analysis of CT Receptor protein in patient samples will lead to improved data for the expression of CT Receptor in GBM and other cancers, and an understanding of the role and activity of the splice variants. This knowledge will aid the effective targeting of this receptor for treatment of GBM.

Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive type of primary brain cancer. With median survival of less than 15 months, identification and validation of new GBM therapeutic targets is of critical importance. RESULTS: In this study we tested expression and performed pharmacological characterization of the calcitonin receptor (CTR) as well as other members of the calcitonin family of receptors in high-grade glioma (HGG) cell lines derived from individual patient tumours, cultured in defined conditions. Previous immunohistochemical data demonstrated CTR expression in GBM biopsies and we were able to confirm CALCR (gene encoding CTR) expression. However, as assessed by cAMP accumulation assay, only one of the studied cell lines expressed functional CTR, while the other cell lines have functional CGRP (CLR/RAMP1) receptors. The only CTR-expressing cell line (SB2b) showed modest coupling to the cAMP pathway and no activation of other known CTR signaling pathways, including ERK1/2 and p38 MAP kinases, and Ca2+ mobilization, supportive of low cell surface receptor expression. Exome sequencing data failed to account for the discrepancy between functional data and expression on the cell lines that do not respond to calcitonin(s) with no deleterious non-synonymous polymorphisms detected, suggesting that other factors may be at play, such as alternative splicing or rapid constitutive receptor internalisation. CONCLUSIONS: This study shows that GPCR signaling can display significant variation depending on cellular system used, and effects seen in model recombinant cell lines or tumour cell lines are not always reproduced in a more physiologically relevant system and vice versa.

Calcitonin receptor expression in medullary thyroid carcinoma.

BACKGROUND: Calcitonin expression is a well-established marker for medullary thyroid carcinoma (MTC); yet the role of calcitonin receptor (CTR), its seven-transmembrane G-protein coupled receptor, remains to be established in C-cells derived thyroid tumors. The aim of this work was to investigate CTR expression in MTC and to correlate such expression with clinicopathological features in order to evaluate its possible role as a prognostic indicator of disease aggressiveness and outcome. METHODS: Calcitonin receptor expression was analyzed in a series of 75 MTCs by immunohistochemistry, and by qPCR mRNA quantification in specimens from four patients. Statistical tests were used to evaluate the correlation between CTR expression and the clinicopathological and molecular characteristics of patients and tumors. RESULTS: Calcitonin receptor expression was detected in 62 out of 75 samples (82.7%), whereas 13 of the 75 samples (17.3%) were completely negative. CTR expression was significantly associated with expression of cytoplasmatic phosphatase and tensin homologue deleted on chromosome 10 and osteopontin, as well as with wild type RET/RAS genes and absence of tumor stroma, suggesting that CTR expression do not associate with clinicopathological signs of worse prognosis. DISCUSSION: Calcitonin receptor expression appears to be associated in MTC with more differentiated status of the neoplastic cells.

Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma.

We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC50 for mAb2C4:dianthin was 10.0 pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5 nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC50 for mAb2C4:dianthin was 20 pM, mAb2C4:gelonin, 20 pM, compared to the ADC (6.3 nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.

Combinatorial approach to increase efficacy of Cetuximab, Panitumumab and Trastuzumab by dianthin conjugation and co-application of SO1861.

The therapeutic relevance of immunotoxins is based on the conjugation of monoclonal antibodies to toxins. In cancer therapies, the conjugated antibodies not only direct the binding of immunotoxins to cancer-specific receptors and mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. In the present study, the therapeutic antibodies Cetuximab (anti-EGFR, Erbitux(®)), Panitumumab (anti-EGFR, Vectibix(®)) and Trastuzumab (anti-HER2, Herceptin(®)) were chemically conjugated to the toxin dianthin. In the first instance, recombinant dianthin was characterized by mass spectrometry and its stability was analyzed by circular dichroism. Dianthin showed increased cytotoxicity on MCF-7 cells when tested in combination with a glycosylated triterpenoid (SO1861) in a real-time impedance-based cytotoxicity assay. In data obtained by live cell imaging, SO1861 specifically mediated the endo/lysosomal escape of dianthin without disrupting the plasma membrane. The purity of immunotoxins was confirmed by SDS-PAGE and Western blot. Their cytotoxicity was evaluated in the presence of SO1861 and dianthin-Cetuximab presented a GI50 (50% growth inhibition) of 5.3pM, dianthin-Panitumumab of 1.5pM, and dianthin-Trastuzumab of 23pM. Finally, the specificity of these immunotoxins was validated in a fluorescence-based real-time assay, where their binding to target cells was prevented by preincubation with an excess of label-free unconjugated antibody. Based on these data, we propose the use of dianthin and SO1861 as a new platform technology to enhance the efficacy of therapeutic antibodies.

