Retinoic acid receptor responder1 promotes development of glomerular diseases via the Nuclear Factor-κB signaling pathway
Mo Ller-Hackbarth, K;Dabaghie, D;Charrin, E;Zambrano, S;Genové, G;Li, X;Wernerson, A;Lal, M;Patrakka, J;
PMID: 34147551 | DOI: 10.1016/j.kint.2021.05.036
Inflammatory pathways are activated in most glomerular diseases but molecular mechanisms driving them in kidney tissue are poorly known. We identified retinoic acid receptor responder 1 (Rarres1) as a highly podocyte-enriched protein in healthy kidneys. Studies in podocyte-specific knockout animals indicated that Rarres1 was not needed for the normal development or maintenance of the glomerulus filtration barrier, and did not modulate the outcome of kidney disease in a model of glomerulonephritis. Interestingly, we detected an induction of Rarres1 expression in glomerular and peritubular capillary endothelial cells in IgA and diabetic kidney disease, as well as in ANCA-associated vasculitis. Analysis of publicly available RNA data sets showed that the induction of Rarres1 expression was a common molecular mechanism in chronic kidney diseases. A conditional knock-in mouse line, overexpressing Rarres1 specifically in endothelial cells, did not show any obvious kidney phenotype. However, the overexpression promoted the progression of kidney damage in a model of glomerulonephritis. In line with this, conditional knock-out mice, lacking Rarres1 in endothelial cells, were partially protected in the disease model. Mechanistically, Rarres1 promoted inflammation and fibrosis via transcription factor Nuclear Factor-κB signaling pathway by activating receptor tyrosine kinase Axl. Thus, induction of Rarres1 expression in endothelial cells is a prevalent molecular mechanism in human glomerulopathies and this seems to have a pathogenic role in driving inflammation and fibrosis via the Nuclear Factor-κB signaling pathway.
Hebsgaard JB, Pyke C, Yildirim E, Knudsen LB, Heegaard S, Kvist PH.
PMID: 29707863 | DOI: 10.1111/dom.13339
Semaglutide is a human glucagon-like peptide-1 (GLP-1) analogue that is in development for the treatment of type 2 diabetes. In the pre-approval cardiovascular outcomes trial SUSTAIN 6, semaglutide was associated with a significant increase in the risk of diabetic retinopathy (DR) complications vs placebo. GLP-1 receptor (GLP-1R) expression has previously been demonstrated in the retina in animals and humans; however, antibodies used to detect expression have been documented to be non-specific and fail to detect the GLP-1R using immunohistochemistry (IHC), a problem common for many G-protein coupled receptors. Using a validated GLP-1R antibody for IHC and in situ hybridization for GLP-1R mRNA in normal human eyes, GLP-1Rs were detected in a small fraction of neurons in the ganglion cell layer. In advanced stages of DR, GLP-1R expression was not detected at the protein or mRNA level. Specifically, no GLP-1R expression was found in the eyes of people with long-standing proliferative DR (PDR). In conclusion, GLP-1R expression is low in normal human eyes and was not detected in eyes exhibiting advanced stages of PDR.
