Reduced G protein signaling despite impaired internalization and ß-arrestin recruitment in patients carrying a CXCR4Leu317fsX3 mutation causing WHIM syndrome.

WHIM syndrome is an inherited immune disorder caused by an autosomal dominant heterozygous mutation in CXCR4. The disease is characterized by neutropenia/leukopenia (secondary to retention of mature neutrophils in bone marrow), recurrent bacterial infections, treatment-refractory warts, and hypogammaglobulinemia. All mutations reported in WHIM patients lead to the truncations in the C-terminal domain of CXCR4, R334X being the most frequent. This defect prevents receptor internalization and enhances both calcium mobilization and ERK phosphorylation, resulting in increased chemotaxis in response to the unique ligand CXCL12. Here, we describe 3 patients presenting neutropenia and myelokathexis, but normal lymphocyte count and immunoglobulin levels, carrying what we believe to be a novel Leu317fsX3 mutation in CXCR4, leading to a complete truncation of its intracellular tail. The analysis of the L317fsX3 mutation in cells derived from patients and in vitro cellular models reveals unique signaling features in comparison with R334X mutation. The L317fsX3 mutation impairs CXCR4 downregulation and ß-arrestin recruitment in response to CXCL12 and reduces other signaling events - including ERK1/2 phosphorylation, calcium mobilization, and chemotaxis - all processes that are typically enhanced in cells carrying the R334X mutation. Our findings suggest that, overall, the L317fsX3 mutation may be causative of a form of WHIM syndrome not associated with an augmented CXCR4 response to CXCL12.

Phosphoproteomic mapping of CCR5 and ACKR2 signaling properties.

ACKR2 is an atypical chemokine receptor which is structurally uncoupled from G proteins and is unable to activate signaling pathways used by conventional chemokine receptors to promote cell migration. Nonetheless, ACKR2 regulates inflammatory and immune responses by shaping chemokine gradients in tissues via scavenging inflammatory chemokines. To investigate the signaling pathways downstream to ACKR2, a quantitative SILAC-based phosphoproteomic analysis coupled with a systems biology approach with network analysis, was carried out on a HEK293 cell model expressing either ACKR2 or its conventional counterpart CCR5. The model was stimulated with the common agonist CCL3L1 for short (3 min) and long (30 min) durations. As expected, many of the identified proteins are known to participate in conventional signal transduction pathways and in the regulation of cytoskeleton dynamics. However, our analyses revealed unique phosphorylation and network signatures, suggesting roles for ACKR2 other than its scavenger activity. In conclusion, the mapping of phosphorylation events at a holistic level indicated that conventional and atypical chemokine receptors differ in signaling properties. This provides an unprecedented level of detail in chemokine receptor signaling and identifying potential targets for the regulation of ACKR2 and CCR5 function.

Plasma small extracellular vesicles from dogs affected by cutaneous mast cell tumors deliver high levels of miR-21-5p.

Small extracellular vesicles (sEV) are a class of extracellular vesicles (30-150 nm), delivering molecules including proteins, metabolites, and microRNAs (miRNAs), involved in physiological intercellular crosstalk and disease pathogenesis. The present pilot study aims are (I) to develop an easy and fast protocol for the isolation of sEV from plasma of mast cell tumor (MCT)-affected dogs; (II) to evaluate if miR-21-5p (sEV-miR-21-5p), a miRNA overexpressed by MCT, is associated with sEV. Seventeen dogs have been enrolled in the study: 4 healthy and 13 (6 with and 7 without nodal metastasis) MCT-affected dogs. sEV were isolated using size exclusion chromatography (SEC) (IZON column 35nm) and were characterized by Western blot, Nanoparticle tracking analysis, and transmission electron microscopy. sEV-miR-21-5p was quantified using digital PCR. sEV expressed the specific markers CD9 and TSG101, and a marker of mast cell tryptase. The sEV mean concentration and size were 2.68E + 10 particles/ml, and 99.6 nm, 2.89E + 10 particles/ml and 101.7 nm, and 3.21E + 10 particles/ml and 124 nm in non-metastatic, nodal metastatic, and healthy samples, respectively. The comparative analysis demonstrated that the level of sEV-miR-21-5p was significantly higher in dogs with nodal metastasis compared to healthy (P = 0.038) and without nodal metastasis samples (P = 0.007). In conclusion, the present work demonstrated that a pure population of sEV can be isolated from the plasma of MCT-affected dogs using the SEC approach and that the level of sEV-miR-21-5p is higher in nodal metastatic MCT-affected dogs compared with healthy and MCT-affected dogs without nodal involvement.

