Uremic Serum Research Articles

Uremic Serum Results in an Activation and Phenotypic Switch of Pig Venous and Arterial Smooth Muscle Cells (SMCs)

Uremic Serum Results in an Activation and Phenotypic Switch of Pig Venous and Arterial Smooth Muscle Cells (SMCs)

Comparison of Autologous and Allogeneic Adipose-Derived Stem Cells in Kidney Transplantation: Immunological Considerations and Therapeutic Efficacy.

Regenerative medicine shows significant potential in treating kidney diseases through the application of various types of stem and progenitor cells, including mesenchymal stem cells (MSCs), renal stem/progenitor cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Stem cells possess the unique ability to repair injured organs and improve impaired functions, making them a key element in the research of therapies for kidney tissue repair and organ regeneration. In kidney transplantation, reperfusion injury can cause tissue destruction, leading to an initially low glomerular filtration rate and long-term impact on function by creating irreversible interstitial fibrosis. MSCs have proven useful in repairing early tissue injury in animal models of kidney, lung, heart, and intestine transplantation. The use of stem cell therapies in solid organ transplantation raises the question of whether autologous or allogeneic cells should be preferred. Adipose-derived stem cells (ASCs), characterized by the lack of HLA Class II molecules and low expression of HLA Class I and co-stimulatory signals, are considered immune-privileged. However, the actual risk of graft rejection associated with allogeneic ASCs remains unclear. It has been demonstrated that donor-derived ASCs can promote the development of Treg cells in vitro, and some degree of tolerance induction has been observed in vivo. Nevertheless, a study comparing the efficacy of autologous and allogeneic ASCs in a rat model with a total MHC mismatch for kidney transplantation showed that donor-derived administration of ASCs did not improve the grafts' survival and was associated with increased mortality through an immunologically mediated mechanism. Given the lack of data, autologous ASCs appear to be a safer option in this research context. The aim of this review was to examine the differences between autologous and allogeneic ASCs in the context of their application in kidney transplantation therapies, considering potential immune reactions and therapeutic efficacy. Some have argued that ASCs harvested from end-stage renal disease (ESRD) patients may have lower regenerative potential due to the toxic effects of uremia, potentially limiting their use in transplantation settings. However, evidence suggests that the beneficial properties of ASCs are not affected by uremia or dialysis. Indeed, some investigators have demonstrated that ASCs harvested from chronic kidney disease (CKD) patients exhibit normal characteristics and function, maintaining consistent proliferative capacity and genetic stability over time, even after prolonged exposure to uremic serum Furthermore, no differences were observed in the response of ASCs to immune activation or their inhibitory effect on the proliferation of alloantigen-activated peripheral blood mononuclear cells between patients with normal or impaired renal function. This review presents the current achievements in stem cell research aimed at treating kidney diseases, highlighting significant progress and ongoing efforts in the development of stem cell-based therapies. Despite the encouraging results, further research is needed to overcome the current limitations and fully realize the potential of these innovative treatments. Advances in this field are crucial for developing effective therapies that can address the complex challenges associated with kidney damage and failure.

Vascular endothelial dysfunction improvements in patients with uraemia using pentoxifylline-suppressing NLRP3 expressions and HMGB1 release.

This study aimed to investigate the protective effect of pentoxifylline (PTX) on vascular endothelial dysfunction in uraemia. The human aortic endothelial cells (HAECs) required for the experiments were all obtained from the National Collection of Authenticated Cell Cultures (Salisbury, UK). The permeability of HAECs was assessed. Each group had six samples. Compared with the healthy volunteer group, HAEC proliferation in the 20% uraemia group was significantly inhibited after 72 h (p < 0.001), co-localisation of nucleotide-binding domain, leucine-rich repeat-containing receptor family pyrin domain-containing 3 (NLRP3) and apoptosis-associated speck-like (ASC) protein induced by uremic serum was enhanced (p < 0.01) and high mobility group box 1 (HMGB1) release was increased (0.594 ± 0.057, p = 0.03). The co-immunoprecipitation of NLRP3, ASC and HMGB1 induced by uremic toxin was also enhanced (p < 0.01), and PTX inhibited this phenomenon. The expression of NLRP3 (0.810 ± 0.032, p = 0.02) and caspase-1 (0.580 ± 0.041, p = 0.03) was increased, whereas the expression of ZO-1 (0.255 ± 0.038, p = 0.03) and VE-cadherin (0.0546 ± 0.053, p = 0.02) was decreased in the uraemia group; compared with the healthy volunteer group, treated with PTX (NLRP3, 0.298 ± 0.042, p = 0.03; caspase-1, 0.310 ± 0.021, p = 0.03; ZO-1, 0.412 ± 0.028, p = 0.02; VE-cadherin, 0.150 ± 0.034, p = 0.02) and MCC950 (NLRP3, 0.432 ± 0.022, p = 0.03; caspase-1, 0.067 ± 0.031, p > 0.05; ZO-1, 0.457 ± 0.026, p = 0.03; VE-cadherin, 0.286 ± 0.017, p = 0.03) lessened this trend. Pentoxifylline promoted the HAEC permeability mediated by uremic toxins (1.507 ± 0.012, p = 0.02). In conclusion, PTX enhances the release of HMGB1, which is dependent on NLRP3 activation, and consequently exerts positive effects on interconnecting proteins, ultimately leading to an improvement in vascular permeability.