Hypertension research : official journal of the Japanese Society of Hypertension
Ochiai, K;Mochida, Y;Nagase, T;Fukuhara, H;Yamaguchi, Y;Nagase, M;
PMID: 36810623 | DOI: 10.1038/s41440-023-01219-9
The recent discovery of mechanosensitive ion channels has promoted mechanobiological research in the field of hypertension and nephrology. We previously reported Piezo2 expression in mouse mesangial and juxtaglomerular renin-producing cells, and its modulation by dehydration. This study aimed to investigate how Piezo2 expression is altered in hypertensive nephropathy. The effects of the nonsteroidal mineralocorticoid receptor blocker, esaxerenone, were also analyzed. Four-week-old Dahl salt-sensitive rats were randomly assigned to three groups: rats fed a 0.3% NaCl diet (DSN), rats fed a high 8% NaCl diet (DSH), and rats fed a high salt diet supplemented with esaxerenone (DSH + E). After six weeks, DSH rats developed hypertension, albuminuria, glomerular and vascular injuries, and perivascular fibrosis. Esaxerenone effectively decreased blood pressure and ameliorated renal damage. In DSN rats, Piezo2 was expressed in Pdgfrb-positive mesangial and Ren1-positive cells. Piezo2 expression in these cells was enhanced in DSH rats. Moreover, Piezo2-positive cells accumulated in the adventitial layer of intrarenal small arteries and arterioles in DSH rats. These cells were positive for Pdgfrb, Col1a1, and Col3a1, but negative for Acta2 (αSMA), indicating that they were perivascular mesenchymal cells different from myofibroblasts. Piezo2 upregulation was reversed by esaxerenone treatment. Furthermore, Piezo2 inhibition by siRNA in the cultured mesangial cells resulted in upregulation of Tgfb1 expression. Cyclic stretch also upregulated Tgfb1 in both transfections of control siRNA and Piezo2 siRNA. Our findings suggest that Piezo2 may have a contributory role in modulating the pathogenesis of hypertensive nephrosclerosis and have also highlighted the therapeutic effects of esaxerenone on salt-induced hypertensive nephropathy. Mechanochannel Piezo2 is known to be expressed in the mouse mesangial cells and juxtaglomerular renin-producing cells, and this was confirmed in normotensive Dahl-S rats. In salt-induced hypertensive Dahl-S rats, Piezo2 upregulation was observed in the mesangial cells, renin cells, and notably, perivascular mesenchymal cells, suggesting its involvement in kidney fibrosis.
Damasceno KA, Ferreira E, Estrela-Lima A, Gamba Cde O, Miranda FF, Alves MR, Rocha RM, de Barros AL, Cassali GD.
PMID: 27490467 | DOI: 10.1371/journal.pone.0160419
Versican expression promotes tumor growth by destabilizing focal cell contacts, thus impeding cell adhesion and facilitating cell migration. It not only presents or recruits molecules to the cell surface, but also modulates gene expression levels and coordinates complex signal pathways. Previously, we suggested that the interaction between versican and human epidermal growth factor receptors may be directly associated with tumor aggressiveness. Thus, the expression of EGFR and HER-2 in these neoplasms may contribute to a better understanding of the progression mechanisms in malignant mammary tumors. The purpose of this study was to correlate the gene and protein expressions of EGFR and HER2 by RNA In Situ Hybridization (ISH) and immunohistochemistry (IHC), respectively, and their relationship with the versican expression in carcinomas in mixed tumors and carcinosarcomas of the canine mammary gland. The results revealed that EGFR mRNA expression showed a significant difference between in situ and invasive carcinomatous areas in low and high versican expression groups. Identical results were observed in HER-2 mRNA expression. In immunohistochemistry analysis, neoplasms with low versican expression showed greater EGFR immunostaining in the in situ areas than in invasive areas, even as the group presenting high versican expression displayed greater EGFR and HER-2 staining in in situ areas. Significant EGFR and HER-2 mRNA and protein expressions in in situ carcinomatous sites relative to invasive areas suggest that these molecules play a role during the early stages of tumor progression.
