Pathophysiology of Physical Exercise in Kidney Patients: Unveiling New Players- The Role of Myokines.

Background Chronic kidney disease (CKD) is a progressive systemic condition characterized by numerous complications. Among these, alterations in skeletal muscle physiology, such as sarcopenia, are particularly significant, as they are associated with poor outcomes and reduced quality of life. Summary Various interventions, including pharmacological approaches and lifestyle modifications have been investigated to slow CKD progression and prevent or treat its complications. Physical exercise, in particular, has emerged as a promising intervention with multiple beneficial effects. These include improvements in physical functioning, increased muscle mass, modulation of metabolic abnormalities, and reduced cardiovascular risk. However, the pathophysiology of physical exercise in patients with kidney disease is complex and remains only partially understood. A crucial advancement in understanding this phenomenon has been the identification of myokines-molecules expressed and released by skeletal muscle in response to physical activity. These myokines can exert both paracrine and systemic effects, influencing not only skeletal muscle physiology but also other processes such as energy metabolism and lipid regulation. Key Messages The interplay among skeletal muscle, physical activity, and myokines may act as a pivotal regulator in various physiological processes, including aging, as well as in pathological conditions like cachexia and sarcopenia, frequently observed in CKD patients at different stages, including patients on dialysis. Despite the potential importance of this relationship, only a limited number of studies have explored the relationship between exercise and myokine, and the effect of this interaction on experimental models or individuals with kidney disease. In the following sections, we review and discuss this topic.

SA-ß-Gal in Kidney Tubules as a Predictor of Renal Outcome in Patients with Chronic Kidney Disease.

Cellular senescence has emerged as an important driver of aging and age-related disease in the kidney. The activity of ß-galactosidase at pH 6 (SA-ß-Gal) is a classic maker of senescence in cellular biology; however, the predictive role of kidney tissue SA-ß-Gal on eGFR loss in chronic kidney disease (CKD) is still not understood. We retrospectively studied the expression of SA-ß-Gal in kidney biopsies obtained in a cohort [n = 22] of incident patients who were followed up for 3 years as standard of care. SA-ß-Gal staining was approximately fourfold higher in the tubular compartment of patients with CKD vs. controls [26.0 ± 9 vs. 7.4 ± 6% positive tubuli in patients vs. controls; p < 0.025]. Tubular expressions of SA-ß-Gal, but not proteinuria, at the time of biopsy correlated with eGFR loss at the follow up; moreover, SA-ß-Gal expression in more than 30% of kidney tubules was associated with fast progressive kidney disease. In conclusion, our study shows that SA-ß-Gal is upregulated in the kidney tubular compartment of adult patients affected by CKD and suggests that tubular SA-ß-Gal is associated with accelerated loss of renal function.

Cyclic fasting bolsters cholesterol biosynthesis inhibitors' anticancer activity.

Identifying oncological applications for drugs that are already approved for other medical indications is considered a possible solution for the increasing costs of cancer treatment. Under the hypothesis that nutritional stress through fasting might enhance the antitumour properties of at least some non-oncological agents, by screening drug libraries, we find that cholesterol biosynthesis inhibitors (CBIs), including simvastatin, have increased activity against cancers of different histology under fasting conditions. We show fasting's ability to increase CBIs' antitumour effects to depend on the reduction in circulating insulin, insulin-like growth factor-1 and leptin, which blunts the expression of enzymes from the cholesterol biosynthesis pathway and enhances cholesterol efflux from cancer cells. Ultimately, low cholesterol levels through combined fasting and CBIs reduce AKT and STAT3 activity, oxidative phosphorylation and energy stores in the tumour. Our results support further studies of CBIs in combination with fasting-based dietary regimens in cancer treatment and highlight the value of fasting for drug repurposing in oncology.

The Contribution of Muscle Innate Immunity to Uremic Cachexia.