Retraction: Functional mechanism of AMPK activation in mitochondrial regeneration of rat peritoneal macrophages mediated by uremic serum.

Retraction: Functional mechanism of AMPK activation in mitochondrial regeneration of rat peritoneal macrophages mediated by uremic serum.

Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease (ESRD).

Trained immunity is the long-term functional reprogramming of innate immune cells, which results in altered responses toward a secondary challenge. Despite indoxyl sulfate (IS) being a potent stimulus associated with chronic kidney disease (CKD)-related inflammation, its impact on trained immunity has not been explored. Here, we demonstrate that IS induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. Mechanistically, the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as arachidonate 5-lipoxygenase (ALOX5) and ALOX5 activating protein (ALOX5AP). Inhibition of AhR during IS training suppresses the induction of IS-trained immunity. Monocytes from end-stage renal disease (ESRD) patients have increased ALOX5 expression and after 6 days training, they exhibit enhanced TNF-α and IL-6 production to lipopolysaccharide (LPS). Furthermore, healthy control-derived monocytes trained with uremic sera from ESRD patients exhibit increased production of TNF-α and IL-6. Consistently, IS-trained mice and their splenic myeloid cells had increased production of TNF-α after in vivo and ex vivo LPS stimulation compared to that of control mice. These results provide insight into the role of IS in the induction of trained immunity, which is critical during inflammatory immune responses in CKD patients.

Uremic toxin indoxyl sulfate induces trained immunity via the AhR-dependent arachidonic acid pathway in end-stage renal disease (ESRD)

Trained immunity is the long-term functional reprogramming of innate immune cells, which results in altered responses toward a secondary challenge. Despite indoxyl sulfate (IS) being a potent stimulus associated with chronic kidney disease (CKD)-related inflammation, its impact on trained immunity has not been explored. Here, we demonstrate that IS induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. Mechanistically, the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as arachidonate 5-lipoxygenase (ALOX5) and ALOX5 activating protein (ALOX5AP). Inhibition of AhR during IS training suppresses the induction of IS-trained immunity. Monocytes from end-stage renal disease (ESRD) patients have increased ALOX5 expression and after 6 days training, they exhibit enhanced TNF-α and IL-6 production to lipopolysaccharide (LPS). Furthermore, healthy control-derived monocytes trained with uremic sera from ESRD patients exhibit increased production of TNF-α and IL-6. Consistently, IS-trained mice and their splenic myeloid cells had increased production of TNF-α after in vivo and ex vivo LPS stimulation compared to that of control mice. These results provide insight into the role of IS in the induction of trained immunity, which is critical during inflammatory immune responses in CKD patients.

#1293 Endothelial calcification induced by high-phosphate is characterized by mesenchymal and osteoblastic transdifferentiation