Pflugers Archiv : European journal of physiology
Heinl, ES;Broeker, KA;Lehrmann, C;Heydn, R;Krieger, K;Ortmaier, K;Tauber, P;Schweda, F;
PMID: 36480070 | DOI: 10.1007/s00424-022-02774-9
The natriuretic peptides (NPs) ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide) mediate their widespread effects by activating the natriuretic peptide receptor-A (NPR-A), while C-type natriuretic peptide (CNP) acts via natriuretic peptide receptor-B (NPR-B). NPs are removed from the circulation by internalization via the natriuretic peptide clearance receptor natriuretic peptide receptor-C (NPR-C). In addition to their well-known functions, for instance on blood pressure, all three NPs confer significant cardioprotection and renoprotection. Since neither the NP-mediated renal functions nor the renal target cells of renoprotection are completely understood, we performed systematic localization studies of NP receptors using in situ hybridization (RNAscope) in mouse kidneys. NPR-A mRNA is highly expressed in glomeruli (mainly podocytes), renal arterioles, endothelial cells of peritubular capillaries, and PDGFR-receptor β positive (PDGFR-β) interstitial cells. No NPR-A mRNA was detected by RNAscope in the tubular system. In contrast, NPR-B expression is highest in proximal tubules. NPR-C is located in glomeruli (mainly podocytes), in endothelial cells and PDGFR-β positive cells. To test for a possible regulation of NPRs in kidney diseases, their distribution was studied in adenine nephropathy. Signal intensity of NPR-A and NPR-B mRNA was reduced while their spatial distribution was unaltered compared with healthy kidneys. In contrast, NPR-C mRNA signal was markedly enhanced in cell clusters of myofibroblasts in fibrotic areas of adenine kidneys. In conclusion, the primary renal targets of ANP and BNP are glomerular, vascular, and interstitial cells but not the tubular compartment, while the CNP receptor NPR-B is highly expressed in proximal tubules. Further studies are needed to clarify the function and interplay of this specific receptor expression pattern.
Underwood, CF;Burke, PGR;Kumar, NN;Goodchild, AK;McMullan, S;Phillips, JK;Hildreth, CM;
PMID: 35654013 | DOI: 10.1159/000525337
Angiotensin (Ang) II signalling in the hypothalamic paraventricular nucleus (PVN) via angiotensin type-1a receptors (AT1R) regulates vasopressin release and sympathetic nerve activity - two effectors of blood pressure regulation. We determined the cellular expression and function of AT1R in the PVN of a rodent model of polycystic kidney disease (PKD), the Lewis Polycystic Kidney (LPK) rat, to evaluate its contribution to blood pressure regulation and augmented vasopressin release in PKD.PVN AT1R gene expression was quantified with fluorescent in-situ hybridisation in LPK and control rats. PVN AT1R function was assessed with pharmacology under urethane anaesthesia in LPK and control rats instrumented to record arterial pressure and sympathetic nerve activity.AT1R gene expression was upregulated in the PVN, particularly in CRH neurons, of LPK versus control rats. PVN microinjection of Ang II produced larger increases in systolic blood pressure in LPK versus control rats (36±5 vs. 17±2 mmHg; P<0.01). Unexpectedly, Ang II produced regionally heterogeneous sympathoinhibition (renal: -33%; splanchnic: -12%; lumbar no change) in LPK and no change in controls. PVN pre-treatment with losartan, a competitive AT1R antagonist, blocked the Ang II-mediated renal sympathoinhibition and attenuated the pressor response observed in LPK rats. The Ang II pressor effect was also blocked by systemic OPC-21268, a competitive V1A receptor antagonist, but unaffected by hexamethonium, a sympathetic ganglionic blocker.Collectively, our data suggest that upregulated AT1R expression in PVN sensitises neuroendocrine release of vasopressin in the LPK, identifying a central mechanism for the elevated vasopressin levels present in PKD.The Author(s).
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism
Vazquez-Liebanas, E;Nahar, K;Bertuzzi, G;Keller, A;Betsholtz, C;Mäe, MA;
PMID: 34689641 | DOI: 10.1177/0271678X211056395
Platelet-derived growth factor B (PDGFB) released from endothelial cells is indispensable for pericyte recruitment during angiogenesis in embryonic and postnatal organ growth. Constitutive genetic loss-of-function of PDGFB leads to pericyte hypoplasia and the formation of a sparse, dilated and venous-shifted brain microvasculature with dysfunctional blood-brain barrier (BBB) in mice, as well as the formation of microvascular calcification in both mice and humans. Endothelial PDGFB is also expressed in the adult quiescent microvasculature, but here its importance is unknown. We show that deletion of Pdgfb in endothelial cells in 2-months-old mice causes a slowly progressing pericyte loss leading, at 12-18 months of age, to ≈50% decrease in endothelial:pericyte cell ratio, ≈60% decrease in pericyte longitudinal capillary coverage and >70% decrease in pericyte marker expression. Similar to constitutive loss of Pdgfb, this correlates with increased BBB permeability. However, in contrast to the constitutive loss of Pdgfb, adult-induced loss does not lead to vessel dilation, impaired arterio-venous zonation or the formation of microvascular calcifications. We conclude that PDFGB expression in quiescent adult microvascular brain endothelium is critical for the maintenance of pericyte coverage and normal BBB function, but that microvessel dilation, rarefaction, arterio-venous skewing and calcification reflect developmental roles of PDGFB.
