Type 2 diabetes mellitus (T2DM) has become a critical health problem, with more than 450 million people living with this disease worldwide [1
]. Diabetic foot syndrome (DFS) is a prevalent complication in this population and a major cause of hospitalizations. This syndrome carries the risk of limb amputation, which represents the most prevalent non-traumatic amputation surgery in the hospital setting [2
]. Mortality associated with DFS is similar to that of breast, prostate, or colon cancer [3
]. Hence, there is an overwhelming need for a better understanding of the molecular mechanisms underlying the development of DFS and foremost to detect early those at the highest risk of this complication and develop more specific and effective therapies.
DFS has a complex and multifactorial pathogenesis where an underlying vascular atherogenic process affecting both the endothelium and the smooth muscle cell layer is a critical factor. Atherosclerosis is a chronic inflammatory phenomenon involving several pathways and molecules, including inflammatory cytokines [4
]. Closely related with the atherogenic phenomena, the alterations of the mineral metabolism have gained special prominence as pathogenic factors in the development and progression of vascular damage [6
The newly described system formed by the fibroblast growth factor (FGF)-23 and Klotho (KL) proteins is recognized as one of the main regulators of mineral metabolism. FGF23 is a phosphaturic hormone that is synthesized in the bone in response to dietary phosphate intake in order to keep a normal phosphate homeostasis [8
]. To this end, it binds to cognate fibroblast growth factor receptors (FGFRs; primarily FGFR1 and 3) in the renal tubular cells in the presence of the obligated co-receptor Klotho, a type 1 trans-membrane protein predominantly expressed in the kidneys [8
]. In addition, a soluble form of Klotho is generated by the shedding of the ectodomain by the A Disintegrin and A Metalloproteinase 17 (ADAM17), or by an alternative RNA splicing [9
]. Beyond this physiological role, the FGF23/Klotho system has been related to various processes associated with cardiovascular damage.
Diverse clinical studies have directly related the increase in serum concentrations of FGF23 with cardiovascular damage and with an increased mortality [12
]. Moreover, recent works suggest that high levels of FGF23 can exert deleterious effects on the cardiovascular system by establishing low affinity bonds with FGFRs, independently of Klotho [14
]. On the other hand, reduced concentrations of soluble Klotho have been involved in processes related to premature aging, including the appearance of vascular damage [16
]. This soluble form of Klotho is detectable in urine, serum, and cerebrospinal fluid, acting as a humoral factor with multiple functions such as anti-oxidation, modulation of ion channels, anti-Wnt signaling or anti-apoptosis, and anti-senescence effects [17
]. Importantly, the expression of KL
has been recently demonstrated in human vascular tissue [16
] and both the levels of systemic and vascular Klotho have been related with cardiovascular complications [16
In this proof-of-concept study, we hypothesize that the levels of FGF23 and Klotho are altered in DFS patients. We determined the serum concentrations and the vascular expression levels of FGF23 and Klotho in a group of diabetic patients, with and without DFS. We also measured the levels of inflammatory mediators, including the cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL10, and analyzed the relationship among these variables and the presence of DFS.
2. Materials and Methods
Eighty-five diabetic patients undergoing an elective open vascular surgery procedure due to established clinical atherosclerotic artery disease were considered for enrollment in this study. Exclusion criteria included hemodynamic instability; history of chronic inflammatory, immunologic, or tumoral disease; positive serology to hepatitis B, hepatitis C, or HIV; acute inflammatory or intercurrent infectious episodes in the previous month; institutionalization; treatment with immunotherapy or immunosuppressive drugs; previous organ transplantation and advanced renal disease (estimated glomerular filtration rate (eGFR) lower than 30 mL/min/1.73m2).
For comparative purposes in the immunohistochemical and gene expression analyses, femoral samples from 12 cadaveric non-diabetic organ donors matched by age, sex, and eGFR, and without any medical history of cardiovascular disease were recovered during organ retrieval surgery.
