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.
McMeekin, LJ;Joyce, KL;Jenkins, LM;Bohannon, BM;Patel, KD;Bohannon, AS;Patel, A;Fox, SN;Simmons, MS;Day, JJ;Kralli, A;Crossman, DK;Cowell, RM;
PMID: 34648866 | DOI: 10.1016/j.neuroscience.2021.10.007
Deficiency in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression or function is implicated in numerous neurological and psychiatric disorders. PGC-1α is required for the expression of genes involved in synchronous neurotransmitter release, axonal integrity, and metabolism, especially in parvalbumin-positive interneurons. As a transcriptional coactivator, PGC-1α requires transcription factors to specify cell-type-specific gene programs; while much is known about these factors in peripheral tissues, it is unclear if PGC-1α utilizes these same factors in neurons. Here, we identified putative transcription factors controlling PGC-1α-dependent gene expression in the brain using bioinformatics, and then validated the role of the top candidate in a knockout mouse model. We transcriptionally profiled cells overexpressing PGC-1α and searched for over-represented binding motifs in the promoters of upregulated genes. Binding sites of the estrogen-related receptor (ERR) family of transcription factors were enriched and blockade of ERRα attenuated PGC-1α-mediated induction of mitochondrial and synaptic genes in cell culture. Localization in the mouse brain revealed enrichment of ERRα expression in parvalbumin-expressing neurons with tight correlation of expression with PGC-1α across brain regions. In ERRα null mice, PGC-1α-dependent genes were reduced in multiple regions, including neocortex, hippocampus, and cerebellum, though not to the extent observed in PGC-1α null mice. Behavioral assessment revealed ambulatory hyperactivity in response to amphetamine and impairments in sensorimotor gating without the overt motor impairment characteristic of PGC-1α null mice. These data suggest that ERRα is required for normal levels of expression of PGC-1α-dependent genes in neurons, but that additional factors may be involved in their regulation. Significance statement The transcription factors with which PGC-1α interacts determine specificity of the transcriptional program it drives across cell populations, but those mediating its functions in parvalbumin-expressing neurons are unknown. Relative to other PGC-1α-interacting transcription factors, ERRα is enriched in parvalbumin-expressing neurons and shows robust spatial and temporal correlation with PGC-1α expression throughout the brain. ERRα is also necessary for PGC-1α-dependent transcription both in vitro and in vivo for metabolic and neuronal transcripts. These data suggest that ERRα is an important player in cell-specific PGC-1α-dependent transcription in the CNS and may play a role in regulating parvalbumin-expressing neuron maturation and function.
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.
Khom, S;Borgonetti, V;Vozella, V;Kirson, D;Rodriguez, L;Gandhi, P;Bianchi, P;Snyder, A;Vlkolinsky, R;Bajo, M;Oleata, C;Ciccocioppo, R;Roberto, M;
| DOI: 10.1016/j.ynstr.2023.100547
Impairments in the function of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced glucocorticoid receptor (GR) activity in the central amygdala (CeA) are critical mechanisms in the pathogenesis of alcohol use disorder (AUD). The GR antagonist mifepristone attenuates craving in AUD patients, alcohol consumption in AUD models, and decreases CeA γ-aminobutyric acid (GABA) transmission in alcohol-dependent rats. Previous studies suggest elevated GR activity in the CeA of male alcohol-preferring Marchigian-Sardinian (msP) rats, but its contribution to heightened CeA GABA transmission driving their characteristic post-dependent phenotype is largely unknown. We determined Nr3c1 (the gene encoding GR) gene transcription in the CeA in male and female msP and Wistar rats using in situ hybridization and studied acute effects of mifepristone (10 μM) and its interaction with ethanol (44 mM) on pharmacologically isolated spontaneous inhibitory postsynaptic currents (sIPSCs) and electrically evoked inhibitory postsynaptic potentials (eIPSPs) in the CeA using ex vivo slice electrophysiology. Female rats of both genotypes expressed more CeA GRs than males, suggesting a sexually dimorphic GR regulation of CeA activity. Mifepristone reduced sIPSC frequencies (GABA release) and eIPSP amplitudes in msP rats of both sexes, but not in their Wistar counterparts; however, it did not prevent acute ethanol-induced increase in CeA GABA transmission in male rats. In msP rats, GR regulates CeA GABAergic signaling under basal conditions, indicative of intrinsically active GR. Thus, enhanced GR function in the CeA represents a key mechanism contributing to maladaptive behaviors associated with AUD.
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.
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.
Molecular Neuropsychiatry
Hu X,. Rocco BR, Fee C, Sibille E.
