Urinary Excretion of Biomolecules Related to Cell Cycle, Proliferation, and Autophagy in Subjects with Type 2 Diabetes and Chronic Kidney Disease

Dysregulation of cell cycle, proliferation, and autophagy plays a pivotal role in diabetic kidney disease. In this study, we assessed urinary excretion of molecular regulators of these processes that mediate their effects via the PI3K/AKT/mTOR pathway in subjects with long-term type 2 diabetes (T2D) and different patterns of chronic kidney disease (CKD). We included 140 patients with T2D and 20 non-diabetic individuals in a cross-sectional study. Urinary PTEN, Beclin-1, sirtuin 1 (SIRT1), Klotho, fibroblast growth factor 21 (FGF21), and connective tissue growth factor (CTGF) were assessed using ELISA. Patients with T2D, when compared to control, demonstrated increased excretion of PTEN, Beclin-1, SIRT1, FGF21, CTGF, and decreased urinary Klotho (all p < 0.05). In the diabetic group, PTEN, FGF21, and CTGF were significantly higher in patients with declined renal function, while Klotho was lower in those with elevated albuminuria. FGF21 and PTEN correlated inversely with the estimated glomerular filtration rate. There was a negative correlation between Klotho and urinary albumin-to-creatinine ratio. In multivariate models, Klotho and PTEN were associated with albuminuric CKD independently. The results provide further support for the role of PTEN, BECN1, FGF21, Klotho, and CTGF in development albuminuric and non-albuminuric CKD in diabetes.


Introduction
The prevalence of chronic kidney disease (CKD), as well as type 2 diabetes (T2D), has been rising globally [1][2][3].Moreover, CKD due to T2D and hypertension accounted for the largest disease burden, with 85% incident cases [4].Recent studies have demonstrated the increasing proportion of declined renal function without preceding or accompanying albuminuria in patients with diabetes and CKD [5][6][7].Albuminuric and non-albuminuric CKD patterns demonstrate some differences in their risk factors, clinical course, and biomarker profiles [7][8][9], which suggests the specificity of the underlying molecular mechanisms.
Diabetic kidney disease is considered to be associated with the proliferation of mesangial cells, activation of apoptosis in podocytes and tubular epithelial cells, and suppression of glomerular and tubular autophagy [10,11].These cellular events are mediated by dysregulation of intracellular signaling pathways induced by hyperglycemia and excessive glucose variability [12,13].
The phosphatidylinositol 3-kinases/protein kinase B/mammalian target of the rapamycin (PI3K/AKT/mTOR) signaling pathway plays an essential role in the implementation of the high glucose effect in diabetic kidney disease and renal fibrosis [14][15][16].The PI3K/AKT/mTOR is a crucial pathways in the regulation of the cell cycle and proliferation [17].Dysregulation of the PI3K/AKT/mTOR has been demonstrated in obesity, and SEA010Hu for CTGF (Cloud Clone Corp., Wuhan, China).The results were adjusted to the urinary creatinine concentrations.

Statistical Analysis
The continuous variables were tested for normal distribution with a Shapiro-Wilk test (SPSS Statistics, IBM, Armonk, NY, USA).As most of the studied parameters were not distributed normally, the data are presented as medians and interquartile ranges (IQRs).The statistical significance of differences between groups were tested with Mann-Whitney U-tests for comparison of two groups.We used Kruskal-Wallis H-test for multiple group comparisons.The χ2 test was applied for categorical data (SPSS Statistics, IBM, Armonk, NY, USA).The differences were noted as significant with p-value below 0.05.
The associations between continuous parameters were assessed with a Spearman correlation analysis (Statistica 13.0, Dell, Round Rock, TX, USA).The associations of urinary excretion of PTEN, BECN1, SIRT1, FGF21, Klotho, and CTGF with declined renal function and elevated albuminuria were tested in receiver operating characteristic (ROC) analysis (SPSS Statistics, IBM, Armonk, NY, USA) and in multiple logistic regression models (Statistica 13.0, Dell, Round Rock, TX, USA).

Clinical Characteristics of the Study Participants
Clinical characteristics of patients are presented in Table 1.We found some differences in age, diabetes duration, and HbA1c levels between the groups.Patients in DRF/NA group were older; those in DRF/EA group had the longest diabetes duration.The highest levels of HbA1c were observed in NRF/EA group.All patients received antihyperglycemic medications, including metformin (n = 105), sulfonylurea (n = 55), dipeptidyl peptidase-4 (DPP4) inhibitors (n = 18), glucagon-like peptide-1 (GPL-1) analogues (n = 2), sodium/glucose cotransporter 2 (SGLT2) inhibitors (n = 35) and insulin (n = 96).Most patients (n = 115) were treated with renin-angiotensin system blockers.Compared to other groups, lower proportion of DF/EA patients received metformin, while calcium channel blockers were used more frequently in this group (Table 2).There were no significant differences in other treatment modalities between diabetic groups.The median age of control subjects was 62.5 years (IQR 59.5-67.4years), and median body mass index (BMI) was 25.7 kg/m 2 (IQR 24.7-28.6kg/m 2 ).There were no significant differences between control and diabetic subjects in these parameters.
We found no associations of the excretion of the studied molecules with coronary artery disease, chronic heart failure, and myocardial infarction in medical history, as well as with smoking status.