Protein energy wasting (PEW) is a common complication both in chronic kidney disease (CKD) and end-stage kidney disease (ESKD). Of note, PEW is one of the stronger predictors of morbidity and mortality in this patient population. The pathogenesis of PEW involves several mechanisms, including anorexia, insulin resistance, acidosis and low-grade inflammation. In addition, "sterile" muscle inflammation contributes to PEW at an advanced CKD stage. Both immune and resident muscle cells can activate innate immunity; thus, they have critical roles in triggering "sterile" tissue inflammation. Toll-like receptor 4 (TLR4) can detect endogenous danger-associated molecular patterns generated or retained in blood in uremia and induce a sterile muscle inflammatory response via NF-κB in myocytes. In addition, TLR4, though the activation of the NLRP3 inflammasome, links the sensing of metabolic uremic stress in muscle to the activation of pro-inflammatory cascades, which lead to the production of IL-1ß and IL-18. Finally, uremia-induced accelerated cell senescence is associated with a secretory phenotype that favors fibrosis in muscle. Targeting these innate immune pathways could lead to novel therapies for CKD-related PEW.

Homocysteine exchange across skeletal muscle in patients with chronic kidney disease.

Sites and mechanisms regulating the supply of homocysteine (Hcy) to the circulation are unexplored in humans. We studied the exchange of Hcy across the forearm in CKD patients (n = 17, eGFR 20 ± 2 ml/min), in hemodialysis (HD)-treated patients (n = 14) and controls (n = 9). Arterial Hcy was ~ 2.5 folds increased in CKD and HD patients (p < 0.05-0.03 vs. controls). Both in controls and in patients Hcy levels in the deep forearm vein were consistently greater (+~7%, p < 0.05-0.01) than the corresponding arterial levels, indicating the occurrence of Hcy release from muscle. The release of Hcy from the forearm was similar among groups. In all groups arterial Hcy varied with its release from muscle (p < 0.03-0.02), suggesting that muscle plays an important role on plasma Hcy levels. Forearm Hcy release was inversely related to folate plasma level in all study groups but neither to vitamin B12 and IL-6 levels nor to muscle protein net balance. These data indicate that the release of Hcy from peripheral tissue metabolism plays a major role in influencing its Hcy plasma levels in humans and patients with CKD, and that folate is a major determinant of Hcy release.

Case Report: Atypical Manifestations Associated With FOXP3 Mutations. The "Fil Rouge" of Treg Between IPEX Features and Other Clinical Entities?

Introduction: The Forkhead box protein P3 (FOXP3) is a transcription factor central to the function of regulatory T cells (Treg). Mutations in the FOXP3 gene lead to a systemic disease called immune dysregulation, polyendocrinopathy, and enteropathy, an X-linked syndrome (IPEX) characterized by the triad of early-onset intractable diarrhea, type 1 diabetes, and eczema. An atypical presentation of IPEX has been reported. Method: We report rare cases with equivocal clinical associations that included inflammatory, kidney, and hematologic involvements screened with massively parallel sequencing techniques. Results: Two patients with hemizygous mutations of FOXP3 [c.779T>A (p.L260Q)] and [c.1087A>G (p.I363V)] presented clinical manifestations not included in typical cases of IPEX: one was a 16-year-old male patient with an initial clinical diagnosis of autoimmune lymphoproliferative syndrome (ALPS) and who developed proteinuria and decreased kidney function due to membranous nephropathy, an autoimmune renal condition characterized by glomerular sub-epithelial antibodies. The second patient was a 2-year-old child with bone marrow failure who developed the same glomerular lesions of membranous nephropathy and received a bone marrow transplantation. High levels of IgG4 in serum, bone marrow, and kidney led to the definition of IgG4-related kidney disease (IgG4 RKD) in this young boy. The circulating Treg levels were normal in the former case and very low in the second. Conclusion: Two atypical associations of functional mutations of FOXP3 that include ALPS and IgG4 RKD are described. Membranous nephropathy leading to renal failure completed in both cases the clinical phenotypes that should be included in the clinical panorama of FOXP3 failure.

Secondary Membranous Nephropathy Due to Benign Tumors in 2 Young Women: A Case Report.

Membranous nephropathy (MN) is one of the most common causes of adult-onset nephrotic syndrome. We describe the cases of 2 young women in their 20s presenting with nephrotic syndrome due to antiphospholipase A2 receptor (anti-PLA2R)-negative MN, that was found to be associated with benign tumors. Both women had no extrarenal symptoms of a connective tissue disease, infection, or malignancy. They both had been previously healthy and were not receiving treatment with any drugs. Both had MN on kidney biopsy. Biopsies were negative for PLA2R antigen, and their serum did not demonstrate the presence of anti-PLA2R antibodies. Both were investigated for a secondary cause on the basis of negative anti-PLA2R serology and biopsy features supportive of secondary MN and were found to have benign tumors on radioimaging: a uterine leiomyoma and mesenteric fibromatosis, respectively. In both instances, the nephrotic syndrome remitted following resection of the tumors. To our knowledge, uterine leiomyoma and mesenteric fibromatosis have not previously been described in association with MN. These cases highlight the importance of pursuing a secondary cause of MN in patients without anti-PLA2R antibodies in serum or PLA2R antigen on kidney biopsy.

