Relationship between Serum Indoxyl Sulfate and Klotho Protein and Vascular Calcification in Patients with Chronic Kidney Disease Stages 3–5

Objective This study aims to explore the relationships between serum indoxyl sulfate (IS) and Klotho protein levels with vascular calcification in patients with chronic kidney disease (CKD) stages 3–5. Methods From December 2021 to January 2023, a total of 108 CKD patients in stages 3–5 were enrolled in this cross-sectional investigation. Demographic information and routine clinical biochemistry test results were gathered. Serum levels of IS and Klotho were quantified through enzyme-linked immunosorbent assays. Furthermore, multislice spiral computed tomography was employed to evaluate vascular calcification. The association between serum IS or Klotho levels and abdominal aorta calcification was assessed using univariate analysis and logistic regression analyses. Results With the progression of CKD stages, serum creatinine, phosphorus, intact parathyroid hormone (iPTH), serum IS, and abdominal aortic calcification exhibited incremental trends, while serum calcium and Klotho protein levels showed a diminishing trend, with statistically significant differences (P < 0.05). Significant differences were observed in age, blood phosphorus, calcium, total parathyroid hormone, serum IS, and Klotho protein levels between patients with and without aortic calcification (P < 0.05). Logistic regression analysis demonstrated that advanced age, high IS level, and low Klotho protein level were independent risk factors for abdominal aortic calcification in CKD patients (P < 0.05). Conclusion This study indicates elevated serum IS levels and decreased Klotho protein levels in CKD patients. High IS level and low Klotho level were independent risk factors for abdominal aortic calcification.


Introduction
Chronic kidney disease (CKD) has become one of the international public health problems that seriously threaten human health.With kidney function progressively diminishing, CKD eventually develops into end-stage renal disease (ESRD).Cardiovascular disease (CVD) is the most common complication in patients with CKD and ESRD and the leading cause of death in patients with ESRD.Vascular calcifcation and its severity have long been recognized as important factors in the development of cardiovascular disease (CVD) in patients with CKD.Te etiology of vascular calcifcation in CKD is multifactorial, infuenced by both conventional and nontraditional elements including malnutrition, smoking, age, diabetes, uremic toxin accumulation, oxidative stress, and infammation [1].
Indoxyl sulfate (IS) has garnered signifcant attention as one of the intestinal protein-binding uremic toxins in recent years.Current studies have shown that [1,2] IS can upregulate the expression of osteoblast-specifc proteins such as runt-related transcription factor 2 and osteopontin, thus promoting calcifcation.Klotho protein, a type I single-channel transmembrane protein, is highly expressed in the kidney.In a mouse model of chronic kidney disease-mineral bone disorder, the release of soluble Klotho alleviates hyperphosphatemia and vascular calcifcation [3].In the late stage of CKD, the diminishing levels of Klotho protein compromise the efective regulation of bone minerals by fbroblast growth factor 23 (FGF23), thus accelerating the occurrence and development of vascular calcifcation in CKD [4].At present, the mechanism underlying vascular calcifcation is not clear, and efective therapeutic strategies are lacking.Consequently, this study aims to investigate the correlation between serum IS level, Klotho protein levels, and vascular calcifcation in patients with CKD stages 3-5, understand the mechanisms underlying vascular calcifcation in CKD, and explore potential therapeutic targets.

Participants.
A total of 108 patients with CKD stages 3-5 admitted to the Department of Nephrology of our hospital from December 2021 to January 2023 were selected.Te inclusion criteria of CKD patients were as follows: (1) CKD stage from 3 to 5 according to the criteria for diagnosis and staging of CKD formulated in the K/DOQI guidelines; (2) age between 18 and 85 years; (3) medical history >6 months; and (4) no administration of phosphate binders or active vitamin D3 before admission.Exclusion criteria were as follows: (1) had previously received kidney transplantation or parathyroidectomy; (2) connective tissue diseases, blood diseases, malignant tumors, severe malnutrition, mental illness, or who are unable to cooperate with the examination for other reasons; (3) long-term use of hormones, immunosuppressants, and antibiotics; (4) severe infection, liver function damage, and cardiovascular and cerebrovascular accidents; (5) sarcomatoid nodular disease, HIV, and other diseases afecting the calcifcation status and soft tissue calcifcation of patients; and (6) pregnant or lactating patients.
All patients were divided into CKD stages 3-4 group (36 cases), CKD stage 5 nondialysis group (38 cases), and CKD stage 5 dialysis group (34 cases).Patients in the CKD stage 5 dialysis group were treated with stable hemodialysis with 3 h each time, 3 times per week, and the total duration of dialysis was 3-6 months.Tis short duration of dialysis can greatly weaken the efect of dialysis procedures on the degree of vascular calcifcation in patients [5].All patients showed good responsiveness and sensitivity to dialysis treatment.
Twenty healthy subjects who underwent physical examination in the physical examination center of our hospital were selected as the healthy control group.Hypertension, diabetes, kidney disease, coronary heart disease, and other diseases were excluded.Tis study was approved by the Ethics Committee of our hospital (Ethics Committee approval no.HDFY-LL-2020-068).All participants signed the written informed consent.

