miR-200c Prevents TGF-ß1-Induced Epithelial-to-Mesenchymal Transition and Fibrogenesis in Mesothelial Cells by Targeting ZEB2 and Notch1.

Peritoneal fibrosis and loss of transport function is a common complication contributing to adverse outcomes in patients on long-term peritoneal dialysis (PD). Epithelial-to-mesenchymal transition (EMT) in mesothelial cells is a salient feature, but its triggering mechanisms remain obscure. Dysregulation of microRNA (miR) expression is implicated in EMT and tissue fibrosis. We investigated the role of miR-200c in EMT and fibrogenesis in a murine PD model and in cultured peritoneal mesothelial cells. PD-fluid-treated mice showed peritoneal miR-200c expression reduced by 76.2% compared with PBS-treated mice, and this was accompanied by increased peritoneal α-smooth muscle actin, fibronectin, and collagen expression. PD fluid and TGF-ß1 both reduced miR-200c expression in cultured mesothelial cells, accompanied by downregulation of E-cadherin and decorin, and induction of fibronectin, collagen I and III, and transcription factors related to EMT. Decorin prevented the suppression of miR-200c by TGF-ß1. Lentivirus-mediated miR-200c overexpression prevented the induction of fibronectin, collagen I, and collagen III by TGF-ß1, independent of decorin, and partially prevented E-cadherin suppression by TGF-ß1. Target genes of miR-200c were identified as ZEB2 and Notch1. Our data demonstrate that miR-200c regulates EMT and fibrogenesis in mesothelial cells, and loss of peritoneal miR-200c contributes to PD-associated peritoneal fibrosis.

Renal stromal miRNAs are required for normal nephrogenesis and glomerular mesangial survival.

MicroRNAs are small noncoding RNAs that post-transcriptionally regulate mRNA levels. While previous studies have demonstrated that miRNAs are indispensable in the nephron progenitor and ureteric bud lineage, little is understood about stromal miRNAs during kidney development. The renal stroma (marked by expression of FoxD1) gives rise to the renal interstitium, a subset of peritubular capillaries, and multiple supportive vascular cell types including pericytes and the glomerular mesangium. In this study, we generated FoxD1(GC);Dicer(fl/fl) transgenic mice that lack miRNA biogenesis in the FoxD1 lineage. Loss of Dicer activity resulted in multifaceted renal anomalies including perturbed nephrogenesis, expansion of nephron progenitors, decreased renin-expressing cells, fewer smooth muscle afferent arterioles, and progressive mesangial cell loss in mature glomeruli. Although the initial lineage specification of FoxD1(+) stroma was not perturbed, both the glomerular mesangium and renal interstitium exhibited ectopic apoptosis, which was associated with increased expression of Bcl2l11 (Bim) and p53 effector genes (Bax, Trp53inp1, Jun, Cdkn1a, Mmp2, and Arid3a). Using a combination of high-throughput miRNA profiling of the FoxD1(+)-derived cells and mRNA profiling of differentially expressed transcripts in FoxD1(GC);Dicer(fl/fl) kidneys, at least 72 miRNA:mRNA target interactions were identified to be suppressive of the apoptotic program. Together, the results support an indispensable role for stromal miRNAs in the regulation of apoptosis during kidney development.

Transmembrane peptides as unique tools to demonstrate the in vivo action of a cross-class GPCR heterocomplex.

Angiotensin (ANGII) and secretin (SCT) share overlapping, interdependent osmoregulatory functions in brain, where SCT peptide/receptor function is required for ANGII action, yet the molecular basis is unknown. Since receptors for these peptides (AT1aR, SCTR) are coexpressed in osmoregulatory centers, a possible mechanism is formation of a cross-class receptor heterocomplex. Here, we demonstrate such a complex and its functional importance to modulate signaling. Association of AT1aR with SCTR reduced ability of SCT to stimulate cyclic adenosine monophosphate (cAMP), with signaling augmented in presence of ANGII or constitutively active AT1aR. Several transmembrane (TM) peptides of these receptors were able to affect their conformation within complexes, reducing receptor BRET signals. AT1aR TM1 affected only formation and activity of the heterocomplex, without effect on homomers of either receptor, and reduced SCT-stimulated cAMP responses in cells expressing both receptors. This peptide was active in vivo by injection into mouse lateral ventricle, thereby suppressing water-drinking behavior after hyperosmotic shock, similar to SCTR knockouts. This supports the interpretation that active conformation of AT1aR is a key modulator of cAMP responses induced by SCT stimulation of SCTR. The SCTR/AT1aR complex is physiologically important, providing differential signaling to SCT in settings of hyperosmolality or food intake, modulated by differences in levels of ANGII.

