OPN deficiency results in severe glomerulosclerosis in uninephrectomized mice.

Osteopontin (OPN) expression has been reported to be elevated in experimental models of renal injury such as arterial hypertension or diabetic nephropathy finally leading to focal segmental glomerulosclerosis (FSGS). FSGS is characterized by glomerular matrix deposition and loss or damage of podocytes that represent the main constituents of the glomerular filtration barrier. To evaluate the role of OPN in the kidney we investigated WT and OPN knockout mice (OPN-/-) without treatment, after uninephrectomy (UNX), as well as after UNX and desoxycorticosterone acetate (DOCA)-salt treatment with respect to urine parameters, glomerular morphology, and expression of podocyte markers. OPN-/- mice showed normal urine parameters while a thickening of the glomerular basement membrane was evident. Intriguingly, following UNX, OPN-/- mice exhibited prominent FSGS, proteinuria, and glomerular matrix deposition. Electron microscopy revealed bulgings of the glomerular basement membrane and occasionally an effacement of podocytes. After UNX and DOCA-salt treatment, severe glomerular lesions as well as proteinuria and albuminuria were seen in WT and OPN-/- mice. Moreover, we found a reduction of specific markers such as Wilm's tumor-1, podocin, and synaptopodin in both experimental groups indicating a loss of podocytes. Podocyte damage was accompanied by increased number of Ki-67-positive cells in the parietal epithelium of Bowman's capsule. We conclude that OPN plays a crucial role in adaptation of podocytes following renal ablation and is renoprotective when glomerular mechanical load is increased.

PodNet, a protein-protein interaction network of the podocyte.

Interactions between proteins crucially determine cellular structure and function. Differential analysis of the interactome may help elucidate molecular mechanisms during disease development; however, this analysis necessitates mapping of expression data on protein-protein interaction networks. These networks do not exist for the podocyte; therefore, we built PodNet, a literature-based mouse podocyte network in Cytoscape format. Using database protein-protein interactions, we expanded PodNet to XPodNet with enhanced connectivity. In order to test the performance of XPodNet in differential interactome analysis, we examined podocyte developmental differentiation and the effect of cell culture. Transcriptomes of podocytes in 10 different states were mapped on XPodNet and analyzed with the Cytoscape plugin ExprEssence, based on the law of mass action. Interactions between slit diaphragm proteins are most significantly upregulated during podocyte development and most significantly downregulated in culture. On the other hand, our analysis revealed that interactions lost during podocyte differentiation are not regained in culture, suggesting a loss rather than a reversal of differentiation for podocytes in culture. Thus, we have developed PodNet as a valuable tool for differential interactome analysis in podocytes, and we have identified established and unexplored regulated interactions in developing and cultured podocytes.

FIMICS: A panel of long noncoding RNAs for cardiovascular conditions.

Cardiovascular disorders such as heart failure are leading causes of mortality. Patient stratification via identification of novel biomarkers could improve management of cardiovascular diseases of complex etiologies. Long-noncoding RNAs (lncRNAs) are highly tissue-specific in nature and have emerged as important biomarkers in human diseases. In this study, we aimed to identify cardiac-enriched lncRNAs as potential biomarkers for cardiovascular conditions. Deep RNA sequencing and quantitative PCR identified differentially expressed lncRNAs between failing and non-failing hearts. An independent dataset was used to evaluate the enrichment of lncRNAs in normal hearts. We identified a panel of 2906 lncRNAs, named FIMICS, that were either cardiac-enriched or differentially expressed between failing and non-failing hearts. Expression of lncRNAs in blood samples differentiated patients with myocarditis and acute myocardial infarction. We hereby present the FIMICS panel, a readily available tool to provide insights into cardiovascular pathologies and which could be helpful for diagnosis, monitoring and prognosis purposes.

Association of miR-144 levels in the peripheral blood with COVID-19 severity and mortality.