The study protocol was approved by the Institutional Ethics Committee of the University Hospital Nuestra Señora de Candelaria (Santa Cruz de Tenerife, Spain) and complied with ethical standards of the Declaration of Helsinki. Written informed consent was obtained from all participants before they participated in the study.
2.2. Samples and Biochemical Markers
During the surgical procedure, a sample of the carotid or femoral arteries, according to the affected vessel, was obtained from the participants. At the same time, serum samples were drawn, aliquoted, and immediately stored at −80 °C for further analysis. Routine biochemical parameters were determined using standard methods. Serum levels of intact FGF23 were measured by specific enzyme-linked immunosorbent assay (ELISA) (EMD Millipore Corporation, Milford, MA, USA), which detects only the intact form of FGF23 with a sensitivity of 3.5 pg/mL and intra- and inter-assay coefficients of 9.5% and 6.85%, respectively. Serum Klotho was measured using the human Klotho ELISA kit (Immuno-Biological Laboratories, Takasaki, Japan), with a sensitivity of 6.15 pg/mL and intra- and inter-assay coefficients of variation of 3.1 % and 6.9 %, respectively. High-sensitivity serum C-reactive protein (hsCRP) was measured by a high-sensitivity particle enhanced immunoturbidimetric fully automated assay (Roche Diagnostics GmbH, Mannheim, Germany) in a Cobas 6000 analyzer from the same manufacturer with a sensitivity of 0.3 mg/L and intra- and inter-assay coefficients of variation of 1.6 and 8.4, respectively. Levels of the inflammatory cytokines TNFα, IL6 and IL10 were also measured by high-sensitivity ELISA methods (Quantikine®, R&D Systems, Abingdon, UK). Minimum detectable concentrations were 0.10 pg/mL, 0.70 pg/mL, and 0.50 pg/mL, respectively. Intra- and inter-assay coefficients of variability were <10.8%.
Sections of blood vessels were fixed in 4% buffered formalin for 24 h and subsequently dehydrated in ascending concentrations of ethanol, cleared in xylene, and embedded in paraffin. Blocks were trimmed and 3 µm sections were processed for immunohistochemistry. Primary antibodies used were: mouse monoclonal anti-FGFR1, 1:400 dilution (Abcam, Cambridge, UK); rabbit polyclonal anti-FGFR3, 1:300 dilution (Abcam); rabbit polyclonal anti-Klotho, 1:100 dilution (Abcam); mouse monoclonal anti-IL6, 1:300 dilution (Santa Cruz Biotechnology Inc., Dallas, TX, USA); and rabbit monoclonal anti-TNFα, 1:100 dilution (Santa Cruz Biotechnology Inc.). For the quantification analysis, a total of five images of each slide that include intima and media layers were captured and processed with a high-resolution video camera (Sony, DF-W-X710, Kōnan, Japan) connected to a light microscope (Nikon Eclipse 50i). The areas of tissue stained by the antibodies were quantified by using ImageJ software (Rasband, W.S., ImageJ, National Institutes of Health, Bethesda, MD, USA). Results are expressed in square microns.
2.4. Quantitative Real-Time PCR
After surgery, vascular tissue fragments were immediately placed in RNAlater® solution (Ambion (Europe) Limited, Cambridge, UK) and stored at 4 °C for subsequent RNA extraction. Total RNA was isolated by homogenization in TRI Reagent® (Sigma-Aldrich, Saint Louis, MO, USA) employing TissueRuptor (QIAGEN, Hilden, Germany). Further purification was performed using RNeasyMini kit (QIAGEN), according to manufacturer’s specifications, and stored at −80 °C. RNA was quantified using a Thermo Scientific NanoDrop Lite Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and its quality was assessed by the A260/A280 ratio measured in this equipment. cDNA was obtained using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) for further use in quantitative RT-PCR (qRTPCR). Transcripts encoding for KL, ADAM17, TNF, IL6, IL10, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured by TaqMan quantitative PCR with TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). TaqMan gene expression assays for each transcript (Hs00183100_m1 (KLOTHO), Hs01041915_m1 (ADAM17), Hs00174128_m1 (TNFα), Hs00985639_ml (IL6), Hs0961622_m1 (IL10), and Hs99999905_m1 (GAPDH)) were analyzed in a 7500 Fast Real-Time PCR System (Applied Biosystems). The level of target mRNA was estimated by relative quantification using the comparative method (2−ΔΔCt) by normalizing to GAPDH expression. mRNA levels were expressed as arbitrary units (a.u.). Quantification of each cDNA sample was tested in triplicate.