Myostatin: Basic biology to clinical application.

Myostatin is a member of the transforming growth factor (TGF)-ß superfamily. It is expressed by animal and human skeletal muscle cells where it limits muscle growth and promotes protein breakdown. Its effects are influenced by complex mechanisms including transcriptional and epigenetic regulation and modulation by extracellular binding proteins. Due to its actions in promoting muscle atrophy and cachexia, myostatin has been investigated as a promising therapeutic target to counteract muscle mass loss in experimental models and patients affected by different muscle-wasting conditions. Moreover, growing evidence indicates that myostatin, beyond to regulate skeletal muscle growth, may have a role in many physiologic and pathologic processes, such as obesity, insulin resistance, cardiovascular and chronic kidney disease. In this chapter, we review myostatin biology, including intracellular and extracellular regulatory pathways, and the role of myostatin in modulating physiologic processes, such as muscle growth and aging. Moreover, we discuss the most relevant experimental and clinical evidence supporting the extra-muscle effects of myostatin. Finally, we consider the main strategies developed and tested to inhibit myostatin in clinical trials and discuss the limits and future perspectives of the research on myostatin.

Ultra-hyper-fractionated radiotherapy for high-grade gliomas.

High-grade gliomas (HGGs; WHO grades III and IV) are invariably lethal brain tumors. Low-dose hyper-radiosensitivity (HRS) of HGG is a well-established phenomenon in vitro. However, possibly linked to the unavailability of accurate animal models of the diseases, this therapeutic effect could not be consistently translated to the animal setting, thus impairing its subsequent clinical development. The purpose of this study was to develop radiotherapeutic (RT) schedules permitting to significantly improve the overall survival of faithful animal models of HGG that have been recently made available. We used primary glioma initiating cell (GIC)-driven orthotopic animal models that accurately recapitulate the heterogeneity and growth patterns of the patients' tumors, to investigate the therapeutic effects of low radiation doses toward HGG. With the same total dose, RT fractions ≤0.5 Gy twice per week [ultra-hyper-fractionation (ultra-hyper-FRT)] started at early stages of tumor progression (a condition that in the clinical setting often occurs at the end of the guidelines treatment) improved the effectiveness of RT and the animal survival in comparison to standard fractions. For the same cumulative dose, the use of fractions ≤0.5 Gy may permit to escape one or more tumor resistance mechanisms thus increasing the effectiveness of RT and the overall animal survival. These findings suggest investigating in the clinical setting the therapeutic effect of an ultra-hyper-FRT schedule promptly extending the conventional RT component of the current guideline ("Stupp") therapeutic protocol.

Renal Ischemia/Reperfusion Early Induces Myostatin and PCSK9 Expression in Rat Kidneys and HK-2 Cells.

During visceral interventions, the transient clampage of supraceliac aorta causes ischemia/reperfusion (I/R) in kidneys, sometime resulting in acute renal failure; preclinical studies identified redox imbalance as the main driver of I/R injury. However, in humans, the metabolic/inflammatory responses seem to prevail on oxidative stress. We investigated myostatin (Mstn) and proprotein convertase subtilisin/kexin type 9 (PCSK9), proatherogenic mediators, during renal I/R. Compared to sham-operated animals, the kidneys of rats who had experienced ischemia (30 min) had higher Mstn and PCSK9 expression after 4 h of reperfusion. After 24 h, they displayed tubular necrosis, increased nitrotyrosine positivity, and nuclear peroxisome proliferator-activated receptor gamma coactivator-1alpha relocation, markers of oxidative stress and mitochondria imbalance. Mstn immunopositivity was increased in tubuli, while PCSK9 immunosignal was depleted; systemically, PCSK9 was higher in plasma from I/R rats. In HK-2 cells, both ischemia and reperfusion enhanced reactive oxygen species production and mitochondrial dysfunction. H2O2 upregulated Mstn and PCSK9 mRNA after 1 and 3.5 h, respectively. Accordingly, ischemia early induced Mstn and PCSK9 mRNA; during reperfusion Mstn was augmented and PCSK9 decreased. Mstn treatment early increased PCSK9 expression (within 8 h), to diminish over time; finally, Mstn silencing restrained ischemia-induced PCSK9. Our study demonstrates that renal I/R enhances Mstn and PCSK9 expression and that Mstn induces PCSK9, suggesting them as therapeutic targets for vascular protection during visceral surgery.

