Pentoxifylline ameliorates subclinical atherosclerosis progression in patients with type 2 diabetes and chronic kidney disease: a randomized pilot trial.

BACKGROUND: Diabetic kidney disease (DKD) is associated with a higher risk of cardiovascular disease (CVD). Pentoxifylline (PTF), a nonselective phosphodiesterase inhibitor with anti-inflammatory, antiproliferative, and antifibrotic actions, has demonstrated renal benefits in both clinical trials and meta-analyses. The present work aimed to study the effects of PTF on the progression of subclinical atherosclerosis (SA) in a population of patients with diabetes and moderate to severe chronic kidney disease (CKD). METHODS: In this open-label, randomized controlled, prospective single-center pilot study the evolution of carotid intima-media thickness (CIMT) and ankle-brachial index (ABI) were determined in 102 patients with type 2 diabetes mellitus and CKD assigned to PTF, aspirin or control groups during 18 months. We also determined the variations in the levels of inflammatory markers and Klotho (KL), a protein involved in maintaining cardiovascular health, and their relationship with the progression of SA. RESULTS: Patients treated with PTF presented a better evolution of CIMT, increased KL mRNA levels in peripheral blood cells (PBCs) and reduced the inflammatory state. The progression of CIMT values was inversely related to variations in KL both in serum and mRNA expression levels in PBCs. Multiple regression analysis demonstrated that PTF treatment and variations in mRNA KL expression in PBCs, together with changes in HDL, were significant determinants for the progression of CIMT (adjusted R2 = 0.24, P < 0.001) independently of traditional risk factors. Moreover, both variables constituted protective factors against a worst progression of CIMT [OR: 0.103 (P = 0.001) and 0.001 (P = 0.005), respectively]. CONCLUSIONS: PTF reduced SA progression assessed by CIMT variation, a beneficial effect related to KL gene expression in PBCs. TRIAL REGISTRATION: The study protocol code is PTF-AA-TR-2009 and the trial was registered on the European Union Drug Regulating Authorities Clinical Trials (EudraCT #2009-016595-77). The validation date was 2010-03-09.

FGF23 as a Potential Pathophysiological Factor in Peripheral Arterial Disease Associated with Chronic Kidney Disease.

Fibroblast growth factor 23 (FGF23) levels are often elevated in chronic kidney disease (CKD). FGF23 and inflammation are common characteristics in CKD, and both are associated with worse disease progression and the occurrence of complications. The existence of an interaction between FGF23 and inflammation has been suggested, each of which influences the expression and activity of the other, leading to a vicious feedback loop with adverse outcomes, including cardiovascular disease and mortality. In this work, we determined circulating FGF23 levels in a group of patients with CKD stages 3 and 4 subjected to elective femoral endarterectomy due to established peripheral artery disease (PAD), a condition resulting from an athero-inflammatory process, and we studied its associations with different inflammatory markers and mediators. We evaluated its association with serum tumor necrosis factor (TNF)α, interleukin (IL) 6, and IL10, as well as with the gene expression levels of these parameters and A disintegrin and metalloproteinase domain-containing protein (ADAM) 17 in femoral vascular tissue and peripheral blood circulating cells (PBCCs). We also analyzed its association with serum concentrations of C-reactive protein (CRP), the systemic immune inflammation index (SII), and the neutrophil-to-lymphocyte ratio (NLR). Finally, we determined the vascular immunoreactivity of protein TNFα in a subgroup of patients. FGF23 concentrations were independently associated with circulating and PBCC mRNA levels of TNFα. Worst kidney function and diabetes were also found to be contributing to FGF23 levels. Patients with higher levels of FGF23 also had greater vascular immunoreactivity for TNFα.

Association of Klotho with Coronary Artery Disease in Subjects with Type 2 Diabetes Mellitus and Preserved Kidney Function: A Case-Control Study.