Magno L, Lessard CB, Martins M, Lang V, Cruz P, Asi Y, Katan M, Bilsland J, Lashley T, Chakrabarty P, Golde TE, Whiting PJ.
PMID: 30711010 | DOI: 10.1186/s13195-019-0469-0
Abstract
BACKGROUND:
Recent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset Alzheimer's disease (LOAD). Amongst these, a polymorphism in phospholipase C-gamma 2 (PLCG2) P522R has been reported to be protective against LOAD. PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function.
METHODS:
We assessed PLCG2 distribution in human and mouse brain tissue via immunohistochemistry and in situ hybridization. We transfected heterologous cell systems (COS7 and HEK293T cells) to determine the effect of the P522R AD-associated variant on enzymatic function using various orthogonal assays, including a radioactive assay, IP-One ELISA, and calcium assays.
RESULTS:
PLCG2 expression is restricted primarily to microglia and granule cells of the dentate gyrus. Plcg2 mRNA is maintained in plaque-associated microglia in the cerebral tissue of an AD mouse model. Functional analysis of the p.P522R variant demonstrated a small hypermorphic effect of the mutation on enzyme function.
CONCLUSIONS:
The PLCG2 P522R variant is protective against AD. We show that PLCG2 is expressed in brain microglia, and the p.P522R polymorphism weakly increases enzyme function. These data suggest that activation of PLCγ2 and not inhibition could be therapeutically beneficial in AD. PLCγ2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLCγ2 may be one potential therapeutic approach.
Heydarian, M;Oak, P;Zhang, X;Kamgari, N;Kindt, A;Koschlig, M;Pritzke, T;Gonzalez-Rodriguez, E;Förster, K;Morty, RE;Häfner, F;Hübener, C;Flemmer, AW;Yildirim, AO;Sudheendra, D;Tian, X;Petrera, A;Kirsten, H;Ahnert, P;Morrell, N;Desai, TJ;Sucre, J;Spiekerkoetter, E;Hilgendorff, A;
PMID: 35580897 | DOI: 10.1136/thoraxjnl-2021-218083
Chronic lung disease, that is, bronchopulmonary dysplasia (BPD) is the most common complication in preterm infants and develops as a consequence of the misguided formation of the gas-exchange area undergoing prenatal and postnatal injury. Subsequent vascular disease and its progression into pulmonary arterial hypertension critically determines long-term outcome in the BPD infant but lacks identification of early, disease-defining changes.We link impaired bone morphogenetic protein (BMP) signalling to the earliest onset of vascular pathology in the human preterm lung and delineate the specific effects of the most prevalent prenatal and postnatal clinical risk factors for lung injury mimicking clinically relevant conditions in a multilayered animal model using wild-type and transgenic neonatal mice.We demonstrate (1) the significant reduction in BMP receptor 2 (BMPR2) expression at the onset of vascular pathology in the lung of preterm infants, later mirrored by reduced plasma BMP protein levels in infants with developing BPD, (2) the rapid impairment (and persistent change) of BMPR2 signalling on postnatal exposure to hyperoxia and mechanical ventilation, aggravated by prenatal cigarette smoke in a preclinical mouse model and (3) a link to defective alveolar septation and matrix remodelling through platelet derived growth factor-receptor alpha deficiency. In a treatment approach, we partially reversed vascular pathology by BMPR2-targeted treatment with FK506 in vitro and in vivo.We identified impaired BMP signalling as a hallmark of early vascular disease in the injured neonatal lung while outlining its promising potential as a future biomarker or therapeutic target in this growing, high-risk patient population.