Dicer function is required in the metanephric mesenchyme for early kidney development.

MicroRNAs (miRNAs) are small, noncoding regulatory RNAs that act as posttranscriptional repressors by binding to the 3'-untranslated region (3'-UTR) of target genes. They require processing by Dicer, an RNase III enzyme, to become mature regulatory RNAs. Previous work from our laboratory revealed critical roles for miRNAs in nephron progenitors at midgestation (Ho J, Pandey P, Schatton T, Sims-Lucas S, Khalid M, Frank MH, Hartwig S, Kreidberg JA. J Am Soc Nephrol 22: 1053-1063, 2011). To interrogate roles for miRNAs in the early metanephric mesenchyme, which gives rise to nephron progenitors as well as the renal stroma during kidney development, we conditionally ablated Dicer function in this lineage. Despite normal ureteric bud outgrowth and condensation of the metanephric mesenchyme to form nephron progenitors, early loss of miRNAs in the metanephric mesenchyme resulted in severe renal dysgenesis. Nephron progenitors are initially correctly specified in the mutant kidneys, with normal expression of several transcription factors known to be critical in progenitors, including Six2, Pax2, Sall1, and Wt1. However, there is premature loss of the nephron progenitor marker Cited1, marked apoptosis, and increased expression of the proapoptotic protein Bim shortly after the initial inductive events in early kidney development. Subsequently, there is a failure in ureteric bud branching and nephron progenitor differentiation. Taken together, our data demonstrate a previously undetermined requirement for miRNAs during early kidney organogenesis and indicate a crucial role for miRNAs in regulating the survival of this lineage.

Vagal afferent mediates the anorectic effect of peripheral secretin.

Secretin (SCT) is a classical peptide hormone that is synthesized and released from the gastrointestinal tract after a meal. We have previously shown that it acts both as a central and peripheral anorectic peptide, and that its central effect is mediated via melanocortin system. As peripheral satiety signals from the gastrointestinal tract can be sent to the brain via the vagal afferent or by crossing the blood-brain barrier (BBB), we therefore sought to investigate the pathway by which peripheral SCT reduces appetite in this study. It is found that bilateral subdiaphragmatic vagotomy and treatment of capsaicin, an excitotoxin for primary afferent neurons, could both block the anorectic effect of peripherally injected SCT. These treatments are found to be capable of blunting i.p. SCT-induced Fos activation in pro-opiomelanocortin (POMC) neurons within the hypothalamic Arcuate Nucleus (Arc). Moreover, we have also found that bilateral midbrain transaction could block feeding reduction by peripheral SCT. Taken together, we conclude that the satiety signals of peripheral SCT released from the gastrointestinal tract are sent via the vagus nerves to the brainstem and subsequently Arc, where it controls central expression of other regulatory peptides to regulate food intake.

Insignificant effect of secretin in rodent models of polycystic kidney and liver disease.

Polycystic kidney (PKD) and liver (PLD) diseases cause significant morbidity and mortality. A large body of evidence indicates that cyclic AMP plays an important role in their pathogenesis. Clinical trials of drugs that reduce cyclic AMP levels in target tissues are now in progress. Secretin may contribute to adenylyl cyclase-dependent urinary concentration and is a major agonist of adenylyl cyclase in cholangiocytes. To investigate the role of secretin in PKD and PLD, we have studied the expression of secretin and the secretin receptor in rodent models orthologous to autosomal recessive (PCK rat) and dominant (Pkd2(-/WS25) mouse) PKD; the effects of exogenous secretin administration to PCK rats, PCK rats lacking circulating vasopressin (PCK(di/di)), and Pkd2(-/WS25) mice; and the impact of a nonfunctional secretin receptor on disease development in Pkd2(-/WS25):SCTR(-/-) double mutants. Renal and hepatic secretin and secretin receptor mRNA and plasma secretin were increased in both models, and secretin receptor protein was increased in the kidneys and liver of Pkd2(-/WS25) mice. However, exogenous secretin administered subcutaneously via osmotic pumps had minimal or negligible effects and the absence of a functional secretin receptor had no influence on the severity of PKD or PLD. Therefore, it is unlikely that by itself secretin plays a significant role in the pathogenesis of PKD and/or PLD.

Origin of secretin receptor precedes the advent of tetrapoda: evidence on the separated origins of secretin and orexin.