Coronavirus disease-2019 (COVID-19) can be asymptomatic or lead to a wide symptom spectrum, including multi-organ damage and death. Here, we explored the potential of microRNAs in delineating patient condition and predicting clinical outcome. Plasma microRNA profiling of hospitalized COVID-19 patients showed that miR-144-3p was dynamically regulated in response to COVID-19. Thus, we further investigated the biomarker potential of miR-144-3p measured at admission in 179 COVID-19 patients and 29 healthy controls recruited in three centers. In hospitalized patients, circulating miR-144-3p levels discriminated between non-critical and critical illness (AUCmiR-144-3p = 0.71; p = 0.0006), acting also as mortality predictor (AUCmiR-144-3p = 0.67; p = 0.004). In non-hospitalized patients, plasma miR-144-3p levels discriminated mild from moderate disease (AUCmiR-144-3p = 0.67; p = 0.03). Uncontrolled release of pro-inflammatory cytokines can lead to clinical deterioration. Thus, we explored the added value of a miR-144/cytokine combined analysis in the assessment of hospitalized COVID-19 patients. A miR-144-3p/Epidermal Growth Factor (EGF) combined score discriminated between non-critical and critical hospitalized patients (AUCmiR-144-3p/EGF = 0.81; p < 0.0001); moreover, a miR-144-3p/Interleukin-10 (IL-10) score discriminated survivors from nonsurvivors (AUCmiR-144-3p/IL-10 = 0.83; p < 0.0001). In conclusion, circulating miR-144-3p, possibly in combination with IL-10 or EGF, emerges as a noninvasive tool for early risk-based stratification and mortality prediction in COVID-19.

AlphaV-integrins mediate the mechanoprotective action of osteopontin in podocytes.

Increased mechanical load in podocytes due to glomerular hypertension is one of the important factors leading to podocyte damage and chronic kidney disease. In previous studies, we have shown that mechanical stretch increases osteopontin (OPN) expression in podocytes and that exogenous OPN is mechanoprotective via facilitating cytoskeletal reorganization of podocytes. In the present study, we asked whether the mechanoprotective effect of OPN in podocytes is mediated through specific integrins and whether endogenous OPN of podocytes is required for mechanoprotection. Conditionally immortalized mouse podocytes and primary podocytes (PP) from OPN-/- and OPN+/+ mice were used. Cyclic biaxial mechanical stretch (0.5 Hz, 7% linear strain) was applied for up to 3 days. Stretch-induced cell loss was ∼30% higher in OPN-/- PP compared with OPN+/+ PP. Increased cell loss of OPN-/- PP was rescued by OPN coating. Analysis of integrin expression by RT-PCR, application of RGD and SLAYGLR peptides and anti-integrin antibodies, small-interfering RNA knockdown of integrins, and application of kinase inhibitors identified αV-integrins (αVß1, αVß3, and αVß5) to mediate the mechano-protective effect of OPN in podocytes involving focal adhesion kinase, Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Our results demonstrate that endogenous OPN of podocytes plays a nonredundant role in podocyte adaptation to mechanical stretch, and that OPN signaling via α(V)-integrins may represent a relevant therapeutical target in podocytes.

Biomarkers Associated with Atrial Fibrillation in Patients with Ischemic Stroke: A Pilot Study from the NOR-FIB Study.

BACKGROUND AND PURPOSE: Cardioembolic stroke due to paroxysmal atrial fibrillation (AF) may account for 1 out of 4 cryptogenic strokes (CS) and transient ischemic attacks (TIAs). The purpose of this pilot study was to search for biomarkers potentially predicting incident AF in patients with ischemic stroke or TIA. METHODS: Plasma samples were collected from patients aged 18 years and older with ischemic stroke or TIA due to AF (n = 9) and large artery atherosclerosis (LAA) with ipsilateral carotid stenosis (n = 8) and age- and sex-matched controls (n = 10). Analyses were performed with the Olink technology simultaneously measuring 184 biomarkers of cardiovascular disease. For bioinformatics, acquired data were analyzed using gene set enrichment analysis (GSEA). Selected proteins were validated using ELISA. Individual receiver operating characteristic (ROC) curves and odds ratios from logistic regression were calculated. A randomForest (RF) model with out-of-bag estimate was applied for predictive modeling. RESULTS: GSEA indicated enrichment of proteins related to inflammatory response in the AF group. Interleukin (IL)-6, growth differentiation factor (GDF)-15, and pentraxin-related protein PTX3 were the top biomarkers on the ranked list for the AF group compared to the LAA group and the control group. ELISA validated increased expression of all tested proteins (GDF-15, PTX3, and urokinase plasminogen activator surface receptor [U-PAR]), except for IL-6. 19 proteins had the area under the ROC curve (AUC) over 0.85 including all of the proteins with significant evolution in the logistic regression. AUCs were very discriminant in distinguishing patients with and without AF (LAA and control group together). GDF-15 alone reached AUC of 0.95. Based on RF model, all selected participants in the tested group were classified correctly, and the most important protein in the model was GDF-15. CONCLUSIONS: Our results demonstrate an association between inflammation and AF and that multiple proteins alone and in combination may potentially be used as indicators of AF in CS and TIA patients. However, further studies including larger samples sizes are needed to support these findings. In the ongoing NOR-FIB study, we plan further biomarker assessments in patients with CS and TIA undergoing long-term cardiac rhythm monitoring with insertable cardiac monitors.