Circulating Klotho levels are significantly reduced in subjects with type 2 diabetes mellitus (T2DM) and in kidney disease patients. In this work, the relationship between Klotho levels and the coronary artery disease (CAD) burden in subjects with T2DM and preserved kidney function was analyzed. For this, we performed a cross-sectional case-control study involving 133 subjects with T2DM and 200 age-, sex- and CAD-incidence-matched, non-diabetic patients undergoing non-emergency diagnostic coronary angiography. All of them were non-albuminuric and with normal glomerular filtration rates. The concentrations of serum Klotho, fibroblast growth factor 23, and inflammatory markers were also measured. As expected, the serum Klotho concentration was lower in the T2DM group (12.3% lower, p = 0.04). However, within the group of patients with T2DM, those subjects with CAD presented significantly higher Klotho levels than those without significant coronary stenosis (314.5 (6.15-562.81) vs. 458.97 (275.2-667.2) pg/mL; p = 0.02). Multiple regression analysis revealed that serum Klotho was positively related with stenosis values exclusively in subjects with T2DM (adjusted R2 = 0.153, p < 0.01). Moreover, logistic regression analysis showed that Klotho was positively associated with the presence of significant CAD in the group of T2DM patients (OR: 1.001; p = 0.041). Our data suggest that higher levels of circulating Klotho in subjects with T2DM and preserved kidney function are associated with the presence of significant CAD.

Associations between Inflammation, Hemoglobin Levels, and Coronary Artery Disease in Non-Albuminuric Subjects with and without Type 2 Diabetes Mellitus.

In this cross-sectional study, we evaluated the associations of inflammation and hemoglobin with coronary artery disease (CAD) in subjects with type 2 diabetes mellitus (T2DM) and preserved kidney function. We recruited 638 participants-254 with T2DM-subjected to coronary angiography with no known cardiovascular disease, normal glomerular filtration rates, and without albuminuria. The hemoglobin and serum levels of inflammatory markers, including high-sensitivity C-reactive protein (hs-CRP), were measured. Multivariable analyses showed that inflammatory markers were not related to the severity of the stenosis in the group of subjects with diabetes. Conversely, inflammatory cytokines and albuminuria were directly related to the percentage of stenosis in subjects without T2DM (R2 = 0.038, p < 0.001). Patients with diabetes presented lower hemoglobin levels, particularly in those who also had significant CAD (14.4 [13.6-15.1] vs. 13.6 [12.2-14.8] g/dL, p = 0.03). Similarly, hemoglobin levels and albuminuria were inversely related to the severity of stenosis exclusively in subjects with diabetes, even after adjusting for multiple confounding factors (R2 = 0.081, p < 0.001). We conclude that reductions in hemoglobin levels in subjects with T2DM and normoalbuminuria may constitute a more relevant risk factor for CAD than inflammation.

MicroRNA-630: A promising avenue for alleviating inflammation in diabetic kidney disease.

Diabetic kidney disease (DKD) is one of the complications of diabetes, affecting millions of people worldwide. The relentless progression of this condition can lead to kidney failure, requiring life-altering interventions such as dialysis or transplants. Accumulating evidence suggests that immunologic and inflammatory elements play an important role in initiating and perpetuating the damage inflicted on renal tissues, exacerbating the decline in organ function. Toll-like receptors (TLRs) are a family of receptors that play a role in the activation of the innate immune system by the recognition of pathogen-associated molecular patterns. Recent data from in vitro and in vivo studies have highlighted the critical role of TLRs, mainly TLR2 and TLR4, in the pathogenesis of DKD. In the diabetic milieu, these TLRs recognize diabetic-associated molecular signals, triggering a proinflammatory cascade that initiates and perpetuates inflammation and fibrogenesis in the diabetic kidney. Emerging non-traditional strategies targeting TLR signaling with potential therapeutic implications in DKD have been pro-posed. One of these approaches is the use of microRNAs, small non-coding RNAs that can regulate gene expression. This editorial comments on the results of this approach carried out in a rat model of diabetes by Wu et al, published in this issue of the World Journal of Diabetes. The results of the experimental study by Wu et al shows that microRNA-630 decreased levels compared to non-diabetic rats. Additionally, microRNA-630 exerted anti-inflammatory effects in the kidneys of diabetic rats through the modulation of TLR4. These findings indicate that the microRNA-630/TLR4 axis might represent a pathological mechanism of DKD and a potential therapeutic target capable of curbing the destructive inflammation characteristic of DKD.