Associations between the Urinary Biomarkers and CKD: Univariate Models
In ROC analysis, PTEN ≥ 1.10 ng/mmol was associated with both eGFR < 60 mL/min × 1.73 m 2 or UACR ≥ 3.0 mg/mmol (Table 3).A reduced eGFR was also associated with high urinary CTGF and FGF21.We found cut-off points for urinary BECN1 and FGF21 as factors associated with DF/NA, as well as for CTGF and FGF21 as factors associated with DF/EA.

Associations between the Urinary Biomarkers and CKD: Multivariate Models
After adjustment for age, sex, BMI, duration of diabetes, and HbA1c, urinary Klotho was significantly associated with UACR ≥ 3.0 mg/mmol in patients with T2D (OR = 0.96, 95% CI 0.93-0.9996for each 10 ng/mmol of urinary Klotho, p = 0.048, Table 4).In a similar model, urinary PTEN was associated with DRF/EA (OR = 6.23, 95% CI 1.43-27.1,p = 0.01, Table 5).We failed to build any model for eGFR < 60 mL/min × 1.73 m 2 , as well as for DRF/NA and NRF/EA patterns of CKD, where urinary excretion of the assessed molecules was significant after adjustment for age, sex, BMI, diabetes duration, and HbA1c.

Discussion
In this study, we have assessed the urinary excretion of biomolecules related to cell cycle, proliferation, and autophagy (PTEN, BECN1, SIRT1, FGF21, Klotho, and CTGF) in patients with long-term T2D and different patterns of CKD.The results indicate that the studied regulators are differently related to albuminuria, renal function, and CKD phenotype.Specifically, urinary Klotho is associated with elevated albuminuria, while high excretion of FGF21, CTGF, and PTEN are associated with decreased renal function.We found elevated excretion of BECN1 in non-albuminuric T2D subjects, while Klotho and PTEN showed independent associations with albuminuric CKD patterns.

PTEN
We found high levels of urinary PTEN in patients with long-term T2D.The increased PTEN excretion adjusted to age, sex, BMI, diabetes duration, and HbA1c was associated with both declined eGFR and elevated UACR.
PTEN is a suppressor of the PI3K/AKT/mTOR signaling pathway and a crucial regulator of cell death, autophagy, and apoptosis [17,23,24].In the human kidney, PTEN is expressed at a low or medium level in the tubular cells, and expressed at a low or zero level in the glomeruli [36].PTEN is considered to be an antagonist of the transforming growth factor beta (TGF-β) signaling pathway [37].The down-regulation of PTEN in podocytes was found in both clinical and experimental diabetes [38][39][40].At the same time, it was demonstrated that high renal PTEN may increase expression of fibrogenic agents, such as TGF-β and CTGF, and promote epithelial-mesenchymal transition [41].
The exact mechanism of the increased urinary excretion of PTEN in diabetes is not clear.Assuming intracellular localization of PTEN, one can speculate that the elevated urinary excretion of this molecule could be a result of renal cell injury.

BECN1
We observed increased urinary excretion of BECN1 in patients with T2D, especially in those with non-albuminuric CKD.
BECN1 is an essential regulator of autophagy that signals the onset of the process [42].A medium level of BECN1 expression in the renal glomeruli and tubules was described [36].Similarly to PTEN, elevation of BECN1 excretion may be a consequence of an injury of the renal cells.Previously, we found a decreased glomerular expression of BECN1 in db/db mice, a model of T2D [43].Accordingly, serum level of BECN1 was reported to be reduced in patients with T2D and CKD [44].
Further studies are needed to clarify changes in the synthesis of PTEN and BECN1 in diabetic kidneys and their role in increasing albuminuria and reducing renal function.

SIRT1
In this study, we revealed elevated urinary SIRT1 excretion in patients with T2D.However, we found no differences in SIRT1 excretion between groups of patients with different patterns of CKD.
In the kidney, SIRT1 is expressed in podocytes and proximal tubular epithelial cells [45].It was demonstrated that renal SIRT1 inhibits cell apoptosis, inflammation, and fibrosis [45,46].Therefore, activation of SIRT1 can be a protective mechanism in diabetic kidney disease [46].The synthesis of SIRT1 in the diabetic kidney and its relationship with the urinary excretion of the molecule requires further research.