Abstract Background and Aims Chronic kidney disease (CKD) is a prevalent condition that affects millions of people. Patients with CKD have an increased cardiovascular risk, mostly due to vascular calcification (VC). By now, it's quite clear how, in vitro and in vivo, vascular smooth muscle cells (VSMCs) actively participate to VC. In contrast to the wide studies on VSMCs, the role of endothelial cells (ECs) has been poorly investigated. It's known that ECs have a dynamic nature and following particular stimuli they can trans-differentiate and acquire several different phenotypes through the endothelial-to-mesenchymal transition (EndMT). Methods Human aortic ECs (HAECs) were challenged, in an in vitro calcification model, with 2, 2.5, 3, 3.5 mM Pi for up to 7 days. Early morphological changes, gene expression and cell migration were evaluated. Afterwards calcium deposition, genes and protein expression and ultrastructural changes were analyzed. Results High-Pi, at a concentration of 3 mM, is able to induce EC calcium deposition. Calcification is dependent on calcium influx without influencing Pit-1 expression. During the first days of high-Pi treatment ECs change their morphology from cobblestone to spindle shape acquiring motility, as demonstrated by the 24% increased closure in the scratch wound test. EndMT master genes such as SNAIL and TWIST resulted up-regulated at day 3 together with an increased protein expression of mesenchymal markers such as N-Cadherin, α-SMA, SM22α, FSP-1 and Fibronectin. Starting from day 5 high-Pi induces osteoblastic differentiation with RUNX2 and BMP2 mRNA up-regulation up to day 7, osteopontin increased protein expression and positive Alcian Blu staining. In calcified ECs mesenchymal and endothelial markers, such as α-SMA, SM22α, FSP-1 and Fibronectin or vWF progressively decrease. Finally, uremic serum is able to exacerbate in vitro calcification, compared to healthy serum characterized by an increase in both mesenchymal and osteoblastic mastergene such as TWIST and RUNX2. Conclusions Our data demonstrate that in vitro high-Pi induces the EndMT process and similar osteoblastic trans-differentiation suggesting a potential role of endothelium in VC. The relevancy of endothelial calcification is suggested by the calcification exacerbation induced by uremic compared to healthy sera.

Activation of BKα channel provented uremic cardiomyopathy by attenuating oxidative stress

Background: Uremic cardiomyopathy and renal fibrosis contribute to chronic kidney disease (CKD)-induced morbidity and mortality. Our previous study found that activation of the large conductance calcium-activated potassium channels (BK channels) attenuated renal fibrosis in mice (Wang et al, Kid Int, 2021). We hypothesized that upregulation of BK channels would suppress cardiac fibrosis in CKD. Methods: CKD mice were induced by 5/6 nephrectomy. To upregulation of BKa channels, BMS-191011 (10 mg/kg BW) was given by IP injection every day for 6-8 weeks. Single channel recordings were used to analyze the activation of BKa channel. Blood pressure was measured by non-invasive photoplethysmography using the tail-cuff method. Echocardiogram was used to assess cardiac function. Cultured H9C2 cardiac myoblasts were used to detect the impact of BK opener (NS1916 20mM) on fibrosis markers and oxidative stress. The mRNA expression of BKa in heart was assayed by qPCR. The Hydrogen peroxide (H2O2) was tested by Amplex Red Hydrogen Peroxide Kit. DHE (Dihydroethidium) was assayed to detect superoxide. Superoxide Dismutase (SOD), a defense of oxidative enzyme, was measured using colorimetric activity kit. Results: The expression of BKa mRNA and protein were decreased and cardiac fibrosis was increased in the heart of CKD mice. Single channel recordings showed that human uremic serum (User) attenuated the activation of BKa channel. Upregulation of BKa channel by BSM-191011 decreased the CKD-induced increase in systolic and diastolic blood pressure. Echocardiogram showed that LV end-diastolic dimension increased in 5/6Nx mice, but back to normal after BK opener treatment. Ejection fraction also improved by this treatment. Fibronectin and vimentin were also significantly increased in CKD heart, whereas BMS treatment attenuated the amount of both proteins. In cultured cardiac myoblasts, User and TGFβ increased fibronectin and collagen I, which were in a dose-dependent manner. BKa openers (NS1916) limited the User and TGF-induced upregulation of both proteins in H9C2 cells. User also significantly increased H2O2 and superoxide production in cultured H9C2 cells. Activation of BKa significantly abolished this upregulation. In addition, User and TGFβ decrease SOD production, NS1916 blocked this decline in a dose dependent manner. Conclusions: Activation of BK channel in CKD animals attenuates cardiac fibrosis likely through reduction of uremia-induced oxidative stress. This study could provide new approaches for developing therapeutic strategies for treating uremic cardiomyopathy in chronic kidney diseases. the Department of Veteran Affairs MERIT Award 5I01BX000994 to HC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Action of 3-Hydroxymethylglutaryl CoA Reductase Inhibitors on ABCA-1 Protein (ATP-Binding Cassette Transporter-1) in Endothelial Cells Stimulated with Uremic Serum

Action of 3-Hydroxymethylglutaryl CoA Reductase Inhibitors on ABCA-1 Protein (ATP-Binding Cassette Transporter-1) in Endothelial Cells Stimulated with Uremic Serum

Expanded Hemodialysis ameliorates uremia-induced impairment of vasculoprotective KLF2 and concomitant proinflammatory priming of endothelial cells through an ERK/AP1/cFOS-dependent mechanism.