The Value of Klotho in Kidney Transplantation.

Kidney transplant recipients have better survival rates and improved quality of life than long-term dialysis patients. However, delayed graft function, immunosuppressive therapy nephrotoxicity, and rejection episodes may compromise graft and patient survival. The KL gene is highly expressed in kidney tubular cells and encodes the antiaging and kidney-protective protein Klotho, which has membrane-anchored and soluble forms and regulates mineral metabolism. Klotho expression decreases during acute kidney injury or chronic kidney disease, and human chronic kidney disease shares features of accelerated aging with murine Klotho deficiency. In this work, we review clinical studies on the relationship between Klotho and kidney transplantation. Specifically, we address the dynamics of serum and kidney Klotho levels in donors and kidney transplant recipients, the role of Klotho as a marker of current graft function and graft outcomes, and the potential impact of Klotho on kidney protection in the transplantation context. A better understanding of the potential biomarker and therapeutic utility of Klotho in kidney transplant recipients may provide new insights into the control of graft function and new therapeutic strategies to preserve allograft function.

Klotho inversely relates with carotid intima- media thickness in atherosclerotic patients with normal renal function (eGFR ≥60 mL/min/1.73m2): a proof-of-concept study.

Introduction: Klotho protein is predominantly expressed in the kidneys and has also been detected in vascular tissue and peripheral blood circulating cells to a lesser extent. Carotid artery intima-media thickness (CIMT) burden, a marker of subclinical atherosclerosis, has been associated with reductions in circulating Klotho levels in chronic kidney disease patients, who show reduced levels of this protein at all stages of the disease. However, the contribution of serum Klotho and its expression levels in peripheral blood circulating cells and in the carotid artery wall on the CIMT in the absence of kidney impairment has not yet been evaluated. Methods: We conducted a single-center study in 35 atherosclerotic patients with preserved kidney function (eGFR≥60 mL/min/1.73m2) subjected to elective carotid surgery. Serum levels of Klotho and cytokines TNFa, IL6 and IL10 were determined by ELISA and transcripts encoding for Klotho (KL), TNF, IL6 and IL10 from vascular segments were measured by qRT-PCR. Klotho protein expression in the intima-media and adventitia areas was analyzed using immunohistochemistry. Results: APatients with higher values of CIMT showed reduced Klotho levels in serum (430.8 [357.7-592.9] vs. 667.8 [632.5-712.9] pg/mL; p<0.001), mRNA expression in blood circulating cells and carotid artery wall (2.92 [2.06-4.8] vs. 3.69 [2.42-7.13] log.a.u., p=0.015; 0.41 [0.16-0.59] vs. 0.79 [0.37-1.4] log.a.u., p=0.013, respectively) and immunoreactivity in the intimal-medial area of the carotids (4.23 [4.15-4.27] vs. 4.49 [4.28-4.63] log µm2 p=0.008). CIMT was inversely related with Klotho levels in serum (r= -0.717, p<0.001), blood mRNA expression (r=-0.426, p=0.011), and with carotid artery mRNA and immunoreactivity levels (r= -0.45, p=0.07; r= -0.455, p= 0.006, respectively). Multivariate analysis showed that serum Klotho, together with the gene expression levels of tumor necrosis factor TNFa in blood circulating cells, were independent determinants of CIMT values (adjusted R2 = 0.593, p<0.001). Discussion: The results of this study in subjects with eGFR≥60mL/min/1.73m2 show that patients with carotid artery atherosclerosis and higher values of CIMT present reduced soluble Klotho levels, as well as decreased KL mRNA expression in peripheral blood circulating cells and Klotho protein levels in the intima-media of the carotid artery wall.