Endogenous modification of the chemoattractant CXCL5 alters receptor usage and enhances its activity toward neutrophils and monocytes.

The inflammatory human chemokine CXCL5 interacts with the G protein-coupled receptor CXCR2 to induce chemotaxis and activation of neutrophils. CXCL5 also has weak agonist activity toward CXCR1. The N-terminus of CXCL5 can be modified by proteolytic cleavage or deimination of Arg9 to citrulline (Cit), and these modifications can occur separately or together. Here, we chemically synthesized native CXCL5(1-78), truncated CXCL5 [CXCL5(9-78)], and the citrullinated (Cit9) versions and characterized their functions in vitro and in vivo. Compared with full-length CXCL5, N-terminal truncation resulted in enhanced potency to induce G protein signaling and ß-arrestin recruitment through CXCR2, increased CXCL5-initiated internalization of CXCR2, and greater Ca2+ signaling downstream of not only CXCR2 but also CXCR1. Citrullination did not affect the capacity of CXCL5 to activate classical or alternative signaling pathways. Administering the various CXCL5 forms to mice revealed that in addition to neutrophils, CXCL5 exerted chemotactic activity toward monocytes and that this activity was increased by N-terminal truncation. These findings were confirmed by in vitro chemotaxis and Ca2+ signaling assays with primary human CD14+ monocytes and human THP-1 monocytes. In vitro and in vivo analyses suggested that CXCL5 targeted monocytes through CXCR1 and CXCR2. Thus, truncation of the N-terminus makes CXCL5 a more potent chemoattractant for both neutrophils and monocytes that acts through CXCR1 and CXCR2.

The kidney, COVID-19, and the chemokine network: an intriguing trio.

On December 30th 2019, some patients with pneumonia of unknown etiology were reported in the Program for Monitoring Emerging Diseases (ProMED), a program run by the International Society for Infectious Diseases (ISID), hypothesized to be related to subjects who had had contact with the seafood market in Wuhan, China. Chinese authorities instituted an emergency agency aimed at identifying the source of infection and potential biological pathogens. It was subsequently named by the World Committee on Virus Classification as 2019-nCoV (2019-novel coronavirus) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A number of studies have demonstrated that 2019-nCoV and the SARS-CoV shared the same cell entry receptor named angiotensin-converting enzyme 2 (ACE2). This is expressed in human tissues, not only in the respiratory epithelia, but also in the small intestines, heart, liver, and kidneys. Here, we examine the most recent findings on the effects of SARS-CoV-2 infection on kidney diseases, mainly acute kidney injury, and the potential role of the chemokine network.

Control of Cytoskeletal Dynamics by ß-Arrestin1/Myosin Vb Signaling Regulates Endosomal Sorting and Scavenging Activity of the Atypical Chemokine Receptor ACKR2.

The atypical chemokine receptor ACKR2, formerly named D6, is a scavenger chemokine receptor with a non-redundant role in the control of inflammation and immunity. The scavenging activity of ACKR2 depends on its trafficking properties, which require actin cytoskeleton rearrangements downstream of a ß-arrestin1-Rac1-PAK1-LIMK1-cofilin-dependent signaling pathway. We here demonstrate that in basal conditions, ACKR2 trafficking properties require intact actin and microtubules networks. The dynamic turnover of actin filaments is required to sustain ACKR2 constitutive endocytosis, while both actin and microtubule networks are involved in processes regulating ACKR2 constitutive sorting to rapid, Rab4-dependent and slow, Rab11-dependent recycling pathways, respectively. After chemokine engagement, ACKR2 requires myosin Vb activity to promote its trafficking from Rab11-positive recycling endosomes to the plasma membrane, which sustains its scavenging activity. Other than cofilin phosphorylation, induction of the ß-arrestin1-dependent signaling pathway by ACKR2 agonists also leads to the rearrangement of microtubules, which is required to support the myosin Vb-dependent ACKR2 upregulation and its scavenging properties. Disruption of the actin-based cytoskeleton by the apoptosis-inducing agent staurosporine results in impaired ACKR2 internalization and chemokine degradation that is consistent with the emerging scavenging-independent activity of the receptor in apoptotic neutrophils instrumental for promoting efficient efferocytosis during the resolution of inflammation. In conclusion, we provide evidence that ACKR2 activates a ß-arrestin1-dependent signaling pathway, triggering both the actin and the microtubule cytoskeletal networks, which control its trafficking and scavenger properties.