A novel population of α-smooth muscle actin-positive cells activated in a rat model of stroke: an analysis of the spatio-temporal distribution in response to ischemia.

In a rat model of stroke, the spatio-temporal distribution of α-smooth muscle actin-positive, (αSMA+) cells was investigated in the infarcted hemisphere (ipsilateral) and compared with the contralateral hemisphere. At day 3 postischemia, αSMA+ cells were concentrated in two main loci within the ipsilateral hemisphere (Area A) in the medial corpus callosum and (Area B) midway through the striatum adjacent to the lateral ventricle. By day 7 and further by day 14, fewer αSMA+ cells remained in Areas A and B but a steady increase in the peri-infarct was observed. αSMA+ cells also expressed glial acidic fibrillary protein [GFAP: αSMA+/GFAP+ (29%); αSMA+/GFAP- (71%) phenotypes] and feline leukemia virus C receptor 2 (FLVCR2), but not ED1(microglia) and established markers of pericytes normally located in vascular wall. αSMA+ cells were also located close to the subventricular zones (SVZ) adjacent to Areas A and B. In conclusion, αSMA+ cells have been identified in a spatial and temporal sequence from the SVZ, at intermediate loci and in the vicinity of the peri-infarct. It is hypothesized that novel populations of αSMA+ precursors of pericytes are born on the SVZ, migrate into the peri-infarct region and are incorporated into new vessels of the peri-infarct regions.

The expression of calcitonin receptor detected in malignant cells of the brain tumour glioblastoma multiforme and functional properties in the cell line A172.

AIM: Previous studies have indicated that expression of calcitonin receptor (CTR) could be induced in a proinflammatory environment. In the present study, CTR-immunoreactivity (CTR-ir) was investigated in brain tissue from patients with glioblastoma multiforme (GBM). METHODS AND RESULTS: In immunohistochemical analysis of GBM samples, tissues with complex glomeruloid structures surrounded by malignant cells were analysed for CTR-ir using anti-human CTR antibodies generated against two separate epitopes of CTR. CTR-ir was associated predominantly with glial cells. Regions with CTR-ir cells were found in 12 of 14 GBM tumours (P < 0.05). Using confocal microscopy, CTR-ir cells were identified that were also positive for glial fibrillary acidic protein, nestin and CD133. Antibodies were verified using immunoblots and confocal microscopy of the Cercopithecus aethiops(COS)-7 transfectants. Immunoblots of membrane preparations from the CTR-positive cell lines demonstrated a major band (≈ 67 kDa) and minor band (≈ 52 kDa), but the intensity was reversed for the GBM cell line A172. In cultured A172 cells, functional studies demonstrated calcitonin stimulation of adenylyl cyclase and inhibition of extracellular-regulated kinase (ERK)1/2 phosphorylation. CONCLUSIONS: The findings that (i) CTR was expressed by glioma cells in a majority of GBM tumours tested, (ii) CTR(+) /CD133(+) cells were identified and (iii) second messenger systems were functionally modified by calcitonin in A172 cells suggest that CTR might be a useful therapeutic target in GBM.

Transient expression of the calcitonin receptor by enteric neurons of the embryonic and early post-natal mouse.

Calcitonin receptor-immunoreactivity (CTR-ir) was found in enteric neurons of the mouse gastrointestinal tract from embryonic day 13.5 (E13.5) to post-natal day 28 (P28). CTR-ir occurred in cell bodies in ganglia of the myenteric plexus extending from the esophagus to the colon and in nerve cells of the submucosal ganglia of the small and large intestines. CTR-ir was also found in vagal nerve trunks and mesenteric nerves. Counts in the ileal myenteric plexus revealed CTR-ir in 80% of neurons. CTR-ir was clearly evident in the cell bodies of enteric neurons by E15.5. The immunoreactivity reached maximum intensity between P1.5 and P12 but was weaker at P18 and barely detectable at P28. The receptor was detected in nerve processes in the intestine for only a brief period around E17.5, when it was present in one to two axonal processes per villus in the small intestine. In late gestation and soon after birth, CTR-ir was also evident in the mucosal epithelium. The perinatal expression of CTR within the ENS suggests that the calcitonin/CTR system may have a role in the maturation of enteric neurons. Signals may reach enteric neurons in milk, which contains high levels of calcitonin.