General Information.
Demographic data for all enrolled patients were meticulously documented, including gender, age, height, body mass index (BMI), smoking history, alcohol consumption history, and other pertinent variables.Detailed etiological records of CKD, including conditions such as diabetic nephropathy, chronic glomerulonephritis, and polycystic kidney disease, were compiled.In addition, histories of concurrent ailments such as diabetes, hypertension, coronary heart disease, and recent medication usage were thoroughly documented.

Laboratory Tests.
Fasting blood samples were procured in the early morning to ensure consistency.For patients undergoing stable hemodialysis, blood samples were obtained before the last hemodialysis session of the week [6].Levels of serum albumin, creatinine (SCr), glucose, calcium, phosphorus, and C-reactive protein (CRP) were measured using standard autoanalyzer techniques (Beckman DxC800).Te iPTH levels were measured by electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Mannheim, Germany).Serum IS and Klotho levels were measured by using an enzyme-linked immunosorbent assay (ELISA) method (Biomedica Co., Vienna, Austria).

Multislice Spiral Computed Tomography.
Te extent of abdominal aortic calcifcation in CKD patients was evaluated through multislice spiral computed tomography (abdominal CT).Tis assessment was conducted independently by two blinded researchers.Te scoring criteria were established as follows: grade 0: the absence of abdominal aortic calcifcation; grade 1: calcifcation occupying less than 1/3 of the abdominal aortic circumference; grade 2: the maximum observed area of calcifcation is greater than 1/3 and less than 1/2 of the abdominal aortic circumference; and grade 3: the maximum observed area of calcifcation on abdominal CT scan was >2/3 of the abdominal aortic circumference.Based on the abdominal aortic calcifcation score, CKD patients were divided into noncalcifcation group (score 0) and calcifcation group (score 1-4).

Statistical Analysis.
Statistical analysis was conducted using SPSS version 26.0 software.Normally distributed measurement data were expressed as mean ± standard deviation (x ± s).Independent sample t-test or one-way analysis of variance was used for intergroup comparison.Te data without normal distribution were represented by median and quartile (Q1-Q3), and intergroup comparisons were performed by using the nonparametric Kruskal-Wallis test.Counting data are expressed as a number of cases (percentage) (cases (%)), and the χ 2 test is used for intergroup comparison.Te risk factors of abdominal aortic calcifcation were analyzed by binary logistic regression analysis.When P < 0.05, the diference was statistically signifcant.3. Te abdominal aortic calcifcation rate of the CKD stages 3-4 group was 25.33%, and that of the CKD stage 5 group was 74.67%, with statistical signifcance (P < 0.01).In addition, the abdominal aortic calcifcation group had higher serum IS levels and lower Klotho levels than those in the nonabdominal aortic calcifcation group, and these diferences were statistically signifcant (P < 0.001).