Expanded hemodialysis (HDx) therapy with improved molecular cut-off dialyzers exerts beneficial effects on lowering uremia-associated chronic systemic microinflammation, a driver of endothelial dysfunction and cardiovascular disease (CVD) in hemodialysis (HD) patients with end-stage renal disease (ESRD). However, studies on the underlying molecular mechanisms are still at an early stage. Here, we identify the (endothelial) transcription factor Krüppel-like factor 2 (KLF2) and its associated molecular signalling pathways as key targets and regulators of uremia-induced endothelial micro-inflammation in the HD/ESRD setting, which is crucial for vascular homeostasis and controlling detrimental vascular inflammation. First, we found that human microvascular endothelial cells (HMECs) and other typical endothelial and kidney model cell lines (e.g. HUVECs, HREC, and HEK) exposed to uremic serum from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation II (PERCI-II) crossover clinical trial - comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes - exhibited strongly reduced expression of vasculoprotective KLF2 with HF dialyzers, while dialysis with MCO dialyzers led to the maintenance and restoration of physiological KLF2 levels in HMECs. Mechanistic follow-up revealed that the strong downmodulation of KLF2 in HMECs exposed to uremic serum was mediated by a dominant engagement of detrimental ERK instead of beneficial AKT signalling, with subsequent AP1-/c-FOS binding in the KLF2 promoter region, followed by the detrimental triggering of pleiotropic inflammatory mediators, while the introduction of a KLF2 overexpression plasmid could restore physiological KLF2 levels and downmodulate the detrimental vascular inflammation in a mechanistic rescue approach. Uremia downmodulates vasculoprotective KLF2 in endothelium, leading to detrimental vascular inflammation, while MCO dialysis with the novel improved HDx therapy approach can maintain physiological levels of vasculoprotective KLF2.

B- and T-lymphocyte attenuator could be a new player in accelerated atherosclerosis associated with chronic kidney disease.

In chronic kidney disease (CKD), cardiovascular morbi-mortality is higher than in general population. Atherosclerotic cardiovascular disease is accelerated in CKD, but specific CKD-related risk factors for atherosclerosis are unknown. CKD patients from the NEFRONA study were used. We performed mRNA array from blood of patients free from atheroma plaque at baseline, with (n=10) and without (n=10) de novo atherosclerotic plaque development 2 years later. Selected mRNA candidates were validated in a bigger sample (n=148). Validated candidates were investigated in vivo in an experimental model of CKD-accelerated atherosclerosis, and in vitro in murine macrophages. mRNA array analysis showed 92 up-regulated and 67 down-regulated mRNAs in samples from CKD patients with de novo plaque development. The functional analysis pointed to a paramount role of the immune response. The validation in a bigger sample confirmed that B- and T-lymphocyte co-inhibitory molecule (BTLA) down-regulation was associated with de novo plaque presence after 2 years. However, BTLA down-regulation was not found to be associated with atherosclerotic progression in patients with plaque already present at baseline. In a model of CKD-accelerated atherosclerosis, mRNA and protein expression levels of BTLA were significantly decreased in blood samples and atheroma plaques. Plaques from animals with CKD were bigger, had more infiltration of inflammatory cells, higher expression of IL6 and IL17 and less presence of collagen than plaques from control animals. Incubation of macrophages with rat uremic serum decreased BTLA expression. BTLA could be a potential biomarker or therapeutic target for atherosclerosis incidence in CKD patients.

Implications of Senescent Cell Burden and NRF2 Pathway in Uremic Calcification: A Translational Study.