Aberrant CXCR4 Signaling at Crossroad of WHIM Syndrome and Waldenstrom's Macroglobulinemia.

Given its pleiotropic functions, including its prominent role in inflammation, immune responses and cancer, the C-X-C chemokine receptor type 4 (CXCR4) has gained significant attention in recent years and has become a relevant target in drug development. Although the signaling properties of CXCR4 have been extensively studied, several aspects deserve deeper investigations. Mutations in the C-term tail of the CXCR4 gene cause WHIM syndrome, a rare congenital immunodeficiency associated by chronic leukopenia. Similar mutations have also been recently identified in 30% of patients affected by Waldenstrom's macroglobulinaemia, a B-cell neoplasia with bone marrow accumulation of malignant cells. An ample body of work has been generated to define the impact of WHIM mutations on CXCR4 signaling properties and evaluate their role on pathogenesis, diagnosis, and response to therapy, although the identity of disease-causing signaling pathways and their relevance for disease development in different genetic variants are still open questions. This review discusses the current knowledge on biochemical properties of CXCR4 mutations to identify their prototypic signaling profile potentially useful to highlighting novel opportunities for therapeutic intervention.

Enhanced myostatin expression and signalling promote tubulointerstitial inflammation in diabetic nephropathy.

Myostatin (MSTN), a family member of the transforming growth factor (TGF)-ß super family, has been detected in the tubuli of pig kidney, but its role in the human kidney is not known. In this study we observed upregulation of MSTN mRNA (~8 to 10-fold increase) both in the glomeruli and tubulointerstitium in diabetic nephropathy (DN). In DN, immunoreactive MSTN was mainly localized in the tubuli and interstitium (∼4-8 fold increase), where it colocalized in CD45+ cells. MSTN was also upregulated in the glomeruli and the arterial vessels. Tubulointerstitial MSTN expression was directly related to interstitial fibrosis (r = 0.54, p < 0.01). In HK-2 tubular epithelial cells, both high (30 mmol) glucose and glycated albumin upregulated MSTN mRNA and its protein (p < 0.05-0.01). MSTN-treated HK-2 cells underwent decreased proliferation, together with NF-kB activation and CCL-2 and SMAD 2,3 overexpression. In addition, MSTN induced intracellular ROS release and upregulated NADPH oxidase, effects which were mediated by ERK activation. In conclusion, our data show that MSTN is expressed in the human kidney and overexpressed in DN, mainly in the tubulointerstitial compartment. Our results also show that MSTN is a strong inducer of proximal tubule activation and suggest that MSTN overexpression contributes to kidney interstitial fibrosis in DN.

New Insights on the Emerging Genomic Landscape of CXCR4 in Cancer: A Lesson from WHIM.

Deciphering the molecular alterations leading to disease initiation and progression is currently crucial to identify the most relevant targets for precision therapy in cancer patients. Cancers express a complex chemokine network influencing leucocyte infiltration and angiogenesis. Moreover, malignant cells also express a selective repertoire of chemokine receptors that sustain their growth and spread. At present, different cancer types have been shown to overexpress C-X-C chemokine receptor type 4 (CXCR4) and to respond to its ligand C-X-C motif chemokine 12 (CXCL12). The CXCL12/CXCR4 axis influences cancer biology, promoting survival, proliferation, and angiogenesis, and plays a pivotal role in directing migration of cancer cells to sites of metastases, making it a prognostic marker and a therapeutic target. More recently, mutations in the C-terminus of CXCR4 have been identified in the genomic landscape of patients affected by Waldenstrom's macroglobulinemia, a rare B cell neoplasm. These mutations closely resemble those occurring in Warts, Hypogammaglobulinemia, Immunodeficiency, and Myelokathexis (WHIM) syndrome, an immunodeficiency associated with CXCR4 aberrant expression and activity and with chemotherapy resistance in clinical trials. In this review, we summarize the current knowledge on the relevance of CXCR4 mutations in cancer biology, focusing on its importance as predictors of clinical presentation and response to therapy.