2.5. Statistical Analysis
All analyses were performed using SPSS software version 25 (IBM Corp. Armonk, NY, USA). Continuous variables are reported as mean ± SD or median with interquartile range (IQR), and categorical data as number and percent frequency. Continuous variables were checked for the normal distribution assumption using the Kolmogorov–Smirnov statistics, and those that did not satisfy the criteria were log-transformed. Comparisons between groups were performed by Chi-square test, Mann–Whitney U test, or Kruskal-Wallis test as appropriate. The Spearman correlation coefficient was calculated to assess the relationship between variables. Partial correlation analysis was performed to measure the association of serum and vascular Klotho expression with inflammatory parameters whilst controlling for the effect of covariates that were selected based on clinical relevance and the results of comparison and correlation analysis. A multiple logistic regression was performed to assess independent predictors of the presence of DFS. For this purpose, we adopted three models: In model 1, we introduced age, sex, uric acid, eGFR, and hsCRP. In model 2, we additionally included serum Klotho, FGF23, IL6, and IL10. Finally, in model 3 we adjusted the analysis for the immunoreactivity and gene expression levels of KL. Regarding gene expression analysis, quantification of each sample was tested in triplicate and data are expressed as arbitrary units. A two tailed p-value < 0.05 was considered statistically significant.
The main results of the present study show that patients with DFS have lower serum concentrations of soluble Klotho and elevated concentrations of FGF23, while at the vascular level, both immunoreactivity and gene expression levels of KL were also diminished. Moreover, these alterations in the components of the FGF23/Klotho system are independently associated with the presence of DFS. These findings have been not reported previously and suggest an intriguing possibility about the potential role of FGF23/Klotho in the development and/or progression of DFS.
Vascular disease is a critical factor underlying the pathogenesis of DFS [22
]. The FGF23/Klotho system has been decisively related to various processes associated with vascular damage. Recent works suggest that FGF23 can exert direct effects on the cardiovascular system. It has been proven that at high concentrations, FGF23 is able to establish low affinity bonds with its receptors, independently of the presence of Klotho, which could cause deleterious effects on multiple organs and tissues [14
]. From a clinical point of view, elevated levels of FGF23 have been associated with the presence of vascular dysfunction [15
], as well as with a greater severity of atherosclerotic vascular damage [23
]. A direct effect of FGF23 on the integrity of vascular tissue has been proposed, a hypothesis that has been reinforced by the recent demonstration of the expression of its receptors, as well as Klotho, in the human vascular wall [16
]. Our results show that patients with DFS present not only higher serum levels of FGF23, but also increased vascular expression of FGFRs. These findings point out the possibility that high FGF23 could exert direct pathogenic actions on the vasculature even though vascular levels of Klotho are diminished.