Increased senescent cell burden and dysregulation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway have been associated with numerous age-related pathologies; however, their role in promoting vascular calcification (VC) in chronic kidney disease (CKD) has yet to be determined. We investigated whether senescence and NRF2 pathways may serve as drivers of uremia-induced VC using three complementary approaches: a novel model of induced VC in 5/6-nephrectomized rats supplemented with high phosphate and vitamin D; epigastric arteries from CKD patients with established medial calcification; and vascular smooth muscle cells (VSMCs) incubated with uremic serum. Expression of p16Ink4a and p21Cip1, as well as γ-H2A-positive cells, confirmed increased senescent cell burden at the site of calcium deposits in aortic sections in rats, and was similarly observed in calcified epigastric arteries from CKD patients through increased p16Ink4a expression. However, uremic serum-induced VSMC calcification was not accompanied by senescence. Expression of NRF2 and downstream genes, Nqo1 and Sod1, was associated with calcification in uremic rats, while no difference was observed between calcified and non-calcified EAs. Conversely, in vitro uremic serum-driven VC was associated with depleted NRF2 expression. Together, our data strengthen the importance of senescence and NRF2 pathways as potential therapeutic options to combat VC in CKD.

Association of circulating MtDNA with CVD in hemodialysis patients and in vitro effect of exogenous MtDNA on cardiac microvascular inflammation

BackgroundChronic kidney disease (CKD) patients sustain a fairly high prevalence of cardiovascular disease (CVD). Microvascular inflammation is an early manifestation of CVD, and the released mitochondrial DNA (MtDNA) has been proposed to be a crucial integrator of inflammatory signals. Herein, the aim of this study was to determine the relationship between CVD, microvessel, and circulating MtDNA in the settings of uremia.MethodsForty-two maintenance hemodialysis (MHD) patients and 36 health controls were enrolled in this study. Plasma cell-free MtDNA was detected by TaqMan-based qPCR assay. CVD risk markers including high-sensitive C-reactive protein (Hs-CRP), monocyte chemoattractant protein-1 (MCP-1), fibrinogen, and erythrocyte sedimentation rate (ESR) were measured by standard assays. Ten-year CVD risk was calculated from the Framingham risk score (FRS) model. In vitro study, human cardiac microvascular endothelial cells (HCMECs) were incubated with normal or uremic serum, with or without exogenous MtDNA. Intracellular toll-like receptor 9 (TLR9), adhesion molecule 1 (ICAM-1), MCP-1 and tumor necrosis factor-α (TNF-α) and cytosolic MtDNA were detected by qPCR.ResultsPlasma MtDNA in MHD patients was significantly higher than healthy controls (4.74 vs. 2.41 × 105 copies/mL; p = 0.000). Subsequently, the MHD patients were classified into two groups based on the MtDNA median (4.34 × 105 copies/mL). In stratified analyses, the levels of Hs-CRP (5.02 vs. 3.73 mg/L; p = 0.042) and MCP-l (99.97 vs. 64.72 pg/mL; p = 0.008) and FRS (21.80 vs. 16.52; p = 0.016) in the high plasma MtDNA group were higher than those in the low plasma MtDNA group. In vitro study, we found that exogenous MtDNA aggravated uremic serum-induced microvascular inflammation (ICAM-1 and TNF-α) in HCMECs (all p < 0.05). Besides, the addition of MtDNA to the medium resulted in a further increase in cytosolic MtDNA and TLR9 levels in uremic serum-treated cells (all p < 0.05). In patients with MHD, MtDNA levels in plasma were significantly reduced after a single routine hemodialysis (pre 4.47 vs. post 3.45 × 105 copies/mL; p = 0.001) or hemodiafiltration (pre 4.85 vs. post 4.09 × 105 copies/mL; p = 0.001). These two approaches seem similar in terms of MtDNA clearance rate (21.26% vs. 11.94%; p = 0.172).ConclusionsOverall, the present study suggests that MtDNA released into the circulation under the uremic toxin environment may adversely affect the cardiovascular system by exacerbating microvascular inflammation, and that reducing circulating MtDNA might be a future therapeutic strategy for the prevention of MHD-related CVD.