Bárez-López, S;Gadd, GJ;Pauža, AG;Murphy, D;Greenwood, MP;
PMID: 37271138 | DOI: 10.1159/000531352
Despite the widespread use of general anaesthetics, the mechanisms mediating their effects are still not understood. Although suppressed in most parts of the brain, neuronal activity, as measured by FOS activation, is increased in the hypothalamic supraoptic nucleus (SON) by numerous general anaesthetics, and evidence points to this brain region being involved in the induction of general anaesthesia and natural sleep. Posttranslational modifications of proteins, including changes in phosphorylation, enable fast modulation of protein function which could be underlying the rapid effects of general anaesthesia. In order to identify potential phosphorylation events in the brain mediating general anaesthesia effects, we have explored the phosphoproteome responses in the rat SON, and compared these to cingulate cortex (CC) which displays no FOS activation is response to general anaesthetics.Adult Sprague-Dawley rats were treated with isoflurane for 15 minutes. Proteins from the CC and SON were extracted and processed for Nano-LC Mass Spectrometry (LC-MS/MS). Phosphoproteomic determinations were performed by LC-MS/MS.We found many changes in the phosphoproteomes of both the CC and SON in response to 15 minutes of isoflurane exposure. Pathway analysis indicated that proteins undergoing phosphorylation adaptations are involved in cytoskeleton remodelling and synaptic signalling events. Importantly, changes in protein phosphorylation appeared to be brain region-specific suggesting that differential phosphorylation adaptations might underlie the different neuronal activity responses to general anaesthesia between the CC and SON.In summary, these data suggest that rapid posttranslational modifications in proteins involved in cytoskeleton remodelling and synaptic signalling events might mediate the central mechanisms mediating general anaesthesia.S. Karger AG, Basel.
Li, K;Shi, Y;Gonye, EC;Bayliss, DA;
PMID: 34732535 | DOI: 10.1523/ENEURO.0212-21.2021
Select neuronal populations display steady rhythmic neuronal firing that provides tonic excitation to drive downstream networks and behaviors. In noradrenergic neurons of the locus coeruleus (LC), circadian neurons of the suprachiasmatic nucleus (SCN), and CO2/H+-activated neurons of the brainstem retrotrapezoid nucleus (RTN), large subthreshold membrane potential oscillations contribute to the pacemaker-like action potential discharge. The oscillations and firing in LC and SCN involve contributions from leak sodium (NALCN) and L-type calcium channels while recent work from RTN suggested an additional pivotal role for a secondary calcium-activated and voltage-gated cationic current sensitive to TRPM4 channel blockers. Here, we tested whether TRPM4 contributes to subthreshold oscillations in mouse LC and SCN. By RNAscope in situ hybridization, Trpm4 transcripts were detected in both cell groups. In whole-cell recordings from acute slice preparations, prominent voltage-dependent membrane potential oscillations were revealed in LC and SCN after blocking action potentials. These oscillations were inhibited by two chemically-distinct blockers of TRPM4, 9-phenanthrol (9-pt) and 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid (CBA). Under whole-cell voltage clamp, inward currents evoked by oscillation voltage waveforms were inhibited in LC by blocking L-type calcium channels and TRPM4. These data implicate TRPM4 in the large subthreshold membrane potential oscillations that underlie tonic action potential discharge in LC and SCN, providing a voltage-dependent and calcium-dependent cationic current to augment the depolarizing inward Na+ and Ca2+ currents previously associated with this distinctive electroresponsive property.
Greenwood MP, Greenwood M, Gillard BT, Chitra Devi R, Murphy D.
PMID: 29311806 | DOI: 10.3389/fnmol.2017.00413
Cyclic AMP (cAMP) inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1) is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH). We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5'UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.