Increased serum uric acid levels are associated to renal arteriolopathy and predict poor outcome in IgA nephropathy.

BACKGROUND AND AIMS: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and a leading cause of end stage renal disease (ESRD). In addition to classical progression factors, other atherosclerosis-related factors, including hyperuricemia (HU), have been associated to the renal progression of IgAN. Increased serum uric acid (SUA) levels are well known to be concomitant of cardiovascular and kidney diseases, and have been proposed to be implicated in the development of arteriolar damage (AD). The aim of the present study was to explore the correlation between SUA levels, renal damage and its implication for outcome in IgAN patients. METHODS AND RESULTS: Clinical, laboratory and histologic data of 145 patients with biopsy proven IgAN were collected and retrospectively analyzed to determine the correlation between SUA levels, renal damage and the primary outcome (death or ESRD). Biopsy-proven AD was defined by the presence of arteriolar hyalinosis and/or intimal thickening. HU, defined as the highest SUA gender-specific tertile, was >7.7 mg/dl for males and >6.2 mg/dl for females. The prevalence of AD increased with the increase in the SUA level tertiles (p = 0.02). At logistic regression analysis SUA was independently related to the presence of AD (OR 1.75 [95%CI 1.10-2.93], p = 0.03). HU and AD had a synergic impact on progression of IgAN. Patients having both AD and HU, showed a reduced survival free from the primary outcome as compared to those having only one risk factor or neither (p = 0.01). CONCLUSIONS: SUA levels are independently associated with AD and poor prognosis in patients with IgAN.

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.

Long-term blood pressure behavior and progression to end-stage renal disease in patients with immunoglobulin A nephropathy: a single-center observational study in Italy.

BACKGROUND: Antihypertensive treatment by the use of RAAS inhibitors (RAAS-is) is of paramount importance in the management of slowly progressive IgA nephropathy (IgAN). With the aim of better understanding the relationship between BP behavior and progression, we looked at time-averaged SBP and time-averaged proteinuria and renal outcome in a single-center cohort of IgAN patients. METHODS: Among 248 consecutive patients referred to the Clinic of Nephrology of San Martino Hospital from 1996 to 2018 for native renal biopsy with a diagnosis of IgAN, we retrospectively analyzed 145 with available data at baseline and during follow-up. All patients received Supportive Care, 39% were on RAAS-is alone, 45% plus steroids, and 16% plus steroids and immunosuppressors. Renal replacing treatment (RRT) was the primary endpoint. RESULTS: During a mean follow-up of 67 ±â€Š6 months, 23% of study patients (n = 33) progressed to RRT and 6% (n = 9) died. Patients who reached the renal endpoint, had lower baseline eGFR and higher proteinuria and proteinuria indexed at baseline. Moreover, they had higher TA-SBP (139 ±â€Š17 vs. 130 ±â€Š13, P = 0.0016). The incidence of RRT was higher in IgAN patients in the highest time-averaged SBP tertile as compared with the others (32 vs. 23 vs. 9%, χ 6.8, P = 0.033). After adjusting for baseline SBP, baseline and time-averaged proteinuria indexed, MEST-C score, and treatment, the association between TA-SBP and RRT persisted. CONCLUSION: Time-averaged low BP values were independently associated to a decreased risk of renal progression in IgAN with no evidence of a J-curve relationship even at SBP levels below 125 mmHg.

Cellular Senescence Is Associated with Faster Progression of Focal Segmental Glomerulosclerosis.

