Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates.

Over 90 years ago, Kolmer and Agduhr identified spinal cerebrospinal fluid-contacting neurons (CSF-cNs) based on their morphology and location within the spinal cord. In more than 200 vertebrate species, they observed ciliated neurons around the central canal that extended a brush of microvilli into the cerebrospinal fluid (CSF). Although their morphology is suggestive of a primitive sensory cell, their function within the vertebrate spinal cord remains unknown. The identification of specific molecular markers for these neurons in vertebrates would benefit the investigation of their physiological roles. PKD2L1, a transient receptor potential channel that could play a role as a sensory receptor, has been found in cells contacting the central canal in mouse. In this study, we demonstrate that PKD2L1 is a specific marker for CSF-cNs in the spinal cord of mouse (Mus musculus), macaque (Macaca fascicularis) and zebrafish (Danio rerio). In these species, the somata of spinal PKD2L1(+) CSF-cNs were located below or within the ependymal layer and extended an apical bulbous extension into the central canal. We found GABAergic PKD2L1-expressing CSF-cNs in all three species. We took advantage of the zebrafish embryo for its transparency and rapid development to identify the progenitor domains from which pkd2l1 (+) CSF-cNs originate. pkd2l1 (+) CSF-cNs were all GABAergic and organized in two rows-one ventral and one dorsal to the central canal. Their location and marker expression is consistent with previously described Kolmer-Agduhr cells. Accordingly, pkd2l1 (+) CSF-cNs were derived from the progenitor domains p3 and pMN defined by the expression of nkx2.2a and olig2 transcription factors, respectively. Altogether our results suggest that a system of CSF-cNs expressing the PKD2L1 channel is conserved in the spinal cord across bony vertebrate species.

(Pro)renin receptor: subcellular localizations and functions.

Since its first report in 1996, the concept of the so-called (Pro)renin receptor ((P)RR/ATP6ap2) has dramactically evolved from a receptor mediating cellular effects of (pro)renin, to a protein with more basic and potentially essential intracellular functions. Among the arguments urging to reconsider the role of (P)RR was the observation that its localization appears mainly intracellular, although this does not preclude potential functions at the cell surface. However, despite about 10 years of research boosted by the generation of genetically modified animal models, the basic mechanisms of action of this protein at the cellular level remain elusive. This review aims at discussing the functions described for (P)RR in relation to its subcellular localization(s).

Control of the Wnt pathways by nephrocystin-4 is required for morphogenesis of the zebrafish pronephros.

Nephronophthisis is a hereditary nephropathy characterized by interstitial fibrosis and cyst formation. It is caused by mutations in NPHP genes encoding the ciliary proteins, nephrocystins. In this paper, we investigate the function of nephrocystin-4, the product of the nphp4 gene, in vivo by morpholino-mediated knockdown in zebrafish and in vitro in mammalian kidney cells. Depletion of nephrocystin-4 results in convergence and extension defects, impaired laterality, retinal anomalies and pronephric cysts associated with alterations in early cloacal morphogenesis. These defects are accompanied by abnormal ciliogenesis in the cloaca and in the laterality organ. We show that nephrocystin-4 is required for the elongation of the caudal pronephric primordium and for the regulation of cell rearrangements during cloaca morphogenesis. Moreover, depletion of either inversin, the product of the nphp2 gene, or of the Wnt-planar cell polarity (PCP) pathway component prickle2 increases the proportion of cyst formation in nphp4-depleted embryos. Nephrocystin-4 represses the Wnt-ß-catenin pathway in the zebrafish cloaca and in mammalian kidney cells in culture. In these cells, nephrocystin-4 interacts with inversin and dishevelled, and regulates dishevelled stability and subcellular localization. Our data point to a function of nephrocystin-4 in a tight regulation of the Wnt-ß-catenin and Wnt-PCP pathways, in particular during morphogenesis of the zebrafish pronephros. Moreover, they highlight common signalling functions for inversin and nephrocystin-4, suggesting that these two nephrocystins are involved in common physiopathological mechanisms.

Physiology of the (pro)renin receptor: Wnt of change?

The (pro)renin receptor is a protein that binds prorenin and renin in tissues, leading to their activation and, at the same time, to the initiation of intracellular signaling. The activation of local renin-angiotensin systems may play an important role in tissue damage induced by cardiovascular diseases and diabetes. However, (pro)renin receptor is also called ATP6ap2 because it has been shown to be associated with vacuolar H(+)-ATPase involvement in vesicular acidification and signaling in cells. Notably, lack of the protein in vertebrates leads to developmental alterations and early embryonic lethality probably as a result of the recently discovered role of the (pro)renin receptor and the vacuolar H(+)-ATPase in Wnt signaling. This review summarizes the current findings about these two functions of (pro)renin receptor/ATP6ap2 pointing out the possible links between both.