Alterations of the podocyte proteome in response to high glucose concentrations.

Diabetic nephropathy is one of the most common complications of diabetes mellitus and the leading cause of end-stage renal disease. A reduction in podocyte number has been documented in the kidneys of these patients. To identify the molecular changes in podocytes that are primarily caused by high glucose (HG) concentrations and not by secondary alterations (e.g. glomerular hypertension), we investigated the protein expression profiles in a podocyte cell line under long-term HG exposure (30 versus 10 mM for 2 wk). Proteins were separated by 2-DE, and we identified 39 different proteins in 48 spots that were differentially regulated by more than twofold in response to HG concentrations using MALDI-TOF MS and MASCOT software. These proteins belong to several protein classes, including cytoskeletal proteins and specific annexins (annexins III and VI). Downregulation of annexins III and VI by HG concentrations was confirmed by qRT-PCR, Western blot, and immunostaining, and was also observed in glomeruli of kidney biopsies from patients with diabetic nephropathy. Our data demonstrate that HG concentrations per se are sufficient to strongly modify the protein expression profile of podocytes, the analysis of which contributes to the identification of novel targets involved in diabetic nephropathy.

Palladin is a dynamic actin-associated protein in podocytes.

Palladin, a cytoskeletal protein with essential functions for stress fiber formation, is found in developing and mature tissues, including the kidney. To define its role in the kidney, we measured its expression in mouse kidney and found it co-localized with F-actin in smooth muscle cells of renal arterial vessels, mesangial cells, and podocytes but not in tubular epithelium. Using immunoelectron microscopy, we confirmed that palladin was present in podocytes. In cultured mouse podocytes, palladin co-localized with F-actin in dense regions of stress fibers, focal adhesions, cell-cell contacts and motile cell margins. Transfection with the N-terminal half of palladin targeted it to F-actin-containing structures in podocytes while the C-terminal half accumulated in the nucleus, a result also found for endogenous palladin in cultured cells after leptomycin B was used to block nuclear export. Green fluorescent protein (GFP)-tagged palladin was found in dynamic ring-like F-actin structures and ruffles in cultured podocytes after stimulation with epidermal growth factor. Inhibition of palladin expression by transfection of an antisense construct reduced the formation of ring-like structures. Photo-bleaching analysis showed that GFP-palladin turned over with a half-time of 10 s in focal adhesions and dense regions of stress fibers, suggesting that palladin is a dynamic scaffolding protein. Our study shows that palladin is expressed in podocytes and plays an important role in actin dynamics.

MicroRNAs: Key Regulators to Understand Osteoclast Differentiation?

MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that represent important posttranscriptional regulators of protein-encoding genes. In particular, miRNAs play key roles in regulating cellular processes such as proliferation, migration, and cell differentiation. Recently, miRNAs emerged as critical regulators of osteoclasts (OCs) biology and have been involved in OCs pathogenic role in several disorders. OCs are multinucleated cells generated from myeloid precursors in the bone marrow, specialized in bone resorption. While there is a growing number of information on the cytokines and signaling pathways that are critical to control the differentiation of osteoclast precursors (OCPs) into mature OCs, the connection between OC differentiation steps and miRNAs is less well-understood. The present review will first summarize our current understanding of the miRNA-regulated pathways in the sequential steps required for OC formation, from the motility and migration of OCPs to the cell-cell fusion and the final formation of the actin ring and ruffled border in the functionally resorbing multinucleated OCs. Then, considering the difficulty of working on primary OCs and on the generation of robust data we will give an update on the most recent advances in the detection technologies for miRNAs quantification and how these are of particular interest for the understanding of OC biology and their use as potential biomarkers.

Synovial-Fluid miRNA Signature for Diagnosis of Juvenile Idiopathic Arthritis.