BACKGROUND: A current, albeit unproven, hypothesis is that an acceleration of cellular senescence is involved in impaired renal repair and progression of glomerular diseases. Focal segmental glomerulosclerosis (FSGS) is a glomerular disease with a substantial risk for progression to ESRD. However, if and to what extent cell senescence predicts a negative outcome in FSGS is still unknown. METHODS: The hypothesis that cell senescence represents a proximate mechanism by which the kidney is damaged in FSGS (NOS phenotype) was investigated in 26 consecutive kidney biopsies from adult FSGS cases (eGFR 72 ± 4 mL/min, proteinuria 2.3 ± 0.6 g/day) who were incident for 2 years in a Northern Italian nephrology center and had a 6-year clinical follow-up. RESULTS: Cell senescence (p16INK4A, SA-ß-galactosidase [SA-ß-Gal]) was upregulated by ∼3- to 4-fold in both glomerular and tubular cells in kidney biopsies of FSGS as compared to age-matched controls (p < 0.05-0.01). Tubular SA-ß-Gal correlated with proteinuria and glomerulosclerosis, while only as a trend, tubular p16INK4A was directly associated with interstitial fibrosis. At univariate analysis, basal eGFR, proteinuria, and tubular expression of SA-ß-Gal and p16INK4A were significantly directly related to the annual loss of eGFR. No correlation was observed between glomerular p16INK4A and eGFR loss. However, at multivariate analysis, eGFR, proteinuria, and tubular p16INK4A, but not SA-ß-Gal, contributed significantly to the prediction of eGFR loss. CONCLUSIONS: The results indicate that an elevated cell senescence rate, expressed by an upregulation of p16INK4A in tubules at the time of initial biopsy, represents an independent predictor of progression to ESRD in adult patients with FSGS.

Signal regulatory protein alpha initiates cachexia through muscle to adipose tissue crosstalk.

BACKGROUND: Muscle wasting from chronic kidney disease (CKD) or from defective insulin signalling results in morbidity and, ultimately, mortality. We have identified an endogenous mediator of insulin resistance, signal regulatory protein alpha (SIRPα), which leads to cachexia in mice and is associated with cachexia in patients with CKD. METHODS: We assessed insulin signalling and mechanisms causing muscle atrophy plus white adipose tissue (WAT) metabolism in mouse models of CKD or acute diabetes (streptozotocin treatment). We then examined these factors in mice with global knockout (KO) of SIRPα and sought mediators of metabolic responses in muscle and adipose tissues of mice with either muscle-specific or adipose tissue-specific KO of SIRPα. Metabolic responses were confirmed in primary cultures of adipose cells. RESULTS: In mice with CKD, SIRPα expression was increased in WAT (three-fold, P < 0.05), and this was associated with precursors of cachexia: 'pathologic browning', thermogenesis, and a two-fold activation of protein kinase A (P < 0.05 vs. control mice) plus loss of adipose tissue mass. In contrast, mice with SIRPα global KO and CKD or acute diabetes experienced improved insulin signalling and activation of pAkt plus 'physiologic browning' of WAT. These mice avoided losses of muscle and adipose tissues and experienced a 31% improvement in survival (P < 0.05) than did wild-type mice with CKD. In muscle-specific SIRPα KO mice with CKD, we uncovered that serum SIRPα levels (P < 0.05) were suppressed and were associated with improved insulin signalling both in skeletal muscles and in WAT. These changes were accompanied by physiologic WAT browning. However, in adipose-specific SIRPα KO mice with CKD, levels of serum SIRPα were increased over two-fold (P < 0.05), while muscle losses were minimally inhibited. Clinical implications of SIRPα signalling are suggested by our findings that include increased SIRPα expression in muscle and adipose tissues (P < 0.05 vs. healthy controls) plus higher SIRPα levels in the serum of patients with CKD (2.4-fold, P=0.000017 vs. healthy controls). CONCLUSIONS: Our results show that SIRPα plays an important role as an anti-insulin mediator regulating pathways to cachexia. In muscle-specific SIRPα KO, changes in SIRPα serum levels seem to improve insulin signalling in muscle and WAT, suggesting crosstalk between muscle and adipose tissue. Therefore, targeting SIRPα may prevent cachexia in patients with CKD or acute diabetes.

Emerging role of myostatin and its inhibition in the setting of chronic kidney disease.