Discussion
Vascular calcifcation, characterized by abnormal deposition of calcium and phosphorus (hydroxyapatite crystals) on the vascular wall, is common in patients with atherosclerosis, diabetes, and kidney disease and even in very young dialysis patients [7].Importantly, vascular calcifcation stands as an independent risk factor for cardiovascular events and mortality within the CKD patients.In this study, the incidence rate of vascular calcifcation was 69.44% (75/108).Tis high prevalence underscores the need for an in-depth exploration of the mechanism underlying vascular calcifcation and possible therapeutic targets to reduce the incidence of cardiovascular events and mortality in patients with CKD.Te mechanism of vascular calcifcation associated with CKD is very complex.Recent investigations have illuminated the complexity of this biological process, revealing its active involvement in multiple cellular signaling pathways and regulatory factors.Te increase in serum phosphorus level is closely related to vascular calcifcation, but the efect of reducing serum phosphorus concentration by taking phosphorus binding agent is not good.Recent studies have shown that uremic toxin IS and kidney protective factor Klotho protein also play the key role in vascular calcifcation, which is expected to become a new target for the treatment of CKD-related vascular calcifcation.
IS is one of the enterogenic protein-binding uremia toxins that has been studied extensively in recent years.Lin et al. [8] found that serum IS levels gradually increased with the decline of renal function and reached a peak in patients with CKD stage 5 dialysis.Current studies have shown [9][10][11] that IS is related to renal tubule cell injury, renal tubule interstitial fbrosis, cardiac fbrosis, vascular calcifcation, and atherosclerosis.Klotho protein is a protein with powerful antiaging properties.It binds with fbroblast growth factor receptor 1 (FGFR1) and FGF23, which can play a role in regulating the synthesis of PTH and promoting phosphorus excretion [12].Mice defcient in the Klotho gene developed high blood phosphorus and calcifed arteries.Khodeir et al. [13] found that the serum Klotho level of CKD patients was signifcantly lower than that of healthy controls, and the serum Klotho content gradually decreased with the progression of CKD stages.It was found [14] that IS can induce CpG hypermethylation of the Klotho gene in smooth muscle cells, thereby decreasing the expression of the Klotho protein.Animal tests have also shown [15] that IS reduces the expression of Klotho protein in the kidney of hypertentic rats and promotes cell senescence, and IS can downregulate the expression of Klotho in proximal renal tubule cells by activating NF-κB through the production of reactive oxygen species [16].In this study, the serum IS concentration International Journal of Endocrinology gradually increased and reached the highest level in maintenance dialysis patients, while the Klotho protein content gradually decreased, which was consistent with the abovementioned research results.
Several studies [2,17] have shown that IS can promote vascular calcifcation in hypertensive rats.Opdebeeck et al. [18] observed increased aortic calcifcation in a rat model of adenine-induced CKD exposed to IS. Notably, higher IS levels were associated with greater vascular calcifcation in patients with end-stage kidney disease [19].In the uremic environment, IS has also been found to induce CpG hypermethylation of the Klotho gene in smooth muscle cells, silencing these complementary calcifcation inhibitors and predisplacing them to vascular calcifcation [14].Research has unveiled that mice with impaired Klotho gene expression exhibited blood vessel calcifcation, growth retardation, and osteoporosis.Hu et al. [20] reported that α-Klotho protein defciency can cause vascular calcifcation in CKD mice.Zhang et al. [21] showed that soluble alpha-Klotho protein could inhibit calcifcation of human bone marrow mesenchymal stem cells.Chang et al. [22] reported that increasing α-Klotho protein expression through Intermedin1-53 could alleviate vascular calcifcation in CKD rats.Tis study indicated that old age, high level of IS, and low level of Klotho protein were independent risk factors for vascular calcifcation.In addition, the incidence of abdominal aortic calcifcation was higher in CKD stage 5 patients than that in CKD stages 3-4 patients, indicating a gradual increase in calcifcation incidence with CKD stage progression.Tis phenomenon may be attributed to the irreversible loss of renal function, CKD exacerbation, elevated serum IS concentrations, decreased Klotho protein levels, and disturbances in calcium, phosphorus, and PTH, ultimately culminating in vascular calcifcation.
In summary, the prevalence of vascular calcifcation is high in patients with CKD, and serum IS and Klotho protein

Table 1 :
Clinical and Biological Characteristics.Twenty healthy controls were enrolled, with an average age of 49.50 ± 12.31 years and an average BMI of 26.79 ± 4.38 kg/ m 2 .All participants in the control group had no hypertension, diabetes, cardiovascular disease, or abdominal aortic calcifcation.All indicators are within the normal range (Table1).A total of 108 patients with CKD, including Comparison of clinical data of CKD groups.7%) and 50 females (46.3%), with an average age of 55.63 ± 13.51 years and an average BMI of 24.72 ± 3.57 kg/m 2 were selected.Te primary nephritis was chronic glomerulonephritis in 71 cases (65.74%), diabetic nephropathy in 27 cases (25.00%), polycystic kidney disease in 5 cases (4.63%), and other causes in 5 cases (4.63%).
* P < 0.05.CKD, chronic kidney disease; BMI, body mass index; CRP, C-reactive protein; iPTH, intact parathyroid hormone; ALB, albumin; Cr, creatinine.aPvalue,between-groupcomparisons for the three CKD groups (excluding the control group).3.2.Comparison of Serum IS and Klotho Levels in CKDPatients.No statistically signifcant diferences in age were observed among the groups (P > 0.05).However, when compared to the healthy control group, the CKD group exhibited higher serum IS levels and lower Klotho levels (P < 0.05).Furthermore, with decreasing renal function, the serum IS levels progressively increased, peaking in the CKD stage 5 dialysis group, while serum Klotho levels gradually declined, with a statistically signifcant diference (P < 0. 05), as shown in Table2.
, while serum Klotho (OR: 0.002, 95% CI: 0.000-0.690,P�0.037) had a negative impact on abdominal aortic calcifcation.Tese results suggested that advanced age, high serum IS level, and low Klotho level were independent risk factors for abdominal aortic calcifcation, as shown in Table4.

Table 3 :
Analysis of clinical indexes in the group with and without abdominal aortic calcifcation.

Table 4 :
Independent risk factor for abdominal aortic calcifcation.

Table 2 :
Comparison of age, IS, and Klotho protein among groups.Compared with CKD stage 5 dialysis group P < 0.05; b compared with CKD stage 5 nondialysis group P < 0.05; c compared with CKD stages 3-4 dialysis group P < 0.05.IS, indoxyl sulfate; CKD, chronic kidney disease.International Journal of Endocrinology levels are closely related to vascular calcifcation.Te increase in serum IS level and the decrease in Klotho protein level are independent risk factors for vascular calcifcation and may become a new target for the treatment of vascular calcifcation in CKD. a