PMID: - | DOI: 10.1159/000495840
Converging evidence suggests that deficits in somatostatin (SST)-expressing neuron signaling contributes to major depressive disorder. Preclinical studies show that enhancing this signaling, specifically at α5 subunit-containing γ-aminobutyric acid subtype A receptors (α5-GABAARs), provides a potential means to overcome low SST neuron function. The cortical microcircuit comprises multiple subtypes of inhibitory γ-aminobutyric acid (GABA) neurons and excitatory pyramidal cells (PYCs). In this study, multilabel fluorescence in situ hybridization was used to characterize α5-GABAAR gene expression in PYCs and three GABAergic neuron subgroups – vasoactive intestinal peptide (VIP)-, SST-, and parvalbumin (PV)-expressing cells – in the human and mouse frontal cortex. Across species, we found the majority of gene expression in PYCs (human: 39.7%; mouse: 54.14%), less abundant expression in PV neurons (human: 20%; mouse: 16.33%), and no expression in VIP neurons (0%). Only human SST cells expressed GABRA5, albeit at low levels (human: 8.3%; mouse: 0%). Together, this localization suggests potential roles for α5-GABAARs within the cortical microcircuit: (1) regulators of PYCs, (2) regulators of PV cell activity across species, and (3) sparse regulators of SST cell inhibition in humans. These results will advance our ability to predict the effects of pharmacological agents targeting α5-GABAARs, which have shown therapeutic potential in preclinical animal models.
Gunduz-Cinar O, Brockway E, Lederle L, Wilcox T, Halladay LR, Ding Y, Oh H, Busch EF, Kaugars K, Flynn S, Limoges A, Bukalo O, MacPherson KP, Masneuf S, Pinard C, Sibille E, Chesler EJ, Holmes A.
PMID: 29311651 | DOI: 10.1038/s41380-017-0003-3
Recent years have seen advances in our understanding of the neural circuits associated with trauma-related disorders, and the development of relevant assays for these behaviors in rodents. Although inherited factors are known to influence individual differences in risk for these disorders, it has been difficult to identify specific genes that moderate circuit functions to affect trauma-related behaviors. Here, we exploited robust inbred mouse strain differences in Pavlovian fear extinction to uncover quantitative trait loci (QTL) associated with this trait. We found these strain differences to be resistant to developmental cross-fostering and associated with anatomical variation in basolateral amygdala (BLA) perineuronal nets, which are developmentally implicated in extinction. Next, by profiling extinction-driven BLA expression of QTL-linked genes, we nominated Ppid (peptidylprolyl isomerase D, a member of the tetratricopeptide repeat (TPR) protein family) as an extinction-related candidate gene. We then showed that Ppid was enriched in excitatory and inhibitory BLA neuronal populations, but at lower levels in the extinction-impaired mouse strain. Using a virus-based approach to directly regulate Ppid function, we demonstrated that downregulating BLA-Ppid impaired extinction, while upregulating BLA-Ppid facilitated extinction and altered in vivo neuronal extinction encoding. Next, we showed that Ppid colocalized with the glucocorticoid receptor (GR) in BLA neurons and found that the extinction-facilitating effects of Ppid upregulation were blocked by a GR antagonist. Collectively, our results identify Ppid as a novel gene involved in regulating extinction via functional actions in the BLA, with possible implications for understanding genetic and pathophysiological mechanisms underlying risk for trauma-related disorders.
Dilz, J;Auge, I;Groeneveld, K;Reuter, S;Mrowka, R;
PMID: 37061575 | DOI: 10.1038/s41598-023-33110-5
Kidneys are complex organs, and reproducing their function and physiology in a laboratory setting remains difficult. During drug development, potential compounds may exhibit unexpected nephrotoxic effects, which imposes a significant financial burden on pharmaceutical companies. As a result, there is an ongoing need for more accurate model systems. The use of renal organoids to simulate responses to nephrotoxic insults has the potential to bridge the gap between preclinical drug efficacy studies in cell cultures and animal models, and the stages of clinical trials in humans. Here we established an accessible fluorescent whole-mount approach for nuclear and membrane staining to first provide an overview of the organoid histology. Furthermore, we investigated the potential of renal organoids to model responses to drug toxicity. For this purpose, organoids were treated with the chemotherapeutic agent doxorubicin for 48 h. When cell viability was assessed biochemically, the organoids demonstrated a significant, dose-dependent decline in response to the treatment. Confocal microscopy revealed visible tubular disintegration and a loss of cellular boundaries at high drug concentrations. This observation was further reinforced by a dose-dependent decrease of the nuclear area in the analyzed images. In contrast to other approaches, in this study, we provide a straightforward experimental framework for drug toxicity assessment in renal organoids that may be used in early research stages to assist screen for potential adverse effects of compounds.