Prorenin and renin-induced extracellular signal-regulated kinase 1/2 activation in monocytes is not blocked by aliskiren or the handle-region peptide.

The recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin prosegment called the "handle-region" peptide (HRP). We tested whether human prorenin and renin induce extracellular signal-regulated kinase (ERK) 1/2 activation and whether the direct renin inhibitor aliskiren or the HRP inhibits the receptor. We detected the (P)RR mRNA and protein in isolated human monocytes and in U937 monocytes. In U937 cells, we found that both human renin and prorenin induced a long-lasting ERK 1/2 phosphorylation despite angiotensin II type 1 and 2 receptor blockade. In contrast to angiotensin II-ERK signaling, renin and prorenin signaling did not involve the epidermal growth factor receptor. A mitogen-activated protein kinase kinase 1/2 inhibitor inhibited both renin and prorenin-induced ERK 1/2 phosphorylation. Neither aliskiren nor HRP inhibited binding of (125)I-renin or (125)I-prorenin to (P)RR. Aliskiren did not inhibit renin and prorenin-induced ERK 1/2 phosphorylation and kinase activity. Fluorescence-activated cell sorter analysis showed that, although fluorescein isothiocyanate-labeled HRP bound to U937 cells, HRP did not inhibit renin or prorenin-induced ERK 1/2 activation. In conclusion, prorenin and renin-induced ERK 1/2 activation are independent of angiotensin II. The signal transduction is different from that evoked by angiotensin II. Aliskiren has no (P)RR blocking effect and did not inhibit ERK 1/2 phosphorylation or kinase activity. Finally, we found no evidence that HRP affects renin or prorenin binding and signaling.

Prorenin is the endogenous agonist of the (pro)renin receptor. Binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor.

OBJECTIVE: Mannose 6-phosphate receptors (M6PR) bind both renin and prorenin, and such binding contributes to renin/prorenin clearance but not to angiotensin generation. Here, we evaluated the kinetics of renin/prorenin binding to the recently discovered human (pro)renin receptor (h(P)RR), and the idea that such binding underlies tissue angiotensin generation. METHODS AND RESULTS: Vascular smooth muscle cells from control rats and transgenic rats with smooth muscle h(P)RR overexpression were incubated at 4 or 37 degrees C with human renin or prorenin. Incubation at 37 degrees C greatly increased binding, suggesting that (pro)renin-binding receptors cycle between the intracellular compartment and the cell surface. Blockade of the M6PR reduced binding by approximately 50%. During M6PR blockade, h(P)RR cells bound twice as much prorenin as control cells, while renin binding was unaltered. Incubation of h(P)RR (but not control) cells with prorenin + angiotensinogen yielded more angiotensin than expected on the basis of the activity of soluble prorenin, whereas angiotensin generation during incubation of both cell types with renin + angiotensinogen was entirely due to soluble renin. The renin + angiotensinogen-induced vasoconstriction of isolated iliac arteries from control and transgenic rats was also due to soluble renin only. The recently proposed (P)RR antagonist 'handle region peptide', which resembles part of the prosegment, blocked neither prorenin binding nor angiotensin generation. CONCLUSIONS: H(P)RRs preferentially bind prorenin, and such binding results in angiotensin generation, most likely because binding results in prorenin activation.

Elevated blood pressure and heart rate in human renin receptor transgenic rats.

Recently, a receptor for renin was described that may be important for vascular uptake and activation of (pro)renin, thus leading to local generation of angiotensin II. To assess the in vivo relevance of this protein, we generated transgenic rats overexpressing the human renin receptor gene in smooth muscle tissue, under the control of a 16-kb fragment of the mouse smooth muscle myosin heavy chain gene [TGR(SMMHC-HRR)]. Four lines of transgenic animals were obtained. The correct pattern of expression of the transgene was confirmed by RNase protection assay and in situ hybridization. TGR(SMMHC-HRR) rats are fertile and develop normally. After 6 months of age, transgenic rats develop a cardiovascular phenotype with an elevated systolic blood pressure (137.8+/-5 versus 118.9+/-3.7 mm Hg; P=0.008), and an augmentation in heart rate (349.1+/-7.7 versus 303.1+/-16.16 bpm; P=0.023) in TGR(SMMHC-HRR) and controls, respectively. These alterations are progressively increasing with aging. Although kidney function and plasma renin were normal in TGR(SMMHC-HRR), an increase in plasma aldosterone [TGR(SMMHC-HRR) 428+/-64.9 versus 207.3+/-73.24 pg/mL in control; P=0.02] and in aldosterone/renin ratio [TGR(SMMHC-HRR) 8.04+/-2.2 versus 2.8+/-0.55 in control; P=0.03] was observed. This suggests that renin receptor overexpression has resulted in increased intraadrenal angiotensin II, thereby provoking enhanced aldosterone generation in the absence of changes in plasma renin. The rise in aldosterone may underlie, at least in part, the observed cardiovascular phenotype of TGR(SMMHC-HRR).