Repurposing drugs for highly prevalent diseases: pentoxifylline, an old drug and a new opportunity for diabetic kidney disease.

Diabetic kidney disease is one of the most frequent complications in patients with diabetes and constitutes a major cause of end-stage kidney disease. The prevalence of diabetic kidney disease continues to increase as a result of the growing epidemic of diabetes and obesity. Therefore, there is mounting urgency to design and optimize novel strategies and drugs that delay the progression of this pathology and contain this trend. The new approaches should go beyond the current therapy focussed on the control of traditional risk factors such as hyperglycaemia and hypertension. In this scenario, drug repurposing constitutes an economic and feasible approach based on the discovery of useful activities for old drugs. Pentoxifylline is a nonselective phosphodiesterase inhibitor currently indicated for peripheral artery disease. Clinical trials and meta-analyses have shown renoprotection secondary to anti-inflammatory and antifibrotic effects in diabetic patients treated with this old known drug, which makes pentoxifylline a candidate for repurposing in diabetic kidney disease.

Klotho expression in peripheral blood circulating cells is associated with vascular and systemic inflammation in atherosclerotic vascular disease.

Cardiovascular disease is the leading cause of death worldwide. New therapeutic strategies are aimed to modulate the athero-inflammatory process that partially orchestrates underlying vascular damage. Peripheral blood circulating cells include different immune cells with a central role in the development of the atherogenic inflammatory response. The anti-aging protein α-Klotho has been related to protective effects against CVD. KL is expressed in monocytes, macrophages, and lymphocytes where it exerts anti-inflammatory effects. In this work, we analyse the relationships of the levels of inflammatory markers with the expression of the KL gene in PBCCs and with the serum levels of soluble KL in atherosclerotic vascular disease. For this, we conducted a cross-sectional single-center case-control study including a study group of 76 CVD patients and a control group of 16 cadaveric organ donors without medical antecedent or study indicating CVD. Vascular artery fragments and whole blood and serum samples were obtained during elective or organ retrieval surgery. Serum levels of sKL, TNFα and IL10, and gene expression levels of KL, TNF, IL10, NFKB1, DNMT1, and DNMT3A in PBCCs were measured. In these cells, we also determined KL promoter methylation percentage. Histological and immunohistochemical analyses were employed to visualize atherosclerotic lesions and to measure IL10 and TNFα levels in vascular fragments. Patients with CVD presented higher values of proinflammatory markers both at systemic and in the vasculature and in the PBCCs, compared to the control group. In PBCCs, CVD patients also presented lower gene expression levels of KL gene (56.4% difference, P < 0.001), higher gene expression levels of DNMT1 and DNMT3A (P < 0.0001, for both) and a higher methylation status of in the promoter region of KL (34.1 ± 4.1% vs. 14.6 ± 3.4%, P < 0.01). In PBCCs and vasculature, KL gene expression correlated inversely with pro-inflammatory markers and directly with anti-inflammatory markers. sKL serum levels presented similar associations with the expression levels of pro- and anti-inflammatory markers in PBCCs. The differences in KL expression levels in PBCCs and in serum sKL levels with respect to control group was even greater in those CVD patients with macroscopically observable atheromatous plaques. We conclude that promoter methylation-mediated downregulation of KL gene expression in PBCCs is associated with the pro-inflammatory status in atherosclerotic vascular disease.

Klotho as a biomarker of subclinical atherosclerosis in patients with moderate to severe chronic kidney disease.