Acta neuropathologica communications
Davis, SE;Cook, AK;Hall, JA;Voskobiynyk, Y;Carullo, NV;Boyle, NR;Hakim, AR;Anderson, KM;Hobdy, KP;Pugh, DA;Murchison, CF;McMeekin, LJ;Simmons, M;Margolies, KA;Cowell, RM;Nana, AL;Spina, S;Grinberg, LT;Miller, BL;Seeley, WW;Arrant, AE;
PMID: 37118844 | DOI: 10.1186/s40478-023-01571-4
Loss of function progranulin (GRN) mutations are a major autosomal dominant cause of frontotemporal dementia (FTD). Patients with FTD due to GRN mutations (FTD-GRN) develop frontotemporal lobar degeneration with TDP-43 pathology type A (FTLD-TDP type A) and exhibit elevated levels of lysosomal proteins and storage material in frontal cortex, perhaps indicating lysosomal dysfunction as a mechanism of disease. To investigate whether patients with sporadic FTLD exhibit similar signs of lysosomal dysfunction, we compared lysosomal protein levels, transcript levels, and storage material in patients with FTD-GRN or sporadic FTLD-TDP type A. We analyzed samples from frontal cortex, a degenerated brain region, and occipital cortex, a relatively spared brain region. In frontal cortex, patients with sporadic FTLD-TDP type A exhibited similar increases in lysosomal protein levels, transcript levels, and storage material as patients with FTD-GRN. In occipital cortex of both patient groups, most lysosomal measures did not differ from controls. Frontal cortex from a transgenic mouse model of TDP-opathy had similar increases in cathepsin D and lysosomal storage material, showing that TDP-opathy and neurodegeneration can drive these changes independently of progranulin. To investigate these changes in additional FTLD subtypes, we analyzed frontal cortical samples from patients with sporadic FTLD-TDP type C or Pick's disease, an FTLD-tau subtype. All sporadic FTLD groups had similar increases in cathepsin D activity, lysosomal membrane proteins, and storage material as FTD-GRN patients. However, patients with FTLD-TDP type C or Pick's disease did not have similar increases in lysosomal transcripts as patients with FTD-GRN or sporadic FTLD-TDP type A. Based on these data, accumulation of lysosomal proteins and storage material may be a common aspect of end-stage FTLD. However, the unique changes in gene expression in patients with FTD-GRN or sporadic FTLD-TDP type A may indicate distinct underlying lysosomal changes among FTLD subtypes.
Ziminski J, Hessler S, Margetts-Smith G, Sieburg MC, Crombag HS, Koya E.
PMID: 28213443 | DOI: 10.1523/JNEUROSCI.3766-16.2017
Cues that predict the availability of food rewards influence motivational states and elicit food-seeking behaviors. If a cue no longer predicts food availability, animals may adapt accordingly by inhibiting food seeking responses. Sparsely activated sets of neurons, coined neuronal ensembles, have been shown to encode the strength of reward-cue associations. While alterations in intrinsic excitability have been shown to underlie many learning and memory processes, little is known about these properties specifically on cue-activated neuronal ensembles. We examined the activation patterns of cue-activated orbitofrontal cortex (OFC) and nucleus accumbens (NAc) shell ensembles using wild-type and Fos-GFP mice following appetitive conditioning with sucrose and extinction learning. We also investigated the neuronal excitability of recently activated, GFP+ neurons in these brain areas using whole-cell electrophysiology in brain slices. Exposure to a sucrose cue elicited activation of neurons in both the NAc shell and OFC. In the NAc shell, but not the OFC, these activated GFP+ neurons were more excitable than surrounding GFP- neurons. Following extinction, the number of neurons activated in both areas was reduced and activated ensembles in neither area exhibited altered excitability. These data suggest that learning-induced alterations in the intrinsic excitability of neuronal ensembles is regulated dynamically across different brain areas. Furthermore, we show that changes in associative strength modulate the excitability profile of activated ensembles in the NAc shell.SIGNIFICANCE STATEMENTSparsely distributed sets of neurons called 'neuronal ensembles' encode learned associations about food and cues predictive of its availability. Widespread changes in neuronal excitability have been observed in limbic brain areas after associative learning, but little is known about the excitability changes that occur specifically on neuronal ensembles that encode appetitive associations. Here we reveal that sucrose cue exposure recruited a more excitable ensemble in the nucleus accumbens, but not orbitofrontal cortex compared to their surrounding neurons. This excitability difference was not observed when the cue's salience was diminished following extinction learning. These novel data provide evidence that the intrinsic excitability of appetitive memory-encoding ensembles is differentially regulated across brain areas and dynamically adapts to changes in associative strength.