The elevated expression of calcitonin receptor by cells recruited into the endothelial layer and neo-intima of atherosclerotic plaque.

Calcitonin receptor-immunoreactive (CTR-ir) endothelial and foam cells were identified in atherosclerotic plaque within the abdominal and thoracic aortas of rabbits fed a cholesterol-supplemented diet. Initially, cells within the endothelial layers of nascent atherosclerotic plaque of arteries were also CD34-positive, a marker of precursor cells of the haematopoietic lineage. In a further rabbit model with more advanced cardiovascular disease, CTR-ir cells were located deeper within the plaque as well as within the endothelial layer overlying the neo-intima. Finally, in the third model, in which the 4-week period on the atherogenic diet was followed by a 12-week period of regression on a normal chow diet, during which serum cholesterol levels returned to the normal range, CTR-ir was markedly reduced in the stabilized fibrous cap of plaque. Thus, the expression of CTR is associated with the early cellular events involved in plaque formation and is down-regulated as stabilisation of plaque progresses in the process of healing.

High dietary taurine reduces apoptosis and atherosclerosis in the left main coronary artery: association with reduced CCAAT/enhancer binding protein homologous protein and total plasma homocysteine but not lipidemia.

We sought to determine whether taurine could specifically protect against coronary artery disease during an atherogenic diet and whether taurine affects the lipid profile, metabolites of methionine, and endothelial atherogenic systems. Rabbits were fed one of the following diets for 4 weeks: (1) control diet; (2) 0.5% cholesterol+1.0% methionine; or (3) 0.5% cholesterol+1.0% methionine+2.5% taurine. Endothelial function was examined, and the left main coronary artery atherosclerosis was quantified by stereology and semiquantitative immunohistochemistry to determine the endothelial expression of proteins related to the NO, renin-angiotensin, endoplasmic reticulum, and oxidative stress systems, as well as apoptosis. Taurine normalized hyperhomocysteinemia (P<0.05) and significantly reduced hypermethioninemia (P<0.05) but not lipidemia. The intima:media ratio was reduced by 28% (P=0.034), and atherosclerosis was reduced by 64% (P=0.012) and endothelial cell apoptosis by 30% (P<0.01). Endothelial cell CCAAT/enhancer binding protein homologous protein was normalized (P<0.05). Taurine failed to improve hyperlipidemia, endothelial function, or endothelial proteins related to the NO, renin-angiotensin, and oxidative stress systems. Taurine reduces left main coronary artery wall pathology associated with decreased plasma total homocysteine, methionine, apoptosis, and normalization of CCAAT/enhancer binding protein homologous protein. These results elucidate the antiapoptotic and antiatherogenic properties of taurine, possibly via normalization of endoplasmic reticulum stress.

Calcitonin gene-related peptide inhibits angiotensin II-mediated vasoconstriction in human radial arteries: role of the Kir channel.

OBJECTIVE: The radial artery is increasingly used for coronary artery bypass grafts, but its potential for spasm increases postoperative risk. Alpha-calcitonin gene-related peptide is a potent antihypertensive peptide. Thus, we set out to determine whether calcitonin gene-related peptide can impair angiotensin II-mediated vasoconstriction in human radial arteries and, if so, to determine its mechanism of action. METHODS: Radial arteries were placed in organ bath chambers and preincubated with 10(-9) to 10(-7) mol/L alpha-calcitonin gene-related peptide for 20 minutes before initiating an angiotensin II dose response curve (10(-10)-10(-6) mol/L). RESULTS: Calcitonin gene-related peptide, 10(-7), 10(-8), 3 x 10(-9), and 10(-9) mol/L, reduced angiotensin II-mediated vasoconstriction to 30.5% +/- 7.2% (P < .001), 32.2% +/- 11.7% (P < .001), 62.6% +/- 8.4% (P < .001), and 77.6% +/- 6.7% (P < .01), respectively, compared with control (normalized to 100%). Calcitonin gene-related peptide also significantly decreased basal vascular tension in human radial arteries (P < .05 in all cases). N-nitro-L-arginine methyl ester, 4-aminopyridine, charybdotoxin, and apamin had no effect on calcitonin gene-related peptide relaxation, but Ba(2+) impaired the effects of alpha-calcitonin gene-related peptide. CONCLUSIONS: Alpha-calcitonin gene-related peptide dose dependently impaired angiotensin II-mediated vasoconstriction in human radial arteries, independent of nitric oxide and all potassium channels except the barium-sensitive Kir channel. Thus, calcitonin gene-related peptide is an endogenous inhibitor of angiotensin II-mediated vasoconstriction in the human radial artery.