Chronic kidney disease (CKD) has been associated with a higher risk of cardiovascular disease (CVD). CKD patients present a decrease in the levels of the protein Klotho that accompanies the decrease in kidney function. This protein has been related to protective effects against CVD. However, it is unclear whether circulating Klotho, and its expression in peripheral blood cells (PBCs) are also associated with subclinical atherosclerosis in CKD. The present study aimed to study the relationship between Klotho and subclinical atherosclerosis in a population of patients with moderate to severe CKD. We determined the serum levels and gene expression in PBCs levels of Klotho and three inflammatory cytokines in 103 patients with CKD and investigated their relationship with two surrogate markers of subclinical atherosclerotis: ankle-brachial index (ABI) and carotid intima-media thickness (CIMT). Patients with subclinical atherosclerosis presented lower serum and PBCs expression levels of Klotho. Both variables were associated with the presence of subclinical atherosclerosis, being directly related with ABI and inversely with CIMT (P < 0.0001 for both). Multiple regression analysis demonstrated that both variables were significant determinants for ABI (adjusted R2 = 0.511, P < 0.0001) and CIMT (adjusted R2 = 0.445, P < 0.0001), independently of traditional and emergent cardiovascular risk factors. Moreover, both constituted protective factors against subclinical atherosclerosis [OR: 0.993 (P = 0.002) and 0.231 (P = 0.025), respectively]. Receiver operating characteristic analysis pointed to the utility of serum Klotho (area under the curve [AUC]: 0.817, 95% CI: 0.736-0.898, P < 0.001) and its gene expression in PBCs (AUC: 0.742, 95% CI: 0.647-0.836, P < 0.001) to distinguish subclinical atherosclerosis. The reductions in serum and PBCs expression levels of Klotho in CKD patients are independently associated with the presence of for subclinical atherosclerosis. Further research exploring whether therapeutic approaches to maintain or elevate Klotho could reduce the impact of CVD in CKD patients is warranted.

Pathophysiological Implications of Imbalances in Fibroblast Growth Factor 23 in the Development of Diabetes.

Observational studies have associated the increase in fibroblast growth factor (FGF) 23 levels, the main regulator of phosphate levels, with the onset of diabetes. These studies open the debate on the plausible existence of undescribed diabetogenic mechanisms derived from chronic supraphysiological levels of FGF23, a prevalent condition in chronic kidney disease (CKD) and end-stage renal disease (ESRD) patients. These maladaptive and diabetogenic responses to FGF23 may occur at different levels, including a direct effect on the pancreatic ß cells, and an indirect effect derived from the stimulation of the synthesis of pro-inflammatory factors. Both mechanisms could be mediated by the binding of FGF23 to noncanonical receptor complexes with the subsequent overactivation of signaling pathways that leads to harmful effects. The canonical binding of FGF23 to the receptor complex formed by the receptor FGFR1c and the coreceptor αKlotho activates Ras/MAPK/ERK signaling. However, supraphysiological concentrations of FGF23 favor non-αKlotho-dependent binding of this molecule to other FGFRs, which could generate an undesired overactivation of the PLCγ/CN/NFAT pathway, as observed in cardiomyocytes and hepatocytes. Moreover, the decrease in αKlotho expression may constitute a contributing factor to the appearance of these effects by promoting the nonspecific activation of the PLCγ/CN/NFAT to the detriment of the αKlotho-dependent Ras/MAPK/ERK pathway. The description of these mechanisms would allow the development of new therapeutic targets susceptible to be modified by dietary changes or by pharmacological intervention.

Inflammatory Cytokines in Diabetic Kidney Disease: Pathophysiologic and Therapeutic Implications.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease and a main contributing factor for cardiovascular morbidity and mortality in patients with diabetes mellitus. Strategies employed to delay the progression of this pathology focus on the control of traditional risk factors, such as hyperglycemia, and elevated blood pressure. Although the intimate mechanisms involved in the onset and progression of DKD remain incompletely understood, inflammation is currently recognized as one of the main underlying processes. Untangling the mechanisms involved in the appearing of a harmful inflammatory response in the diabetic patient is crucial for the development of new therapeutic strategies. In this review, we focus on the inflammation-related pathogenic mechanisms involved in DKD and in the therapeutic utility of new anti-inflammatory strategies.