Klotho
The obtained results demonstrate the association between a decrease in the urinary excretion of Klotho and the elevation of albuminuria in patients with T2D.
Previously, decreased serum levels of Klotho were found in patients with T2D [47,48].However, a study including 13,751 subjects from the National Health and Nutrition Examination Survey (NHANES) database revealed higher prevalence of T2D in people with serum levels of Klotho in the lower or upper quartiles (Q1, Q4) compared to those with Klotho in Q2 or Q3.This study also demonstrated lower eGFR in participants with serum levels of Klotho below 993.25 pg/mL [49].The independent associations between serum levels of Klotho and eGFR as well as HbA1c were describes previously [50].
In the kidney, Klotho expresses in tubular cells predominantly [36].The downregulation of the renal Klotho was reported in kidney diseases [51][52][53].In patients with CKD, a reduction of renal α-Klotho expression was associated with CKD progression [51,54].Epigenetic modifications of Klotho gene promoters under the influence of serum uremic toxins can be responsible for the Klotho down-regulation [55].
Klotho protein is considered to have renoprotective activity as an antagonist of the TGF-β signaling pathway [37].In animal models and cell cultures, Klotho deficiency enhanced renal inflammation and promoted fibrosis, while treatment with Klotho analogues mitigated these changes [56][57][58].
The associations between low serum and urinary Klotho and albuminuria in patients with T2D were noted in previous studies [48,59,60].Some experimental data may help explain the mechanism of this relationship.It was demonstrated that albumin directly decreased Klotho mRNA and protein expression in cultured murine and human tubular cells [60].This effect is mediated by endoplasmic reticulum stress and epigenetic modifications [60,61].In albumiuria models, Klotho-ameliorated endoplasmic reticulum stress, decreased Ca 2+ influx, and reduced cytoskeleton remodeling in podocytes [62,63].
Therefore, decreased urinary excretion of Klotho can be considered as a possible risk factor for albuminuric CKD in diabetes.

FGF21
We found associations between increased excretion of FGF21 and declined renal function.
FGF21 is expressed in the liver predominantly, and is secreted to blood; FGF21 expression was not detected in the kidney [36].However, elevated serum FGF21 was found in patients with diabetic CKD [64,65].
In diabetes, FGF21 is commonly considered to be a protective factor due to its insulinsensitizing and anti-inflammatory effects [66,67].Recent studies have demonstrated antifibrotic activity of FGF21 in the kidney [68][69][70].It was revealed that FGF21 needs to interact with Klotho in order to activate the FGF receptor [28].Therefore, it can be assumed that a decrease in the renal production of Klotho in CKD contributes to FGF21 resistance and diminishes its protective effect.

CTGF
In our study, patients with declined renal function demonstrated higher urinary CTGF, regardless of the elevation of albuminuria.
Previously, elevated serum levels of CTGF were found in patients with diabetic CKD [71,72].Under normal conditions, CTGF is expressed in the glomerular and tubular cells at a low level [36].High glucose up-regulates CTGF in the proximal tubular cells [73], as well as in the mesangial and mesenchymal cells [74][75][76].CTGF is considered to be a fibrogenic agent in diabetic kidney disease [77][78][79].
Therefore, the elevated urinary excretion of CTGF could be an indicator of renal fibrogenesis in T2D patients with decreased renal function.

Dysregulation of the PI3K/AKT/mTOR Pathway in the Pathogenesis of Diabetic CKD
A growing body of evidence indicates the role of the PI3K/AKT/mTOR signaling pathway in the pathogenesis of diabetic kidney disease [14,80,81].Specifically, this signaling pathway is responsible for the activation of apoptosis, suppression of autophagy, and the epithelial-mesenchymal transformation of podocytes and tubular cells, proliferation of mesangial cells, and fibrogenesis.In agreement with these data, some novel pharmacological agents modulating the PI3K/AKT/mTOR pathway showed protective effects in diabetic kidney [82][83][84].
In this study, we tested hypothesis that molecules influencing the PI3K/AKT/mTOR signaling can be perspective biomarkers of albuminuric and/or non-albuminuric CKD in T2D.The results indicate that decreased urinary excretion of Klotho is related to increased albuminuria, FGF21, and CTGF are associated with declined renal function, PTEN is associated with both albuminuria and decreased eGFR, and BECN1 is associated with non-albuminuric CKD.It can be assumed that the pathogenetic significance of the studied regulators for increasing albuminuria and decreasing renal function is not the same.