Myostatin in the Arterial Wall of Patients with End-Stage Renal Disease.

AIM: Myostatin (Mstn) has been described as a trigger for the progression of atherosclerosis. In this study, we evaluated the role of Mstn in arterial remodeling in patients with end-stage renal disease (ESRD). METHODS: Vascular specimens were collected from 16 ESRD patients (56.4±7.9 years) undergoing renal transplant (recipients) and 15 deceased kidney non-uremic donors (55.4±12.1 years). We studied gene and protein expression of Mstn, ubiquitin ligases, Atrogin-1, and muscle ring finger protein-1 (MuRF-1), inflammatory marker CCL2, cytoskeleton components, and Klotho by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Moreover, we assessed vascular calcification and collagen deposition. Finally, we studied the effects of recombinant Mstn on rat vascular smooth muscle cells (VSMCs, A7r5) and evaluated the effects of uremic serum (US) on primary human VSMCs. RESULTS: Myostatin mRNA was upregulated in the arterial vascular wall of recipients compared with donors (~15- folds, p＜0.05). This response was accompanied by the upregulation of gene expression of Atrogin-1 and MuRF-1 (＋2.5- and ＋10-fold) and CCL2 (＋3-fold). Conversely, we found downregulation of protein expression of Smoothelin, α-smooth muscle actin (α-SMA), vimentin, and Klotho (-85%, -50%, -70%, and -80%, respectively; p＜0.05) and gene expression of vimentin and Klotho. Exposition of A7r5 to Mstn induced a time-dependent SMAD 2/SMAD 3 phosphorylation and expression of collagen-1 and transforming growth factor ß (TGFß) mRNA, while US induced overexpression of Mstn and Atrogin-1 and downregulation of Smoothelin and Klotho. CONCLUSIONS: Our data suggest that uremia might induce vascular Mstn gene expression together with a complex pathway of molecular and structural changes in the vascular wall. Myostatin, in turn, can translate the metabolic alterations of uremia into profibrotic and stiffness inducing signals.

The Organ Handling of Soluble Klotho in Humans.

BACKGROUND: Chronic kidney disease (CKD) reduces both Klotho expression and its shedding into circulation, an effect that accelerates progression and cardiovascular complications. However, the mechanisms that regulate Klotho release by the human kidney are still unknown. METHODS: We measured plasma Klotho across the kidney, splanchnic organs and lung in 22 patients (71 ± 2 years, estimated glomerular filtration rate [eGFR] 60 ± 5.4 mL/min 1.73 m2) during elective diagnostic cardiac catheterizations. RESULTS: Although the Klotho average renal vein concentrations were remarkably higher (by ∼9%) than arterial values, the kidney removed Klotho (or was at zero balance) in 7 subjects, indicating that the kidney contribution to systemic Klotho is not constant. Klotho fractional enrichment across the kidney was inversely related to plasma sodium (r = 0.43, p = 0.045) and acid uric acid levels (r = 0.38, p = 0.084) and directly, to renal oxygen extraction (r = 0.56, p = 0.006). In multivariate analysis, renal oxygen extraction was the only predictor of the enrichment of Klotho across the kidney, suggesting the dependence of renal Klotho release on tubular hypoxia or oxidative metabolism. Klotho balance was neutral across the lung. In patients with eGFR <60 mL/min, Klotho was also removed by splanchnic organs (single pass fractional extraction ∼11%). CONCLUSIONS: The present study identifies kidney oxygen uptake as a predictor of Klotho release, and splanchnic organs as a site for Klotho removal. This study provides new understanding of kidney Klotho release and suggests that modulating kidney oxygen metabolism could increase Klotho delivery, as an option to slow disease progression and blunt organ damage.