Amylin in the periphery II: An updated mini-review.

Amylin is a polypeptide that is cosecreted with insulin from the beta cells of the pancreas. Therefore, in states of diabetes in which the beta-cell mass is largely depleted or dysfunctional, insulin and amylin secretion are also lost or dysregulated. While the soluble monomeric form of amylin acts as a hormone that alters physiological responses related to feeding and acts as a specific growth factor, there has been renewed interest in the less-soluble oligomeric and insoluble polymeric forms of human (also monkey and cat) amylin that may contribute to the establishment of a pathophysiological pathway to overt diabetes. With this discovery has grown the hope of minimizing, with appropriate therapy, these toxic forms to preserve the functional (c) not-cell mass. Human beta cells may also be more vulnerable to these forms and one risk factor, a higher fat diet, may promote toxic forms. The generation and utilities of transgenic rodent models, which express enhanced levels of human amylin, have been accompanied by strategies that may lead to the reduction of toxic forms and associated risk factors. The successful definition and faithful expression of the physiological receptors (and complexes) for amylin that may differ for each target organ is an important development in the field of amylin research generally. Besides the heuristic value for the understanding of the molecular biology of receptors, the opportunity to screen and identify nonpeptide analogues that bind the physiological receptors has important implications for biomedicine and clinical practice in relation to treatments for diabetic complications, bone diseases, and eating disorders. In particular, in their capacities to mimic the effects of amylin as a growth factor, amylin analogues may prove useful in the stimulation of beta-cell mass (in conjunction with other factors), reduce the activity of the osteoclast population, and stimulate the regeneration of proximal tubules following toxic insult (and thus avoid the development of renal insufficiency).

Improved islet morphology after blockade of the renin- angiotensin system in the ZDF rat.

The renin-angiotensin system (RAS) has an important role in the endocrine pancreas. Although angiotensin II has significant effects on cell proliferation and apoptosis, the contribution of the RAS to changes in islet structure and function associated with type 2 diabetes is yet to be defined. This study examined the specific effects of RAS blockade on islet structure and function in diabetes. Thirty-six male Zucker diabetic fatty (ZDF) rats, 10 weeks of age, were randomized to receive the angiotensin-converting enzyme inhibitor perindopril (8 mg/l in drinking water; n = 12), irbesartan (15 mg/kg via gavage; n = 12), or no treatment (n = 12) for 10 weeks. Results were compared with lean littermates (ZL) (n = 12) studied concurrently. ZDF rats had increased intra-islet expression of components of the RAS correlating with increased intraislet fibrosis, apoptosis, and oxidative stress. Disordered islet architecture, seen in ZDF rats, was attenuated after treatment with perindopril or irbesartan. Islet fibrogenesis was also diminished, as measured by picrosirius staining and expression of collagens I and IV. Gene expression of transforming growth factor-beta1 was increased in the ZDF pancreas (ZL, 1.0 +/- 0.1; ZDF, 2.0 +/- 0.3; P < 0.05) and reduced after blockade of the RAS (ZDF + P, 1.3 +/- 0.2; ZDF + I, 1.5 +/- 0.1; vs. ZDF, both P < 0.05). Improvements in structural parameters were also associated with functional improvements in first-phase insulin secretion. These findings provide a possible mechanism for the reduced incidence of new-onset diabetes that has been observed in clinical trials of RAS blockade.

Novel hexad repeats conserved in a putative transporter with restricted expression in cell types associated with growth, calcium exchange and homeostasis.

A transport protein is described with 12 transmembrane spans. Within the cytoplasmic amino-terminal domain, several novel hexad repeats are conserved in human, mouse, rat and pig, four to six of which had the canonical form PS_S_H(+). In the carboxyl-terminal domain, a polyglutamate sequence (5-8) is conserved. Restricted expression of the transporter was identified in acidophil cells of the adult pituitary that secrete growth hormone and prolactin. In the fetus, expression was restricted to osteoclasts, chondrocytes, thyroid, pituitary, central nervous system, eye, liver and heart. In particular, expression was found in structures associated with rapid calcium exchange including the retina, cardiomyocytes and in the intraplacental yolk sac that expresses calcitropic molecules. Furthermore, expression found in osteoclasts and kidney, within the distal portions of nephrons and collecting ducts, was consistent with a role in calcium homeostasis. In human pituitary, four mRNA transcripts, and in mouse kidney, three mRNA transcripts were expressed. In developing mouse kidney, the amount of each transcript varied that suggested the multiple transcripts might be differentially expressed in different physiological states. We propose that the transporter is specific for a calcium-chelator complex and is important for growth and calcium metabolism.