At present, secretin and its receptor have only been identified in mammals, and the origin of this ligand-receptor pair in early vertebrates is unclear. In addition, the elusive similarities of secretin and orexin in terms of both structures and functions suggest a common ancestral origin early in the vertebrate lineage. In this article, with the cloning and functional characterization of secretin receptors from lungfish and X. laevis as well as frog (X. laevis and Rana rugulosa) secretins, we provide evidence that the secretin ligand-receptor pair has already diverged and become highly specific by the emergence of tetrapods. The secretin receptor-like sequence cloned from lungfish indicates that the secretin receptor was descended from a VPAC-like receptor prior the advent of sarcopterygians. To clarify the controversial relationship of secretin and orexin, orexin type-2 receptor was cloned from X. laevis. We demonstrated that, in frog, secretin and orexin could activate their mutual receptors, indicating their coordinated complementary role in mediating physiological processes in non-mammalian vertebrates. However, among the peptides in the secretin/glucagon superfamily, secretin was found to be the only peptide that could activate the orexin receptor. We therefore hypothesize that secretin and orexin are of different ancestral origins early in the vertebrate lineage.

Secretin and body fluid homeostasis.

Body fluid homeostasis is critical for the survival of living organisms and hence is tightly controlled. From initial studies on the effects of secretin (SCT) on renal water reabsorption in the 1940s and recent investigations of its role in cardiovascular and neuroendocrine functions, it has now become increasingly clear that this peptide is an integral component of the homeostatic processes that maintain body fluid balance. This review, containing some of our recent findings of centrally expressed SCT on water intake, focuses on the actions of SCT in influencing the physiological, neuroendocrine, and cardiovascular processes that subserve body fluid homeostasis.

An indispensable role of secretin in mediating the osmoregulatory functions of angiotensin II.

Fluid balance is critical to life and hence is tightly controlled in the body. Angiotensin II (ANGII), one of the most important components of this regulatory system, is recognized as a dipsogenic hormone that stimulates vasopressin (VP) expression and release. However, detailed mechanisms regarding how ANGII brings about these changes are not fully understood. In the present study, we show initially that the osmoregulatory functions of secretin (SCT) in the brain are similar to those of ANGII in mice and, more important, we discovered the role of SCT as the link between ANGII and its downstream effects. This was substantiated by the use of two knockout mice, SCTR(-/-) and SCT(-/-), in which we show the absence of an intact SCT/secretin receptor (SCTR) axis resulted in an abolishment or much reduced ANGII osmoregulatory functions. By immunohistochemical staining and in situ hybridization, the proteins and transcripts of SCT and its receptor are found in the paraventricular nucleus (PVN) and lamina terminalis. We propose that SCT produced in the circumventricular organs is transported and released in the PVN to stimulate vasopressin expression and release. In summary, our findings identify SCT and SCTR as novel elements of the ANGII osmoregulatory pathway in maintaining fluid balance in the body.

Functional identification of an intronic promoter of the human glucose-dependent insulinotropic polypeptide gene.

Glucose-dependent insulinotropic polypeptide (GIP), a physiological incretin and enterogastrone, plays a vital role in regulating glucose-dependent insulin release from the pancreas and gastric acid secretion from the stomach. By using a transgenic mouse approach, we previously reported that the distal 1.2kb promoter region of the human GIP (hGIP) gene (-2545/-346, relative to the ATG) was able to target the transgene expression in the stomach but not in the small intestine where the majority of GIP-producing cells are located. In the present study, in order to identify the cis-acting element(s) that is/are required for intestinal expression, a 1.6kb (-1580/-) DNA fragment within the first intron of the hGIP gene was isolated and characterized in three GIP-expressing cell lines including HuTu80 (duodenal cells), PANC-1 (pancreatic ductal cells) and Hs746T (stomach cells). By 5' and 3' deletion analysis, a proximal promoter element was confined within the nucleotides -102/-1. This promoter element, functions in an orientation-dependent manner, was able to drive 15.1 and 18.3 fold increases in promoter activities in HuTu80 and PANC-1 cells, respectively. Site-directed mutation analysis indicated that the region -54/-23 was essential for promoter function while the region -22/-1 might possess opposite effects in HuTu80 and PANC-1 cells. In competitive and antibody supershift assays, interactions of the progesterone receptor (PR) and some unknown protein factors from HuTu80 and PANC-1 with the motif(s) at -54/-23 were evident. Consistent with this finding, we demonstrated the transcriptional regulation of the hGIP promoter by progesterone via the PR-B isoform and that progesterone treatment in both HuTu80 and PANC-1 cells resulted in an increase in hGIP transcript level. In addition, a sequence motif (ACATGT) residing -48/-43 was found to be responsible for the binding of potential TFII regulator(s). Taken together, our results suggest that the proximal intronic sequences contain essential cis-acting elements for the cell-specific expression of the hGIP gene.