Indoxyl Sulfate Induces Renal Fibroblast Activation through a Targetable Heat Shock Protein 90-Dependent Pathway.

Indoxyl sulfate (IS) accumulation occurs early during chronic kidney disease (CKD) progression and contributes to renal dysfunction by inducing fibrosis, inflammation, oxidative stress, and tissue remodeling. Renal toxicity of high IS concentrations (250 µM) has been widely explored, particularly in resident tubular and glomerular cells, while the effect of a moderate IS increase on kidneys is still mostly unknown. To define the effects of IS accumulation on renal fibroblasts, we first analyzed kidneys of C57BL/6 mice receiving IS (0.1%) in drinking water for 12 weeks. As a next step, we treated renal fibroblasts (NRK-49F) with IS (20 µM) with or without the HSP90 inhibitor 17-AAG (1 µM). In mouse kidneys, IS increased the collagen deposition and HSP90 and α-SMA expression (immunohistochemistry) in interstitial fibroblasts and caused tubular necrosis (histological H&E and picrosirius red staining). In NRK-49F cells, IS induced MCP1, TGF-ß, collagen I, α-SMA, and HSP90 gene/protein expression and Smad2/3 pathway activation. IS had no effects on fibroblast proliferation and ROS production. 17-AAG counteracted IS-induced MCP1, TGF-ß, collagen I, and α-SMA expression and Smad2/3 phosphorylation. Our study demonstrates that the IS increase promotes renal fibroblast activation by a HSP90-dependent pathway and indicates HSP90 inhibition as a potential strategy to restrain IS-induced kidney inflammation and fibrosis in CKD.

Immunohistochemical Staining of TLR4 in Human Skeletal Muscle Samples.

Growing evidence suggests the involvement of TLR4, a receptor in the innate immune system, in muscle loss in uremia. Recently, we have evaluated TLR4 in human skeletal muscle from chronic kidney disease patients, by immunohistochemistry and image analysis. Unlike the commonly-used Western blot method, immunohistochemistry allows for the observation of protein distribution in the intact tissue while, image analysis, its quantification. In fact, our data highlighted our hypothesis that an enhanced TLR4 skeletal muscle cell expression contributes to the activation of the downward inflammatory pathway in uremic sarcopenia. In this protocol, we describe the procedure for immunostaining TLR4 in human skeletal muscle and for quantifying it by image analysis.

Uric acid and angiotensin II additively promote inflammation and oxidative stress in human proximal tubule cells by activation of toll-like receptor 4.

Renal proximal tubular cells (PTECs) participate in several mechanisms of innate immunity, express toll-like receptors (TLRs), and proinflammatory cytokines. Hyperuricemia may be a promoter of inflammation and renal damage. Angiotensin II (Ang II) modulate immune and inflammatory responses in renal tubular cells. With the aim to evaluate the effect of uric acid (UA) and Ang II on oxidative stress and inflammation mediated by toll-like receptor 4 (TLR4) activation in human PTECs, human kidney 2 (HK2) were incubated for 24 hr with UA (12 mg/dl) and Ang II (10 -7 M). HK2 were pretreated with an antagonist of TLR4 (TAK 242), valsartan or losartan. The genic expression of TLR4, monocyte chemoattractant protein-1 (MCP1), and Nox4 was quantified with reverse transcription polymerase chain reaction, proteins were evaluated with Western blot. The incubation of HK2 either with UA or with Ang II determines an increased expression of TLR4, production of proinflammatory cytokines as MCP1 and pro-oxidants as Nox4 ( p < 0.05). TAK 242 attenuates the expression of MCP1 induced both by UA and Ang II. Valsartan attenuated all the effects we described after exposure to Ang II but not those observed after UA exposure. At variance, pretreatment with losartan, which inhibits UA internalization, attenuates the expression of TLR4, MCP1, and Nox4 in cells previously treated with UA, Ang II, and UA plus Ang II. Proinflammatory pathways are induced in an additive manner by UA and Ang II ( p < 0.05) and might be mediated by TLR4 in PTECs. Renin-angiotensin-aldosterone system (RAAS) activation, hyperuricemia, and innate immunity interplay in the development of chronic tubular damage and the interaction of several nephrotoxic mechanisms blunt the protective effect of RAAS inhibition.