Amylin in the periphery.

Amylin (islet amyloid polypeptide) is a peptide synthesized principally in the beta-cells of the pancreatic islets together with insulin and has actions as a hormone, growth factor, and modifier of behavior. As a hormone, amylin acts to modify gastric motility, renal resorption, and has metabolic actions. It is postulated that the principal function of amylin as a hormone is the activation of physiological processes associated with feeding. As a growth factor, amylin acts on bone cells, renal proximal tubular cells, and islet beta-cells. Amylin has important targets in the brain that mediate its actions in the modification of behavior, including thirst and satiety. In man, amylin can form islet amyloid deposits, an event linked to the reduction of b-cell mass and loss of signal-secretion coupling. Recent evidence has defined a new role for monomeric amylin as a growth factor and regulator of beta-cell mass that is postulated to be a key factor in pathophysiological processes that result in overt diabetes.

Calcitonin receptor isoforms expressed in the developing rat kidney.

BACKGROUND: Development in the metanephric-kidney transition period involves the precise expression of paracrine and autocrine events in an ordered spatio-temporal manner. Expression of these molecular events is tightly controlled and includes positive and negative growth factors and cognate receptors within close proximity in developing structures in the expanding renal cortex and medulla. The expression of calcitonin receptor (CTR) isoforms C1a and C1b in this context has not previously been described. Our current study also explored the relationship between the expression of CTR isoforms and amylin binding sites. METHODS: Techniques included immunohistochemistry with novel antibodies that detect CTR isoforms, real time PCR for the quantification of CTR isoforms, Western blot and in vitro autoradiography, on tissues from embryo day 18 to postnatal day 30. RESULTS: The CTR C1a isoform is expressed in the ureteric ducts of the metanephros and both isoforms are expressed in the developing distal convoluted tubules, ascending limbs of the loop of Henle and collecting ducts in the postnatal rat kidney. There was a 60-fold excess of C1a versus C1b isoforms. An apparent molecular weight of 63 kD was found. In vitro autoradiography demonstrated that while amylin binding sites were predominantly in the cortex, CTR expression was largely localized in the medulla in an earlier event, followed by cortical expression. CONCLUSIONS: CTR C1a protein expression has been identified in the ureteric ducts in the metanephros and both isoforms expressed in the distal portions of the developing nephrons and collecting ducts. Since amylin binding sites have been localized on the proximal tubules of the cortex, it is unlikely that amylin receptors can be represented by modification of CTR affinity with receptor activity modifying proteins in the kidney.

Nephrin expression in the post-natal developing kidney in normotensive and hypertensive rats.

Nephrin is a slit diaphragm protein and its expression in the developing kidney is largely unknown. In this study, we explored the expression of nephrin in the developmental kidney in spontaneously hypertensive (SHR) and in Wistar-Kyoto (WKY) rats at different time points, from day 5 after birth to adulthood. Real time RT-PCR, in situ hybridization and immunohistochemistry were used to assess and quantify gene and protein expression of nephrin in the kidney. SHR had hypertension at week 10 and albuminuria at week 20. Nephrin expression in both SHR and WKY increased from day 5 to adulthood. Furthermore, both gene and protein expression of nephrin were significantly lower in SHR after birth when compared to WKY at the same age. These findings suggest that both in normotensive and hypertensive rats, nephrin expression increased from birth to the adult age and that down-regulation of nephrin in SHR evident from the early developmental kidney to adulthood may contribute to the development of albuminuria in adult SHR.

Calcitropic gene expression suggests a role for the intraplacental yolk sac in maternal-fetal calcium exchange.

The expression of calcitropic genes and proteins was localized within murine placenta during late gestation (the time frame of active calcium transfer) with an analysis of several gene-deletion mouse models by immunohistochemistry and in situ hybridization. Parathyroid hormone-related protein (PTHrP), the PTH/PTHrP receptor, calcium receptor, calbindin-D(9k), Ca(2+)-ATPase, and vitamin D receptor were all highly expressed in a localized structure of the murine placenta, the intraplacental yolk sac, compared with trophoblasts. In the PTHrP gene-deleted or Pthrp-null placenta in which placental calcium transfer is decreased, calbindin-D(9k) expression was downregulated in the intraplacental yolk sac but not in the trophoblasts. These observations indicated that the intraplacental yolk sac contains calcium transfer and calcium-sensing capability and that it is a probable route of maternal-fetal calcium exchange in the mouse.