Limitations of the Study and Future Remarks
This study is not without limitations.First of all, due to the variability of the eGFR and UACR, some patients may have been misclassified with CKD patterns.The limited sample size and cross-sectional design are other obvious limitations.Morphological verification of kidney pathology was not carried out.
the activation of the PI3K/AKT/mTOR pathway.A schematic representation of the role of PI3K/AKT/mTOR signaling in diabetic kidney disease is shown in Figure 2. In this study, we tested hypothesis that molecules influencing the PI3K/AKT/mTOR signaling can be perspective biomarkers of albuminuric and/or non-albuminuric CKD in T2D.The results indicate that decreased urinary excretion of Klotho is related to increased albuminuria, FGF21, and CTGF are associated with declined renal function, PTEN is associated with both albuminuria and decreased eGFR, and BECN1 is associated with non-albuminuric CKD.It can be assumed that the pathogenetic significance of the studied regulators for increasing albuminuria and decreasing renal function is not the same.

Limitations of the Study and Future Remarks
This study is not without limitations.First of all, due to the variability of the eGFR and UACR, some patients may have been misclassified with CKD patterns.The limited sample size and cross-sectional design are other obvious limitations.Morphological verification of kidney pathology was not carried out.
However, to the best of our knowledge, this is the first study examining the excretion of biomolecules influencing the PI3K/AKT/mTOR signaling pathway in albuminuric and non-albuminuric CKD in T2D.Future translational studies are needed to clarify the pathogenetic role of the studied molecules in albuminuric and non-albuminuric diabetic CKD.The significance of the studied molecules as predictors of decreased renal function and increased albuminuria deserves testing in prospective study.However, to the best of our knowledge, this is the first study examining the excretion of biomolecules influencing the PI3K/AKT/mTOR signaling pathway in albuminuric and non-albuminuric CKD in T2D.Future translational studies are needed to clarify the pathogenetic role of the studied molecules in albuminuric and non-albuminuric diabetic CKD.The significance of the studied molecules as predictors of decreased renal function and increased albuminuria deserves testing in prospective study.
High glucose, reactive oxygen species (ROS), fibrogenic growth factors (TGF-β, CTGF), elevated SIRT1, decreased Klotho, PTEN, and BECN1, as well as impaired FGF21 signaling, can contribute to the activation of PI3K/AKT/mTOR.In its turn, activation of the PI3K/AKT/mTOR is important for apoptosis promoting, autophagy suppression, and epithelial-mesenchymal transition in the podocyte and tubular cells, proliferation of mesangial cells, and fibrosis.

Conclusions
In patients with long-term T2D, urinary PTEN, BECN1, FGF21, Klotho, and CTGF, the molecules that are involved in the regulation of cell cycle, proliferation, and autophagy, are associated differently with albuminuric and non-albuminuric patterns of CKD.Specifically, decreased urinary Klotho is related to increased albuminuria, FGF21 and CTGF are associated with declined renal function, PTEN is associated with both albuminuria and decreased eGFR, and BECN1 is associated with non-albuminuric CKD.The results provide further support for the role of PTEN, BECN1, FGF21, Klotho, and CTGF in the pathogenesis of diabetic kidney disease, and highlight the differences in the molecular pathways of albuminuric and non-albuminuric CKD in diabetes.

Figure 2 .
Figure 2. A schematic representation of the possible involvement of PTEN, BECN1, FGF21, Klotho, CTGF, and SIRT1 in the pathogenesis of diabetic kidney disease.PTEN, phosphatase and tensin homolog; BECN1, Beclin-1; FGF21, fibroblast growth factor 21; CTGF, connective tissue growth factor; SIRT1, sitruin 1; TGF-beta, transforming growth factor beta; ROS, reactive oxygen species; PI3K/AKT/mTOR, phosphatidylinositol 3-kinases/protein kinase B/mammalian target of the rapamycin.Red upward arrow indicates increased production of a molecule or activation of a process.Red downward arrow indicates decreased production of a molecule or suppression of a processes.

Figure 2 .
Figure 2. A schematic representation of the possible involvement of PTEN, BECN1, FGF21, Klotho, CTGF, and SIRT1 in the pathogenesis of diabetic kidney disease.PTEN, phosphatase and tensin homolog; BECN1, Beclin-1; FGF21, fibroblast growth factor 21; CTGF, connective tissue growth factor; SIRT1, sitruin 1; TGF-beta, transforming growth factor beta; ROS, reactive oxygen species; PI3K/AKT/mTOR, phosphatidylinositol 3-kinases/protein kinase B/mammalian target of the rapamycin.Red upward arrow indicates increased production of a molecule or activation of a process.Red downward arrow indicates decreased production of a molecule or suppression of a processes.

Table 1 .
Clinical characteristics of patients with T2D depending on CKD status.

Table 2 .
Treatment of patients with T2D and different CKD status.

Table 3 .
Associations of urinary biomarkers with CKD in patients with T2D: The results of ROCanalysis.

Table 5 .
The logistic regression model for DRF/EA pattern of CKD.