On the other hand, Klotho deficiency has been also associated with vascular damage. In murine models, absence of Klotho causes a syndrome of accelerated aging, which included arteriosclerosis and vascular calcifications [24
], alterations that could be reversed by administration of the Kl
gene or by parabiosis with the wild mice [25
]. More recent studies have confirmed the protective effects of Klotho on the vascular system, including its participation in the maintenance of endothelial homeostasis and vascular functionality [26
]. In the clinical setting, low serum Klotho concentrations in adult subjects without known risk factors for cardiovascular disease have been related to a reduced capacity of flow-mediated dilation of the brachial artery and higher values of epicardial fat thickness and carotid artery intima-media thickness, suggesting that Klotho deficiency may be an early predictor of subclinical atherosclerosis [27
]. Moreover, recent works have related the presence of established clinical atherosclerotic disease with low serum Klotho levels [19
]. To the best of our knowledge, only one previous study has evaluated the serum Klotho concentrations in T2DM in relation to diabetic complications. In that study, Zhang et al. [29
] studied 102 T2DM patients with DFS and observed that these individuals presented a 44.79% reduction in serum Klotho as compared with non-diabetic controls, with no differences with respect to patients with other complications but no diabetic foot. In the present work, patients with DFS presented a median percent decrease of 37.4% in serum Klotho concentrations as compared with diabetic subjects without DFS. These differences could be explained by the distinct subjects’ characteristics between the studies, since in the work by Zhang et al. the patients were younger (mean age, 56.1 vs. 71.8 years), with a greater female proportion (43.1% vs. 25%), lower mean BMI (27.4 vs. 30.8 kg/m2
), and more importantly, all of our patients had severe DFS with lower limb amputation, whereas in the study by Zhang et al. the criteria for the DFS group included foot ulcers, deformity, infection, or gangrene. Therefore, it is possible to speculate that reduction in serum Klotho may reflect a progressive process that is associated with the severity of DFS.
Importantly, the endogenous expression of KL
in human arteries has been recently demonstrated, an issue of special relevance since the vascular wall may constitute a source of soluble Klotho, with putative autocrine and/or paracrine protective beneficial effects on vascular homeostasis [16
]. Clinical data regarding the relationship between vascular KL
expression and atherosclerotic disease are scarce. In previous works, our group reported that low vascular KL
gene expression was associated with the presence and severity of coronary artery disease [21
] and clinical atherosclerotic disease [19
] independently of established cardiovascular risk factors. The present study reports for the first-time data about vascular expression of Klotho, both at gene and protein levels, in patients with DFS. In particular, we have detected a 51.4% lower gene expression and a 52.1% lower protein expression of Klotho in vascular samples of patients with DFS as compared with diabetic subjects without this complication. Overall, these findings may be relevant from a mechanistic perspective since Klotho has been involved in maintaining the health of the vasculature, and thus, disruption of Klotho homeostasis may be an important factor in the development and progression of atherosclerotic disease in general, and DFS in particular.
Finally, it is important to note the interplay between inflammation and Klotho, since inflammation induces the reduction of KL
expression, both in the kidney as well as in the vessels [16
]. Our results show that subjects with DFS presented higher serum concentrations of hsCRP and IL6, and reduced levels of the anti-inflammatory cytokine IL10. In addition, the immunoreactivity levels of TNFα and IL6 were increased in the two groups of diabetic patients as compared with non-diabetic subjects. Regarding gene expression, diabetic subjects presented a higher expression of TNF
and lower levels of IL10
compared to control subjects, whereas IL6 expression was significantly higher in patients with DFS as compared with non-diabetic individuals and diabetic subjects without DFS. Importantly, after adjusting for the effect of other variables, arterial KL
mRNA levels showed an independent negative relationship with IL6
expression, while vascular Klotho immunoreactivity was also negatively associated with immunoreactivity levels for IL6, supporting the role of inflammation as an inducer of vascular Klotho downregulation in DFS.
Although this study provides novel information about the relationship between DFS and FGF23/Klotho, several limitations need to be acknowledged. First, the small sample size since this work was designed as a proof-of-concept study, which might imply that some of our results did not reach statistical significance and limit the ability to adequately describe the closeness of the relationship between the variables; however, the results indicate that the initial assumptions can be valid for generating new hypotheses. Second, given the cross-sectional nature of the study, we can only demonstrate associations without definitive inferences on their direction or causality. Third, serum levels of other factors related to Klotho and FGF23, such as vitamin D, which may potentially impact DFS, were not measured, and therefore, their potential influence on the results may not be completely ruled out.