The past two decades have witnessed tremendous progress in our understanding of the mechanisms underlying wasting and cachexia in chronic kidney disease (CKD) and in other chronic illnesses, such as cancer and heart failure. In all these conditions wasting is an effect of the activation of protein degradation in muscle, a response that increases the risk of morbidity and mortality. Major recent advances in our knowledge on how CKD and inflammation affect cellular signaling include the identification of the myostatin (MSTN)/activin system, and its related transcriptional program that promotes protein degradation. In addition, the identification of the role of MSTN/activin in the vascular wall shows premise that its inhibition can better control or prevent some effects of CKD on vessels, such as accelerated atherosclerosis and vascular calcifications. In this review, we summarize the expanding role of MSTN activation in promoting muscle atrophy and the recent clinical studies that investigated the efficacy of MSTN/activin pathway antagonism in sarcopenic patients. Moreover, we also review the utility of MSTN inhibition in the experimental models of CKD and its potential advantages in CKD patients. Lessons learned from clinical studies on MSTN antagonism in sarcopenic patients tell us that the anabolic intervention is likely better if we use a block of the two ActRII receptors. At the same time, however, it is becoming clear that MSTN-targeted therapies should not be seen as a substitute for physical activity and nutritional supplementation which are mandatory to successfully manage patients with wasting.

[mTOR inhibitors in kidney transplantation].

A changing paradigm of treatment of kidney transplant recipients is a new, wider approach to immunosuppression, which should take into account both antiviral and anticancer effects, in addition to cardiovascular protection. Recent observations suggest that the early introduction of mammalian target of rapamycin inhibitors (mTORi) in association with low dose CNI may offer many of these effects. The present manuscript summarizes benefits and contraindications of combinations with mTORi in kidney transplant immunosuppressive strategies.

Toll-like receptor-4 signaling mediates inflammation and tissue injury in diabetic nephropathy.

Toll-like receptors (TLRs) are a class of receptors of the innate immune system which detect pathogen-associated and danger-associated molecular patterns in order to initiate an inflammatory response. TLR2 and TLR4 downward signaling causes the production of proinflammatory cytokines that can induce insulin resistance and cardiovascular damage in obesity and type 2 diabetes mellitus. In diabetic nephropathy, TLR4, nucleotide-binding oligomerization domain-containing protein 2 (NOD2), and NLRP3 inflammasome are involved in the production and persistence of inflammation. The activation of TLRs stimulates the expression of several inflammatory cytokines and chemokines such as CCL2 and tumor necrosis factor (TNF)-α, which are associated with the progression of diabetic nephropathy. Different inflammatory mechanisms seem to take place in the early and late stages of diabetic kidney disease, with activation of the innate immunity response and enhanced chemiotactic effects in native kidney cells at an early stage, followed by tubulointerstitial monocyte infiltration at a more advanced disease state. Overall, available data indicate that the upregulated TLR4 response in the kidney translates the metabolic alterations of diabetes into kidney damage.

Toll-like receptor 4 signalling mediates inflammation in skeletal muscle of patients with chronic kidney disease.

BACKGROUND: Inflammation in skeletal muscle is implicated in the pathogenesis of insulin resistance and cachexia but why uremia up-regulates pro-inflammatory cytokines is unknown. Toll-like receptors (TLRs) regulate locally the innate immune responses, but it is unknown whether in chronic kidney disease (CKD) TLR4 muscle signalling is altered. The aim of the study is to investigate whether in CKD muscle, TLRs had abnormal function and may be involved in transcription of pro-inflammatory cytokine. METHODS: TLR4, phospho-p65, phospho-ikBα, tumour necrosis factor (TNF)-α, phospho p38, Murf 1, and atrogin were studied in skeletal muscle from nondiabetic CKD stage 5 patients (n = 29) and controls (n = 14) by immunohistochemistry, western blot, and RT-PCR. Muscle cell cultures (C2C12) exposed to uremic serum were employed to study TLR4 expression (western blot and RT-PCR) and TLR-driven signalling. TLR4 signalling was abrogated by a small molecule chemical inhibitor or TLR4 siRNA. Phospho AKT and phospho p38 were evaluated by western blot. RESULTS: CKD subjects had elevated TLR4 gene and protein expression. Also expression of NFkB, p38 MAPK and the NFkB-regulated gene TNF-α was increased. At multivariate analysis, TLR4 protein content was predicted by eGFR and Subjective Global Assessment, suggesting that the progressive decline in renal function and wasting mediate TLR4 activation. In C2C12, uremic serum increased TLR4 as well as TNF-α and down-regulated pAkt. These effects were prevented by blockade of TLR4. CONCLUSIONS: CKD promotes muscle inflammation through an up-regulation of TLR4, which may activate downward inflammatory signals such as TNF-α and NFkB-regulated genes.