A Toll‐like receptor‐4/NLRP3 inflammasome pathway promotes inflammation in skeletal muscle of chronic kidney disease patients

AbstractBackgroundAn emerging hypothesis is that the activation of innate immunity in the muscle of patients with chronic kidney disease (CKD) is implicated in the development and progression of wasting and cachexia. We previously observed that Toll‐like receptor‐4 (TLR4) and its downward NF‐κB‐dependent pro‐inflammatory pathways are activated in CKD muscle. It is however unknown if TLR4 can activate the TLR4/NOD, LRR, and pyrin domain‐containing protein 3 (NLRP3) inflammasome pathway, which is implicated in cardiovascular disease and frailty, clinical settings that are commonly observed in CKD patients.MethodsIn a case–control cohort study, we hypothesized that a TLR4/NLRP3 inflammasome pathway is activated in skeletal muscle in uraemia. First, we studied the regulation TLR4/NLRP3/caspase‐1 in skeletal muscle biopsies (20M/11F) obtained from 31 non‐diabetic CKD5 patients (eGFR 8 ± 1 mL/min 1.73 m2) scheduled for peritoneal dialysis catheter insertion and 15 controls (10M/5F, eGFR 99 ± 6 mL/min 1.73 m2). Second, the effects of uraemic serum on the TLR4/NLRP3 inflammasome pathway were studied in C2C12 cells.ResultsIn the muscle of CKD subjects, NLRP3 mRNA as well as its protein were overexpressed (by ~16‐fold, respectively, P &lt; 0.05 both vs. controls). Both IL‐1β and IL‐18 mRNA expressions were also up‐regulated (~11.8–3.2‐fold, respectively, P &lt; 0.05). Also, cleaved caspase‐1 was overexpressed in CKD muscle samples (P &lt; 0.001 vs. controls). Both muscle NLRP3 mRNA (n = 22, r = −0.606, P &lt; 0.01) and logIL‐1 β protein (n = 26, r = −0.460, P &lt; 0.02) were inversely associated with residual renal function, which suggests that the inflammasome is progressively activated in skeletal muscle of CKD patients as the residual renal function deteriorates. In addition, we observed that in C2C12 myotubes, uraemic serum up‐regulates NLRP3 mRNA (~11‐fold increase, P &lt; 0.05), cleaved caspase‐1 (by ~5‐fold, P &lt; 0.05), Il‐1β mRNA (~3‐fold increase, P &lt; 0.05) and oxidative stress markers respect to normal serum. These effects were prevented by TAK‐242, a selective TLR4 inhibitor.ConclusionsOverall, our data demonstrate the activation of TLR4/NLRP3/caspase‐1 inflammasome and its downward inflammatory cascade in the muscle of subjects with advanced‐stage CKD and suggest targeting TLR4/NLRP3 inflammasome as a new therapeutic strategy to blunt muscle inflammation in CKD.

Hydrogen sulfide alleviates uremic cardiomyopathy by regulating PI3K/PKB/mTOR-mediated overactive autophagy in 5/6 nephrectomy mice.

The gasotransmitter hydrogen sulfide (H2S) plays important physiological and pathological roles in the cardiovascular system. However, the involvement of H2S in recovery from uremic cardiomyopathy (UCM) remains unclear. This study aimed to determine the therapeutic efficacy and elucidate the underlying mechanisms of H2S in UCM. A UCM model was established by 5/6 nephrectomy in 10-week-old C57BL/6 mice. Mice were treated with sodium hydrosulfide (NaHS, H2S donor), L-cysteine [L-Cys, cystathionine gamma-lyase (CSE) substrate], and propargylglycine (PPG, CSE inhibitor). Treatment of H9C2 cardiomyocytes utilized different concentrations of uremic serum, NaHS, PPG, and PI3K inhibitors (LY294002). Mouse heart function was assessed by echocardiography. Pathological changes in mouse myocardial tissue were identified using hematoxylin and eosin and Masson's trichrome staining. Cell viability was assessed using the Cell Counting Kit-8. The protein expressions of CSE, p-PI3K, PI3K, p-PKB, PKB, p-mTOR, mTOR, and autophagy-related markers (Beclin-1, P62, and LC3) were detected using Western blotting. We found that NaHS and L-Cys treatment attenuated myocardial disarray, fibrosis, and left ventricular dysfunction in UCM mice. These abnormalities were further aggravated by PPG supplementation. Enhanced autophagy and decreased phosphorylation of PI3K, PKB, and mTOR protein expression by UCM were altered by NaHS and L-Cys treatment. In vitro, uremic serum increased overactive autophagy and decreased the phosphorylation levels of PI3K, PKB, and mTOR in cardiomyocytes, which was substantially exacerbated by endogenous H2S deficiency and attenuated by pre-treatment with 100µm NaHS. However, the protective effects of NaHS were completely inhibited by LY294002. These findings support a protective effect of H2S exerted against UCM by reducing overactive autophagy through activation of the PI3K/PKB/mTOR pathway.