Juvenile idiopathic arthritis (JIA) is the most common chronic inflammatory rheumatism in childhood; microRNAs (miRNAs) have been proposed as diagnostic biomarkers. Although joints are the primary targets for JIA, a synovial fluid-based miRNA signature has never been studied. We aim to identify miRNA biomarkers in JIA by comparing synovial fluid and serum samples from children with JIA and K.kingae septic arthritis (SA). With next-generation high-throughput sequencing, we measured the absolute levels of 2083 miRNAs in synovial fluid and serum from an exploratory cohort of children and validated differentially expressed miRNAs in a replication study by using RT-qPCR. We identified a 19-miRNA signature only in synovial fluid samples that was significantly deregulated, with at least 2-fold change in expression, in JIA versus SA (p < 0.01). The combination of miR-6764-5p, miR-155, and miR-146a-5p expression in synovial fluid yielded an area under the receiver operating characteristic curve of 1 (95% CI 0.978 to 1), thereby perfectly differentiating JIA from SA in children. We propose, for the first time, a synovial fluid-specific miRNA signature for JIA and associated signaling pathways that may indicate potential biomarkers to assist in the classification and differential diagnosis of JIA and help in understanding JIA pathogenesis.

The phosphoinositide 3-kinase/Akt pathway: a new target in human renal cell carcinoma therapy.

Metastatic renal cell carcinoma is resistant to current therapies. The phosphoinositide 3-kinase (PI3K)/Akt signaling cascade induces cell growth, cell transformation, and neovascularization. We evaluated whether targeting this pathway could be of therapeutic value against human renal cell carcinoma. The activation of the PI3K/Akt pathway and its role in renal cell carcinoma progression was evaluated in vitro in seven human cell lines by Western blot, cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and fluorescence-activated cell sorting analysis, using two PI3K inhibitors, LY294002 and wortmannin, as well as by transfection with various Akt constructs and through Akt knockdown by small interfering RNA (siRNA). In vivo nude mice bearing human renal cell carcinoma tumor xenografts were treated with LY294002 (75 mg/kg/wk, 4 weeks, i.p.). Tumor growth was measured and tumors were subjected to Western blot and immunohistochemical analysis. Akt was constitutively activated in all cell lines. Constitutive phosphorylation of glycogen synthase kinase-3 (GSK-3) was observed in all cell lines, whereas forkhead transcription factor and mammalian target of rapamycin, although expressed, were not constitutively phosphorylated. Exposure to LY294002 or wortmannin decreased Akt activation and GSK-3 phosphorylation and reduced cell growth by up to 70% through induction of cell apoptosis. These effects were confirmed by transfection experiments with Akt constructs or Akt siRNA. Importantly, LY294002 induced up to 50% tumor regression in mice through tumor cell apoptosis. Tumor neovascularization was significantly increased by LY294002 treatment. Blood chemistries showed no adverse effects of the treatment. Our results suggest an important role of PI3K/Akt inhibitors as a potentially useful treatment for patients with renal cell carcinoma.

Abnormal renovascular parathyroid hormone-1 receptor in hypertension: Primary defect or secondary to angiotensin ii type 1 receptor activation?

We previously reported that PTHrP-induced renal vasodilation is impaired in mature spontaneously hypertensive rats (SHR) through down-regulation of the type 1 PTH/PTHrP receptor (PTH1R), a feature that contributes to the high renal vascular resistance in SHR. Here we asked whether this defect represents a prime determinant in genetic hypertension or whether it is secondary to angiotensin II (Ang II) and/or the mechanical forces exerted on the vascular wall. We found that the treatment of SHR with established hypertension by the Ang II type 1 receptor antagonist, losartan, reversed the down-regulation of PTH1R expression in intrarenal small arteries and restored PTHrP-induced vasodilation in ex vivo perfused kidneys. In contrast, the PTH1R deregulation was not found in intrarenal arteries isolated from prehypertensive SHR. Moreover, this defect, which is not seen in extrarenal vessels (aorta, mesenteric arteries) from mature SHR appeared kidney specific in accordance with the acknowledged enrichment of interstitial Ang II in this organ and its enhancement in SHR. In deoxycorticosterone-acetate-salt rats, an Ang II-independent model of hypertension, renovascular PTH1R expression and related vasodilation were not altered. In SHR-derived renovascular smooth muscle cells (RvSMCs), the PTH1R was spontaneously down-regulated and its transcript destabilized, compared with Wistar RvSMCs, both effects being antagonized by losartan. Exogenous Ang II elicited down-regulation of PTH1R mRNA in RvSMCs from Wistar rats. Together, these data demonstrate that Ang II acts via the Ang II type 1 receptor to destabilize PTH1R mRNA in the renal vessel in the SHR model of genetic hypertension. This process is kidney specific and independent from blood pressure increase.

Parathyroid hormone-related protein is an essential growth factor for human clear cell renal carcinoma and a target for the von Hippel-Lindau tumor suppressor gene.