Soluble Toll-like Receptor 4: A New Player in Subclinical Inflammation and Malnutrition in Hemodialysis Patients.

OBJECTIVE: Toll-like receptor 4 (TLR4) promotes inflammation in hemodialysis patients (HD). A soluble form of extracellular TLR4 (sTLR4) has been recently characterized, which showed the ability to attenuate TLR4 signalling. In this study, we describe the sTLR4 profile in regular HD patients. SUBJECTS: In a cross-sectional study we enrolled forty prevalent HD patients (68.2 ± 16.3 years, twenty-five males) with a median dialysis vintage of 41 months. Nineteen patients were undergoing standard bicarbonate HD (BHD) and 21 patients on-line hemodiafiltration (HDF). Ten healthy sex-matched subjects constituted the controls (C). INTERVENTION: Before and after the HD session, serum was tested for sTLR4 levels by ELISA. Moreover, clinical and biochemical data were collected, including body mass index, albumin, and C-reactive protein (CRP) levels. Body composition was expressed as a 3-compartment model, providing lean tissue index and fat tissue index (FTI). MAIN OUTCOME MEASURE: Describe the profile of sTLR4 in HD patients, evaluating the correlations among sTLR4 levels and the main clinical characteristics, inflammatory and nutritional parameters. RESULTS: Patients with subclinical inflammation (i.e., high CRP levels without clinical symptomatology) presented higher sTLR4 levels (0.42 ± 0.25 ng/mL) with respect to both C and not inflamed HD patients (0.23 ± 0.19 ng/mL, P < .05). There was a significant direct correlation between predialysis sTLR4 and body mass index, FTI (r = 0.55), and CRP levels (r = 0.52) and inverse correlation with lean tissue index and albumin (r = -0.4). In multivariate analysis, sTLR4 resulted directly associated with FTI (P = .038). Notably, sTLR4 levels resulted higher in bicarbonate hemodialysis versus hemodiafiltration (0.37 ± 0.18 vs. 0.19 ± 0.21 ng/mL, P < .05). CONCLUSIONS: sTLR4 correlates with inflammatory and nutritional parameters, presenting as a new potential player in modulating subclinical inflammation in HD patients.

Perfusion of isolated rat kidney with Mesenchymal Stromal Cells/Extracellular Vesicles prevents ischaemic injury.

Kidney donation after circulatory death (DCD) is a less than ideal option to meet organ shortages. Hypothermic machine perfusion (HMP) with Belzer solution (BS) improves the viability of DCD kidneys, although the graft clinical course remains critical. Mesenchymal stromal cells (MSC) promote tissue repair by releasing extracellular vesicles (EV). We evaluated whether delivering MSC-/MSC-derived EV during HMP protects rat DCD kidneys from ischaemic injury and investigated the underlying pathogenic mechanisms. Warm ischaemic isolated kidneys were cold-perfused (4 hrs) with BS, BS supplemented with MSC or EV. Renal damage was evaluated by histology and renal gene expression by microarray analysis, RT-PCR. Malondialdehyde, lactate, LDH, glucose and pyruvate were measured in the effluent fluid. MSC-/EV-treated kidneys showed significantly less global ischaemic damage. In the MSC/EV groups, there was up-regulation of three genes encoding enzymes known to improve cell energy metabolism and three genes encoding proteins involved in ion membrane transport. In the effluent fluid, lactate, LDH, MDA and glucose were significantly lower and pyruvate higher in MSC/EV kidneys as compared with BS, suggesting the larger use of energy substrates by MSC/EV kidneys. The addition of MSC/EV to BS during HMP protects the kidney from ischaemic injury by preserving the enzymatic machinery essential for cell viability and protects the kidney from reperfusion damage.

Modulation of Myostatin/Hepatocyte Growth Factor Balance by Different Hemodialysis Modalities.

