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.
Mochida, Y;Ochiai, K;Nagase, T;Nonomura, K;Akimoto, Y;Fukuhara, H;Sakai, T;Matsumura, G;Yamaguchi, Y;Nagase, M;
PMID: 35273307 | DOI: 10.1038/s41598-022-07987-7
The kidney plays a central role in body fluid homeostasis. Cells in the glomeruli and juxtaglomerular apparatus sense mechanical forces and modulate glomerular filtration and renin release. However, details of mechanosensory systems in these cells are unclear. Piezo2 is a recently identified mechanically activated ion channel found in various tissues, especially sensory neurons. Herein, we examined Piezo2 expression and regulation in mouse kidneys. RNAscope in situ hybridization revealed that Piezo2 expression was highly localized in mesangial cells and juxtaglomerular renin-producing cells. Immunofluorescence assays detected GFP signals in mesangial cells and juxtaglomerular renin-producing cells of Piezo2GFP reporter mice. Piezo2 transcripts were observed in the Foxd1-positive stromal progenitor cells of the metanephric mesenchyme in the developing mouse kidney, which are precursors of mesangial cells and renin-producing cells. In a mouse model of dehydration, Piezo2 expression was downregulated in mesangial cells and upregulated in juxtaglomerular renin-producing cells, along with the overproduction of renin and enlargement of the area of renin-producing cells. Furthermore, the expression of the renin coding gene Ren1 was reduced by Piezo2 knockdown in cultured juxtaglomerular As4.1 cells under static and stretched conditions. These data suggest pivotal roles for Piezo2 in the regulation of glomerular filtration and body fluid balance.
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.
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.
Histopathology. 2018 Oct 4.
Sun C, Jia Y, Wang W, Bi R, Wu L, Bai Q, Zhou X.
PMID: 30286249 | DOI: 10.1111/his.13765
Abstract AIMS: The Protein expression of Programmed Death-Ligand 1 (PD-L1) has been recognized a poor prognostic biomarker in diffuse large B-cell lymphoma (DLBCL). We aim to detect PD-L1 DNA and mRNA status, and explore whether they contribute to protein expression and their clinicopathological correlation in DLBCL. METHODS AND RESULTS: In the study, we detected PD-L1 status in three different levels by Fluorescence in situ hybridization, RNA in situ hybridization and immunohistochemistry in 287 DLBCL samples with follow-ups, respectively. Their correlation and clinical pathological relevance was further analyzed. Our results showed that 1.7% (3/175) patients had PD-L1 amplification, 19.9% (57/287) PD-L1 mRNA high expression and 11.8% (34/287) high protein expression. Both mRNA and protein high expression of PD-L1 was significantly elevated in non-GCB than that in GCB DLBCL (P<0.05). In addition, the patients with PD-L1 mRNA or protein high expression but not DNA amplification have significantly poorer overall survival (OS) than that with PD-L1 low expression (P<0.05). Furthermore, we found that PD-L1 mRNA and protein expression are highly correlated (P=0.012), which was observed in all three samples with PD-L1 DNA amplification. CONCLUSIONS: PD-L1 DNA amplification is a rare event, PD-L1 mRNA mainly contribute to the protein high expression, and the latter two will serve as important biomarkers for predicting prognosis and selecting patients for immunotherapy in DLBCL.
Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys
Blessing, NA;Wu, Z;Madhavan, SM;Choy, JW;Chen, M;Shin, MK;Hoek, M;Sedor, JR;O'Toole, JF;Bruggeman, LA;
PMID: 34138902 | DOI: 10.1371/journal.pone.0253197
The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases.
Journal of Hematopathology
Shi, Y;Mi, L;Lai, Y;Zhao, M;Jia, L;Du, T;Song, Y;Li, X;
| DOI: 10.1007/s12308-023-00530-1
Overexpression of PD-L1 can be a predictive marker for anti-PD-1 therapeutic efficacy in classic Hodgkin lymphoma (CHL); however, harmonization of different IHC assays remains to be accomplished, and interpretations of PD-L1 immunostaining results remain controversial in CHL. In this study, we sought to optimize the PD-L1 immunohistochemistry (IHC) assay in CHL. All tests were performed on a tumour tissue microarray established from 54 CHL cases. Three IHC antibodies (405.9A11, SP142, 22C3) for detecting PD-L1 expression were compared semi quantitatively with the RNAscope assay (No. 310035, ACD), and the difference in the expression in background immune cells (ICs) between assays and the associations of expression levels with densities of TILs/TAMs were also analysed. 405.9A11 demonstrated best specificity in HRS cells and best sensitivity in ICs. Positive expression of PD-L1 was more frequent in ICs (85.2%) than in HRS cells (48.1%). Different subgroups of background ICs, including tumour-associated macrophages (TAMs), were assessed and scored for CD4, CD8, FOXP3, and CD163 expression. PD-L1 expression on ICs was the factor most associated with the density of TAMs. 405.9A11 provided the most convincing PD-L1 expression results. Pathologists should report PD-L1 expression in a combined manner, including both the status of HRS cells and the percentage of PD-L1-positive ICs.