Clear cell renal carcinoma (CCRC) is responsible for 2% of cancer-related deaths worldwide and is resistant to virtually all therapies, indicating the importance of a search for new therapeutic targets. Parathyroid hormone-related protein (PTHrP) is a polyprotein derived from normal and malignant cells that regulates cell growth. In the current study, we show that blocking PTHrP with antibodies or antagonizing the common parathyroid hormone (PTH)/PTHrP receptor, the PTH1 receptor, dramatically blunts the expansion of human CCRC in vitro by promoting cell death. Importantly, in nude mice, anti-PTHrP antibodies induced complete regression of 70% of the implanted tumors by inducing cell death. In addition, we demonstrate that the von Hippel-Lindau tumor suppressor protein, which functions as a gatekeeper for CCRC, negatively regulates PTHrP expression at the post-transcriptional level. These studies indicate that PTHrP is an essential growth factor for CCRC and is a novel target for the von Hippel-Lindau tumor suppressor protein. Taken together, these results strongly suggest that targeting the PTHrP/PTH1 receptor system may provide a new avenue for the treatment of this aggressive cancer in humans.

Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis.

Rapidly progressive glomerulonephritis (RPGN) is a life-threatening clinical syndrome and a morphological manifestation of severe glomerular injury that is marked by a proliferative histological pattern ('crescents') with accumulation of T cells and macrophages and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the epidermal growth factor receptor (EGFR, also known as ErbB1) in mice with RPGN. In HB-EGF-deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started 4 d after the induction of experimental RPGN. This suggests that targeting the HB-EGF-EGFR pathway could also be beneficial in treatment of human RPGN.

Antitumor effect of parathyroid hormone-related protein neutralizing antibody in human renal cell carcinoma in vitro and in vivo.

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene occurs in 40-80% of human conventional renal cell carcinomas (RCCs). We showed recently that VHL-deficient RCCs expressed large amounts of parathyroid hormone-related protein (PTHrP), and that PTHrP, acting through the PTH1 receptor (PTH1R), plays an essential role in tumor growth. We also showed that PTHrP expression is negatively regulated by the VHL gene products (pVHL). Our goal was to determine whether blocking the PTHrP/PTH1R system might be of therapeutic value against RCC, independent of VHL status and PTHrP expression levels. The antitumor activity of PTHrP neutralizing antibody and of PTH1R antagonist were evaluated in vitro and in vivo in a panel of human RCC lines expressing or not pVHL. PTHrP is upregulated compared with normal tubular cells. In vitro, tumor cell growth and viability was decreased by up to 80% by the antibody in all cell lines. These effects resulted from apoptosis. Exogenously added PTHrP had no effect on cell growth and viability, but reversed the inhibitory effects of the antibody. The growth inhibition was reproduced by a specific PTH1R antagonist in all cell lines. In vivo, the treatment of nude mice bearing the Caki-1 RCC tumor with the PTHrP antibody inhibited tumor growth by 80%, by inducing apoptosis. Proliferation and neovascularization were not affected by the antiserum. Anti-PTHrP treatment induced no side effects as assessed by animal weight and blood chemistries. Current therapeutic strategies are only marginally effective against metastatic RCC, and adverse effects are common. This study provides a rationale for evaluating the blockade of PTHrP signaling as therapy for human RCC in a clinical setting.

Role of parathyroid hormone-related protein in the regulation of stretch-induced renal vascular smooth muscle cell proliferation.

In vivo, vascular smooth muscle cells (VSMC) are continuously exposed to mechanical cyclic stretch as a result of the pulsatile blood flow from the cardiac contractile cycle. Stretch is altered in pathologic conditions and contributes to vascular remodeling by modulating VSMC proliferation and death. Parathyroid hormone-related protein (PTHrP) is a locally produced poly-protein that regulates cell growth. It was shown previously that PTHrP inhibits VSMC proliferation through the auto/paracrine pathway by interacting with its receptor, the PTH1R, but stimulates VSMC proliferation through the intracrine pathway by translocating into the nucleus. In the current study, VSMC that were isolated from both resistance and compliance vessels were used to study the role of PTHrP in VSMC proliferation under experimental stretch. It is shown that PTHrP gene expression is upregulated by stretch and that PTHrP opposes the inhibitory effect induced by stretch on VSMC proliferation through the intracrine pathway. In addition, it is demonstrated that PTHrP expression is controlled at the post-transcriptional level by stretch. Taken together, these results strongly suggest that PTHrP plays a critical role in the modulation of VSMC proliferation in response to stretch. Thus, in conditions in which stretch is increased, such as in hypertension or in restenosis after angioplasty, PTHrP may contribute to vessel hyperplasia.