Secretin as a neurohypophysial factor regulating body water homeostasis.

Hypothalamic magnocellular neurons express either one of the neurohypophysial hormones, vasopressin or oxytocin, along with different neuropeptides or neuromodulators. Axonal terminals of these neurons are generally accepted to release solely the two hormones but not others into the circulation. Here, we show that secretin, originally isolated from upper intestinal mucosal extract, is present throughout the hypothalamo-neurohypophysial axis and that it is released from the posterior pituitary under plasma hyperosmolality conditions. In the hypothalamus, it stimulates vasopressin expression and release. Considering these findings together with our previous findings that show a direct effect of secretin on renal water reabsorption, we propose here that secretin works at multiple levels in the hypothalamus, pituitary, and kidney to regulate water homeostasis. Findings presented here challenge previous understanding regarding the neurohypophysis and could provide new concepts in treating disorders related to osmoregulation.

Vasopressin-independent mechanisms in controlling water homeostasis.

The maintenance of body water homeostasis depends on the balance between water intake and water excretion. In the kidney, vasopressin (Vp) is a critical regulator of water homeostasis by controlling the insertion of aquaporin 2 (AQP2) onto the apical membrane of the collecting duct principal cells in the short term and regulating the gene expression of AQP2 in the long term. A growing body of evidence from both in vitro and in vivo studies demonstrated that both secretin and oxytocin are involved as Vp-independent mechanisms regulating the renal water reabsorption process, including the translocation and expression of AQP2. This review focuses on how these two hormones are potentially involved as Vp-independent mechanisms in controlling water homeostasis.

Multiple actions of secretin in the human body.

The discovery of secretin initiated the field of endocrinology. Over the past century, multiple gastrointestinal functions of secretin have been extensively studied, and it was discovered that the principal function of this peptide in the gastrointestinal system is to facilitate digestion and to provide protection. In view of the late identification of secretin and the secretin receptor in various tissues, including the central nervous system, the pleiotropic functions of secretin have more recently been an area of intense focus. Secretin is a classical hormone, and recent studies clearly showed secretin's involvement in neural and neuroendocrine pathways, although the neuroactivity and neural regulation of its release are yet to be elucidated. This chapter reviews our current understanding of the pleiotropic actions of secretin with a special focus on the hormonal and neural interdependent pathways that mediate these actions.

Phenotypes developed in secretin receptor-null mice indicated a role for secretin in regulating renal water reabsorption.

Aquaporin 2 (AQP2) is responsible for regulating the concentration of urine in the collecting tubules of the kidney under the control of vasopressin (Vp). Studies using Vp-deficient Brattleboro rats, however, indicated the existence of substantial Vp-independent mechanisms for membrane insertion, as well as transcriptional regulation, of this water channel. The Vp-independent mechanism(s) is clinically relevant to patients with X-linked nephrogenic diabetes insipidus (NDI) by therapeutically bypassing the dysfunctional Vp receptor. On the basis of studies with secretin receptor-null (SCTR(-/-)) mice, we report here for the first time that mutation of the SCTR gene could lead to mild polydipsia and polyuria. Additionally, SCTR(-/-) mice were shown to have reduced renal expression of AQP2 and AQP4, as well as altered glomerular and tubular morphology, suggesting possible disturbances in the filtration and/or water reabsorption process in these animals. By using SCTR(-/-) mice as controls and comparing them with wild-type animals, we performed both in vivo and in vitro studies that demonstrated a role for secretin in stimulating (i) AQP2 translocation from intracellular vesicles to the plasma membrane in renal medullary tubules and (ii) expression of this water channel under hyperosmotic conditions. The present study therefore provides information for at least one of the Vp-independent mechanisms that modulate the process of renal water reabsorption. Future investigations in this direction should be important in developing therapeutic means for treating NDI patients.

Signaling mechanisms of secretin receptor.

Secretin, a 27-amino acid gastrointestinal peptide, was initially discovered based on its activities in stimulating pancreatic juice. In the past 20 years, secretin was demonstrated to exhibit pleiotropic functions in many different tissues and more importantly, its role as a neuropeptide was substantiated. To carry out its activities in the central nervous system and in peripheral organs, secretin interacts specifically with one known receptor. Secretin receptor, a member of guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) in the secretin/VIP/glucagon subfamily, possesses the characteristics of GPCR with seven conserved transmembrane domains, a relatively large amino-terminal extracellular domain and an intracellular carboxyl terminus. The structural features and signal transduction pathways of the secretin receptor in various tissues are reviewed in this article.