A unique exonic splice enhancer mutation in a family with X-linked mental retardation and epilepsy points to a novel role of the renin receptor.

The renin-angiotensin system (RAS) is essential for blood pressure control and water-electrolyte balance. Until the discovery of the renin receptor, renin was believed to be mainly a circulating enzyme with a unique function, the cleavage of angiotensinogen. We report a unique mutation in the renin receptor gene (ATP6AP2) present in patients with X-linked mental retardation and epilepsy (OMIM no. 300423), but absent in 1200 control X-chromosomes. A silent mutation (c.321C>T, p.D107D) residing in a putative exonic splicing enhancer site resulted in inefficient inclusion of exon 4 in 50% of renin receptor mRNA, as demonstrated by quantitative RT-PCR. Analysis of membrane associated-receptor molecular forms showed the presence of full-length and truncated proteins in the patient. Functional analysis demonstrated that the mutated receptor could bind renin and increase renin catalytic activity, similar to the wild-type receptor, but resulted in a modest and reproducible impairment of ERK1/2 activation. Thus, our findings confirm the importance of the RAS in cognitive processes and indicate a novel specific role for the renin receptor in cognitive functions and brain development.

[The (pro)renin receptor: biology and functional significance]. / Le récepteur de la (pro)rénine: propriétés biologiques et fonctions potentielles.

Our model of the renin-angiotensin system has become increasingly complex with the identification of new components and additional roles for angiotensin peptides and their receptors. A functional (pro)renin receptor has been cloned. It acts as (pro)renin co-factor on the cell surface, enhancing the efficiency of angiotensinogen cleavage by (pro)renin and unmasking prorenin catalytic activity. Binding of (pro)renin to the receptor mediates (pro)renin cellular effects by activating MAP kinases ERK1/2. Immunofluorescence studies have located the receptor on mesangial and vascular smooth-muscle cells in human heart and kidney. This suggests that the renin receptor may represent a mean of capturing (pro)renin from the circulation and concentrating it at the interface between smooth-muscle and endothelial cells. This recent discovery of a functional (pro)renin receptor forces the emergence of a new concept that casts renin as potentially playing a direct role in tissue damage, especially in situations where the tissue RAS is activated.

Renin/prorenin-receptor biochemistry and functional significance.

The renin-angiotensin system (RAS) has become increasingly complex. New components have been identified, and additional roles for angiotensin peptides and their receptors are being uncovered. A functional (pro)renin receptor has been cloned that acts as (pro)renin cofactor on cell surface, enhancing the efficiency of angiotensinogen cleavage by (pro)renin and unmasking prorenin catalytic activity. Binding of (pro)renin to the receptor mediates (pro)renin cellular effects by activating mitogen-activating protein (MAP) kinases, extracellular signal-regulated kinases (ERK)1/2. Immunofluorescence studies have localized the receptor on mesangial and vascular smooth muscle cells in human heart and kidney. This suggests that the renin receptor might represent a means to capture (pro)renin from the circulation and to concentrate (pro)renin at the interface between smooth muscle and endothelial cells. In this article, we review the biochemical characteristics of this receptor and of other renin-binding proteins, and discuss their physiologic significance.

The renin receptor: the facts, the promise and the hope.

PURPOSE OF REVIEW: The renin-angiotensin system plays a major role in the control of blood pressure and of salt balance, but it is also involved in physiological and pathological processes, development, inflammation and cardiac hypertrophy. A concept has emerged suggesting that these effects are due to a local activation of the renin-angiotensin system. The search for a receptor of renin was based on the idea that tissue (pro)renin is taken up from the circulation and on data suggesting that renin has cellular effects independent of angiotensin II. RECENT FINDINGS: Endothelial cells and cardiac myocytes bind (pro)renin via the mannose-6-phosphate receptor, mainly a clearance receptor as no cellular effect has been specifically attributed to prorenin binding. A functional receptor was cloned recently. It mediates intracellular signalling by activating the mitogen activated protein kinases, extracellular signal regulated kinases 1 and 2, and acts as a co-factor by increasing the efficiency of angiotensinogen cleavage by receptor-bound (pro)renin. The receptor is abundantly expressed in heart, brain, placenta and eye, compared with a lower expression in liver and kidney. In normal human kidney and heart, it is localized in the mesangium and in the coronary and kidney artery, associated with smooth-muscle cells and co-localized with renin. SUMMARY: This receptor provides a functional role for prorenin and may help to understand the physiological and pathological role of elevated levels of prorenin and of local activation of the renin-angiotensin system. From a practical point of view, it questions the need for a pharmacological compound blocking (pro)renin binding and activity as an alternative to the classical inhibitors of the renin-angiotensin system.

Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.

Renin is an aspartyl protease essential for the control of blood pressure and was long suspected to have cellular receptors. We report the expression cloning of the human renin receptor complementary DNA encoding a 350-amino acid protein with a single transmembrane domain and no homology with any known membrane protein. Transfected cells stably expressing the receptor showed renin- and prorenin-specific binding. The binding of renin induced a fourfold increase of the catalytic efficiency of angiotensinogen conversion to angiotensin I and induced an intracellular signal with phosphorylation of serine and tyrosine residues associated to an activation of MAP kinases ERK1 and ERK2. High levels of the receptor mRNA are detected in the heart, brain, placenta, and lower levels in the kidney and liver. By confocal microscopy the receptor is localized in the mesangium of glomeruli and in the subendothelium of coronary and kidney artery, associated to smooth muscle cells and colocalized with renin. The renin receptor is the first described for an aspartyl protease. This discovery emphasizes the role of the cell surface in angiotensin II generation and opens new perspectives on the tissue renin-angiotensin system and on renin effects independent of angiotensin II.

Diagnosis of pheochromocytoma and laparoscopic adrenalectomy in two anephric patients with von Hippel-Lindau disease.

This study reports the first two cases of laparoscopic treatment of functional pheochromocytomas in patients with von Hippel-Lindau (VHL) disease receiving regular dialysis treatment. The genetic changes predisposing to pheochromocytoma, diagnostic procedures, and surgical management were analyzed. Both patients were considered at low risk of developing pheochromocytoma because they belonged to VHL families with 10 and 25 affected relatives without pheochromocytoma (VHL type 1 families). The mutation responsible for the disease was a stop codon in one case and a missense change in the other. Multiple renal cell carcinomas had required removal of the kidneys at age 37 in one patient and age 25 in the other patient. Computed tomography scan was crucial for diagnosis, showing a unilateral enlarged adrenal gland after 3 and 6 years on regular dialysis. No change in blood pressure was observed. MIBG scintigraphy was negative. Plasma epinephrine in one case and dopamine in the other were increased compared with patients on hemodialysis without pheochromocytoma. Pheochromocytomas were removed successfully by laparoscopic adrenalectomy. Lifelong follow-up of all affected tissues is required in all VHL patients, and pheochromocytoma should be screened for even in the absence of family history in large kindreds. Computed tomography scan allows early recognition, and patients on hemodialysis are responsive to laparoscopic adrenalectomy.

[Proteases and antiproteases in the progression of chronic renal insufficiency lesions. The role of the tissue renin-angiotensin system and the renin receptor]. / Protéases et antiprotéases dans la progression des lésions de l'insuffisance rénale chronique. Rôle du système rénine-angiotensine tissulaire et du récepteur de la rénine?

The role of proteases and of antiproteases in the progression of renal disease is well established. Most studies have focused on the serine-proteases of the plasmin/plasminogen activator system and on matrix metalloproteases. Recently, renin, an aspartyl-protease, has attracted much attention because of the role of angiotensin II in the progression of renal lesions and because of the discovery of a functional renin receptor. This receptor is a 45 kDa membrane-protein that binds specifically renin and prorenin. The binding of renin induces an increase of the catalytic efficiency of angiotensinogen conversion into angiotensin I by receptor-bound renin compared to renin in soluble phase, and a rapid phosphorylation of the receptor on serine and tyrosine residues associated with an activation of MAP kinases ERK1/2. Immunofluorescence and confocal analyses on normal human kidney and cardiac biopsies show that the receptor is localized within the mesangial area of glomeruli and in the sub-endothelium of kidney and coronary arteries, associated to smooth-muscle cells. In summary, this receptor exerts dual effects, mediating renin cellular response and increasing the efficiency of angiotensinogen cleavage by membrane-bound renin. These observations emphasizes the importance of angiotensin II generation at the cell surface and the cellular effects of renin add new dimensions (and complexity) to the classical dogma that angiotensin II is the only effector of the RAS.