Association Between Uremic Pruritus and Serum Level of Fibroblast Growth Factor 23 in Hemodialysis Patients in North of Iran: An Observational Study

Background: Chronic itch (CI) in hemodialysis (HD), also called uremic pruritus (UP), is a common, distressing, and unpleasant symptom. Fibroblast growth factor 23 (FGF23) is a bone-secreted phosphaturic factor. Several bone diseases can accompany kidney diseases. It is unclear whether the common disturbance of calcium/phosphate homeostasis can cause CI in chronic kidney disease. Objectives: This study investigated the association between FGF23 and UP among HD patients in northern Iran. Methods: Patients undergoing maintenance HD at four referral medical centers were recruited in this cross-sectional survey. The enzyme-linked immunosorbent assay was used to evaluate serum FGF23 levels. An interview questionnaire was used to analyze the various aspects of pruritus. Results: Of 237 subjects, 54.01% had UP. There was no difference in serum FGF23 levels between patients with and without UP (413.17 ± 416.97 vs. 410.81 ± 444.49, P = 0.85). Those with UP required dialysis for a longer period (P = 0.02). FGF23 was shown to be associated with parathyroid hormone in UP (P = 0.02, r = 0.41). Conclusions: There was no linear correlation between FGF23 and UP in this study.

The impact of senescence on muscle wasting in chronic kidney disease.

Muscle wasting is a common complication of chronic kidney disease (CKD) that is associated with higher mortality. Although the mechanisms of myofibre loss in CKD has been widely studied, the contribution of muscle precursor cell (MPC) senescence remains poorly understood. Senescent MPCs no longer proliferate and can produce proinflammatory factors or cytokines. In this study, we tested the hypothesis that the senescence associated secretory phenotype (SASP) of MPCs contributes to CKD-induced muscle atrophy and weakness. CKD was induced in mice by 5/6th nephrectomy. Kidney function, muscle size, and function were measured, and markers of atrophy, inflammation, and senescence were evaluated using immunohistochemistry, immunoblots, or qPCR. To study the impact of senescence, a senolytics cocktail of dasatinib+quercetin (D&Q) was given orally to mice for 8weeks. To investigate CKD-induced senescence at the cellular level, primary MPCs were incubated with serum from CKD or control subjects. The roles of specific proteins in MPC senescence were studied using adenoviral transduction, siRNA, and plasmid transfection. In the hindlimb muscles of CKD mice, (i) the senescence biomarker SA-β-gal was sharply increased (~30-fold); (ii) the DNA damage response marker γ-H2AX was increased 1.9-fold; and (iii) the senescence pathway markers p21 and p16INK4a were increased 1.99-fold and 2.82-fold, respectively (all values, P<0.05), whereas p53 was unchanged. γ-H2AX, p21, and p16INK4A were negatively correlated at P<0.05 with gastrocnemius weight, suggesting a causal relationship with muscle atrophy. Administration of the senolytics cocktail to CKD mice for 8weeks eliminated the disease-related elevation of p21, p16INK4a , and γ-H2AX, abolished positive SA-β-gal, and depressed the high levels of the SASP cytokines, TNF-α, IL-6, IL-1β, and IFN (all values, P<0.05). Skeletal muscle weight, myofibre cross-sectional area, and grip function were improved in CKD mice receiving D&Q. Markers of protein degradation, inflammation, and MPCs dysfunction were also attenuated by D&Q treatment compared with the vehicle treatment in 5/6th nephrectomy mice (all values, P<0.05). Uraemic serum induced senescence in cultured MPCs. Overexpression of FoxO1a in MPCs increased the number of p21+ senescent cells, and p21 siRNA prevented uraemic serum-induced senescence (P<0.05). Senescent MPCs are likely to contribute to the development of muscle wasting during CKD by producing inflammatory cytokines. Limiting senescence with senolytics ameliorated muscle wasting and improved muscle strength in vivo and restored cultured MPC functions. These results suggest potential new therapeutic targets to improve muscle health and function in CKD.

Uremic Myopathy and Mitochondrial Dysfunction in Kidney Disease.