Lab Invest. 2014 Jan;94(1):107-16.
Velcheti V, Schalper KA, Carvajal DE, Anagnostou VK, Syrigos KN, Sznol M, Herbst RS, Gettinger SN, Chen L, Rimm DL (2014).
PMID: 24217091doi
Recent strategies targeting the interaction of the programmed cell death ligand-1 (PD-L1, B7-H1, CD274) with its receptor, PD-1, resulted in promising activity in early phase clinical trials. In this study, we used various antibodies and in situ mRNA hybridization to measure PD-L1 in non-small cell lung cancer (NSCLC) using a quantitative fluorescence (QIF) approach to determine the frequency of expression and prognostic value in two independent populations. A control tissue microarray (TMA) was constructed using PD-L1-transfected cells, normal human placenta and known PD-L1-positive NSCLC cases. Only one of four antibodies against PD-L1 (5H1) validated for specificity on this TMA. In situ PD-L1 mRNA using the RNAscope method was similarly validated. Two cohorts of NSCLC cases in TMAs including 340 cases from hospitals in Greece and 204 cases from Yale University were assessed. Tumors showed PD-L1 protein expression in 36% (Greek) and 25% (Yale) of the cases. PD-L1 expression was significantly associated with tumor-infiltrating lymphocytes in both cohorts. Patients with PD-L1 (both protein and mRNA) expression above the detection threshold showed statistically significant better outcome in both series (log-rank P=0.036 and P=0.027). Multivariate analysis showed that PD-L1 expression was significantly associated with better outcome independent of histology. Measurement of PD-L1 requires specific conditions and some commercial antibodies show lack of specificity. Expression of PD-L1 protein or mRNA is associated with better outcome. Further studies are required to determine the value of this marker in prognosis and prediction of response to treatments targeting this pathway.
Coy S, Rashid R, Lin JR, Du Z, Donson AM, Hankinson TC, Foreman NK, Manley PE, Kieran MW, Reardon DA, Sorger PK, Santagata S.
PMID: 29509940 | DOI: 10.1093/neuonc/noy035
Abstract
BACKGROUND:
Craniopharyngiomas are neoplasms of the sellar/parasellar region that are classified into adamantinomatous (ACP) and papillary (PCP) subtypes. Surgical resection of craniopharyngiomas is challenging, and recurrence is common, frequently leading to profound morbidity. BRAF V600E mutations render PCP susceptible to BRAF/MEK inhibitors, but effective targeted therapies are needed for ACP. We explored the feasibility of targeting the PD-1/PD-L1 immune checkpoint pathway in ACP and PCP.
METHODS:
We mapped and quantified PD-L1 and PD-1 expression in ACP and PCP resections using immunohistochemistry, immunofluorescence, and RNA in situ hybridization. We used tissue-based cyclic immunofluorescence (t-CyCIF) to map the spatial distribution of immune cells and characterize cell cycle and signaling pathways in ACP tumor cells which intrinsically express PD-1.
RESULTS:
All ACP (15±14% of cells, n=23, average±S.D.) and PCP (35±22% of cells, n=18) resections expressed PD-L1. In ACP, PD-L1 was predominantly expressed by tumor cells comprising the cyst-lining. In PCP, PD-L1 was highly-expressed by tumor cells surrounding the stromal fibrovascular cores. ACP also exhibited tumor cell-intrinsic PD-1 expression in whorled epithelial cells with nuclear-localized beta-catenin. These cells exhibited evidence of elevated mTOR and MAPK signaling. Profiling of immune populations in ACP and PCP showed a modest density of CD8+ T-cells.
CONCLUSIONS:
ACP exhibit PD-L1 expression in the tumor cyst-lining and intrinsic PD-1 expression in cells proposed to comprise an oncogenic stem-like population. In PCP, proliferative tumor cells express PD-L1 in a continuous band at the stromal-epithelial interface. Targeting PD-L1 and/or PD-1 in both subtypes of craniopharyngioma might therefore be an effective therapeutic strategy.