Background. In this study we investigated the relevance of myostatin and Hepatocyte Growth Factor (HGF) in patients undergoing hemodialysis HD and the influence of different HD modalities on their levels. Methods. We performed a prospective crossover study in which HD patients were randomized to undergo 3-month treatment periods with bicarbonate hemodialysis (BHD) followed by online hemodiafiltration (HDF). Clinical data, laboratory parameters, and myostatin and HGF serum levels were collected and compared. Results. Ten patients and six controls (C) were evaluated. In any experimental condition myostatin and HGF levels were higher in HD than in C. At enrollment and after BHD there were not significant correlations, whereas at the end of the HDF treatment period myostatin and HGF were inversely correlated (r -0.65, p < 0.05), myostatin serum levels inversely correlated with transferrin (r -0.73, p < 0.05), and HGF levels that resulted positively correlated with BMI (r 0.67, p < 0.05). Moving from BHD to HDF, clinical and laboratory parameters were unchanged, as well as serum HGF, whereas myostatin levels significantly decreased (6.3 ± 4.1 versus 4.3 ± 3.1 ng/ml, p < 0.05). Conclusions. Modulation of myostatin levels and myostatin/HGF balance by the use of different HD modalities might represent a novel approach to the prevention and treatment of HD-related muscle wasting syndrome.

Interorgan handling of fibroblast growth factor-23 in humans.

Fibroblast growth factor-23 (FGF-23) accumulates in blood of patients with chronic kidney disease (CKD) and is associated both with cardiovascular complications and disease progression. However, our knowledge of the sites and mechanisms that regulate plasma FGF-23 is still incomplete. We measured plasma intact FGF-23 across the kidney, splanchnic organs, and lung in 11 patients [estimated glomerular filtration rate (eGFR) 60 ± 6 ml/min] during elective diagnostic cardiac catheterizations. In these patients FGF-23 was removed by the kidney, with a fractional extraction (FE) of ∼22%. The FE of FGF-23 across the kidney was similar to that of creatinine (∼17%, P = NS). In addition, the FGF-23 FE by the kidney was significantly directly related to eGFR (r = 0.709 P = 0.018) and to kidney creatinine FE (r = 0.736 P = 0.013) but only as a trend to plasma phosphate levels (r = 0.55, P = 0.18). There was no difference in FGF-23 levels in blood perfusing splanchnic organs and cardiopulmonary bed. However, the arterial-venous difference of FGF-23 across the lung was directly related to FGF-23 pulmonary artery levels, suggesting that the lung, and possibly the heart, participate in the homeostasis of plasma FGF-23 when its systemic levels are increased. Our data show that the human kidney is the only site for FGF-23 removal from blood and suggest that FGF-23 is predominantly removed by glomerular filtration. The kidney ability to remove FGF-23 from the circulation likely accounts for the early increase in blood of FGF-23 in patients with CKD.

Mesenchymal Stromal Cells Prevent Renal Fibrosis in a Rat Model of Unilateral Ureteral Obstruction by Suppressing the Renin-Angiotensin System via HuR.

We studied Mesenchymal Stromal Cells (MSC) effects in experimental Unilateral Ureteral Obstruction (UUO), a fibrogenic renal disease. Rats were divided in 5 groups: sham, UUO, MSC treated-UUO, ACEi treated-UUO, MSC+ACEi treated- UUO. Data were collected at 1, 7, 21 days. UUO induced monocyte renal infiltration, tubular cell apoptosis, tubular atrophy, interstitial fibrosis and overexpression of TGFß, Renin mRNA (RENmRNA), increase of Renin, Angiotensin II (AII) and aldosterone serum levels. Both lisinopril (ACEi) and MSC treatment prevented monocyte infiltration, reduced tubular cell apoptosis, renal fibrosis and TGFß expression. Combined therapy provided a further suppression of monocyte infiltration and tubular injury. Lisinopril alone caused a rebound activation of Renin-Angiotensin System (RAS), while MSC suppressed RENmRNA and Renin synthesis and induced a decrease of AII and aldosterone serum levels. Furthermore, in in-vitro and in-vivo experiments, MSC inhibit Human antigen R (HuR) trascription, an enhancer of RENmRNA stability by IL10 release. In conclusion, we demonstrate that in UUO MSC prevent fibrosis, by decreasing HuR-dependent RENmRNA stability. Our findings give a clue to understand the molecular mechanism through which MSC may prevent fibrosis in a wide and heterogeneous number of diseases that share RAS activation as common upstream pathogenic mechanism.