Alterations in muscle structure and function in chronic kidney disease (CKD) patients are associated with poor outcomes. As key organelles in muscle cell homeostasis, mitochondrial metabolism has been studied in the context of muscle dysfunction in CKD. We conducted a study to determine the contribution of oxidative metabolism, glycolysis and fatty acid oxidation to the muscle metabolism in CKD. Mice developed CKD by exposure to adenine in the diet. Muscle of CKD mice showed significant weight loss compared to non-CKD mice, but only extensor digitorum longus (EDL) muscle showed a decreased number of fibers. There was no difference in the proportion of the various muscle fibers in CKD and non-CKD mice. Muscle of CKD mice had decreased expression of proteins associated with oxidative phosphorylation but increased expression of enzymes and transporters associated with glycolysis. In cell culture, myotubes exposed to uremic serum demonstrated decreased oxygen consumption rates (OCR) when glucose was used as substrate, conserved OCR when fatty acids were used and increased lactate production. In conclusion, mice with adenine-induced CKD developed sarcopenia and with increased glycolytic metabolism but without gross changes in fiber structure. In vitro models of uremic myopathy suggest fatty acid utilization is preserved compared to decreased glucose utilization.

Effect of hypoxia and uremia on oxidative stress on erythrocytes from hemodialysis patients.

Oxidative stress (OS) is essential in uremia-associated comorbidities, including renal anemia. Complications experienced by hemodialysis (HD) patients, such as hypoxemia and uremic toxins accumulation, induce OS and premature death of red blood cells (RBC). We aimed to characterize reactive oxygen species (ROS) production and antioxidant pathways in HD-RBC and RBC from healthy controls (CON-RBC) and evaluate the role of uremia and hypoxia in these pathways. ROS production, xanthine oxidase (XO) and superoxide dismutase (SOD) activities, glutathione (GSH), and heme oxygenase-1 (HO-1) levels were measured using flow cytometry or spectrophotometry in CON-RBC and HD-RBC (pre- and post-HD), at baseline and after 24 h incubation with uremic serum (S-HD) and/or under hypoxic conditions (5% O2 ). Ketoprofen was used to inhibit RBC uremic toxins uptake. HD-RBC showed higher ROS levels and lower XO activity than CON-RBC, particularly post-HD. GSH levels were lower, while SOD activity and HO-1 levels of HD-RBC were higher than control. Hypoxia per setriggered ROS production in CON-RBC and HD-RBC. S-HD, on top of hypoxia, increased ROS levels. Inhibition of uremic toxins uptake attenuated ROS of CON and HD-RBC under hypoxia and uremia. CON-RBC in uremia and hypoxia showed lower GSH levels than cells in normoxia and non-uremic conditions. Redox mechanisms of HD-RBC are altered and prone to oxidation. Uremic toxins and hypoxia play a role in unbalancing these systems. Hypoxia and uremia participate in the pathogenesis of OS in HD-RBC and might induce RBC death and thus compound anemia.

Effects of Chronic Kidney Disease on Nanomechanics of the Endothelial Glycocalyx Are Mediated by the Mineralocorticoid Receptor.

Endothelial mechanics control vascular reactivity and are regulated by the mineralocorticoid receptor (MR) and its downstream target, the epithelial Na+ channel (ENaC). Endothelial dysfunction is a hallmark of chronic kidney disease (CKD), but its mechanisms are poorly understood. We hypothesized that CKD disrupts endothelial mechanics in an MR/ENaC-dependent process. Methods: Primary human endothelial cells were cultured with uremic serum derived from children with stage 3–5 (predialysis) CKD or adult hemodialysis (HD) patients or healthy controls. The height and stiffness of the endothelial glycocalyx (eGC) and cortex were monitored by atomic force microscopy (AFM) using an ultrasensitive mechanical nanosensor. Results: In a stage-dependent manner, sera from children with CKD induced a significant increase in eGC and cortex stiffness and an incremental reduction of the eGC height. AFM measurements were significantly associated with individual pulse wave velocity and serum concentrations of gut-derived uremic toxins. Serum from HD patients increased MR expression and mechanical stiffness of the endothelial cortex, an effect reversed by MR and ENaC antagonists, decreased eNOS expression and NO bioavailability, and augmented monocyte adhesion. Conclusion: These data indicate progressive structural damage of the endothelial surface with diminishing kidney function and identify the MR as a mediator of CKD-induced endothelial dysfunction.