Du Z, Abedalthagafi M, Aizer AA, McHenry AR, Sun HH, Bray MA, Viramontes O, Machaidze R, Brastianos PK, Reardon DA, Dunn IF, Freeman GJ, Ligon KL, Carpenter AE, Alexander BM, Agar NY, Rodig SJ, Bradshaw EM, Santagata S
PMID: 25609200
There are no effective medical treatments for WHO grade III (anaplastic) meningioma. Patients with this high-grade malignancy have a median survival of less than two years. Therapeutics that modulate the mechanisms that inhibit local immune responses in the tumor microenvironment are showing significant and durable clinical responses in patients with treatment refractory high-grade tumors. We examined the immune infiltrate of 291 meningiomas including WHO grade I-III meningiomas using immunohistochemistry and we examined the expression of PD-L1 mRNA by RNAscope in situ hybridization and PD-L1 protein by immunohistochemistry. In meningioma, the tumor infiltrating lymphocytes are predominantly T cells. In anaplastic meningioma, there is a sharp decrease in the number of T cells, including the numbers of CD4+ and CD8+ T cells and cells expressing PD-1 and there is also an increase in the number of FOXP3 expressing immunoregulatory (Treg) cells. PD-L1 expression is increased in anaplastic meningioma - both mRNA and protein. Using patient derived meningioma cell, we confirm that PD-L1 is expressed in meningioma cells themselves, and not solely in infiltrating immune cells. This work indicates that high-grade meningioma harbor an immunosuppressive tumor microenviroment and that increased Treg cells and elevated PD-L1 may contribute to the aggressive phenotype of these tumors.
Yuan J, Zhang J, Zhu Y, Li N, Tian T, Li Y, Li Y, Li Z, Lai Y, Gao J, Shen L.
PMID: 27191996 | DOI: 10.18632/oncotarget.9381
PD-L1 expression may be a predictive marker for anti-PD-1 therapeutic efficacy. No standard detection method of PD-L1 expression was available for advanced gastric cancer (AGC), which would be investigated in this study using RNA in situ hybridization and immunohistochemistry. Patients (N = 165) with AGC treated at Peking University Cancer Hospital from October 2008 to February 2013 were retrospectively studied. Tissue samples prior to chemotherapy were assessed for PD-L1 expression using RNA in situ hybridization (an RNAscope assay) and immunohistochemistry (IHC). The correlations of PD-L1 expression to patient characteristics and clinical outcomes were statistically analyzed. PD-L1 mRNA signals were located in tumor compartments or the mesenchyme in a brown dotted or clustered pattern, and PD-L1 mRNA expression in gastric cancer was heterogeneous. PD-L1-positive expressions were observed in 33.9% (56/165) and 35.1% (46/131) patients in mRNA level and protein level, respectively. A positive relationship was found between PD-L1 mRNA and PD-L1 protein, and compared to IHC, RNAscope assay could provide an intuitional and quantitative data with potential clinical application. No statistically significant differences occurred between PD-L1 expression and clinical response to chemotherapy, or survival. However, we found that PD-L1 expression was higher in intestinal type than in diffuse type. These findings suggested that the RNAscope assay may be a promising method for patient assessment in gastric cancer clinical trials, which would be illustrated in further study.
Terranova-Barberio M, Thomas S, Ali N, Pawlowska N, Park J, Krings G, Rosenblum MD, Budillon A, Munster PN.
PMID: - | DOI: 10.18632/oncotarget.23169
ABSTRACT
Triple-negative breast cancer (TNBC) represents a more aggressive and difficult subtype of breast cancer where responses to chemotherapy occur, but toxicity is significant and resistance often follows. Immunotherapy has shown promising results in various types of cancer, including breast cancer. Here, we investigated a new combination strategy where histone deacetylase inhibitors (HDACi) are applied with immune checkpoint inhibitors to improve immunotherapy responses in TNBC.
Testing different epigenetic modifiers, we focused on the mechanisms underlying HDACi as priming modulators of immunotherapy. Tumor cells were co-cultured with human peripheral blood mononuclear cells (PBMCs) and flow cytometric immunophenotyping was performed to define the role of epigenetic priming in promoting tumor antigen presentation and immune cell activation. We found that HDACi up-regulate PD-L1 mRNA and protein expression in a time-dependent manner in TNBC cells, but not in hormone responsive cells. Focusing on TNBC, HDACi up-regulated PD-L1 and HLA-DR on tumor cells when co-cultured with PBMCs and down-regulated CD4+ Foxp3+ Treg in vitro. HDACi significantly enhanced the in vivo response to PD-1/CTLA-4 blockade in the triple-negative 4T1 breast cancer mouse model, the only currently available experimental system with functional resemblance to human TNBC. This resulted in a significant decrease in tumor growth and increased survival, associated with increased T cell tumor infiltration and a reduction in CD4+Foxp3+ T cells in the tumor microenvironment. Overall, our results suggest a novel role for HDAC inhibition in combination with immune checkpoint inhibitors and identify a promising therapeutic strategy, supporting its further clinical evaluation for TNBC treatment.
