Association of TGF-β1 with cystatin-C in patients with diabetic nephropathy

Ali Hassan Jasem1, Nazar S. Haddad2, Nassar Taha Yaseen3                   
Author affiliations:
  1. Ali Hassan Jasem, Al-Faiha’a Teaching Hospital, Basra, Iraq; E-mail: alihassan04@yahoo.com
  2. Nazar S. Haddad, Iraqi Board of Medical Specializations In Clinical Chemistry (FIBMS. Chem. Path.), American Association for Clinical Chemistry (FAACC), College of Medicine, University of Basrah, Basrah, Iraq; E-mail: NazarHaddad00@gmail.com
  3. Nassar Taha Yaseen, Consultant Endocrinologist, Faiha Specialized Diabetes, Endocrine and Metabolism Center (FDEMC). TahaYaseen29@yahoo.com
Correspondence: Mohauman Mohammed Majeed, E-mail: muhaimin.alrufaie@uokufa.edu.iq;  Phone: 07809086646

 

ABSTRACT

 

Background & Objective: Diabetes is considered a condition that is characterized by an increase in oxidative stress and inflammation. Transforming growth factor-beta (TGF-β) belongs to the TGF-β subfamily of the TGF-β superfamily. Numerous biological processes, such as cell growth, differentiation, adhesion, proliferation, tissue repair, morphogenesis, and apoptosis, are regulated by the TGF-β subfamily. Five TGF-β isoforms in this subfamily have been found in vertebrates; however, only TGF-β 1-3 has been demonstrated to be expressed in mammals.

Methodology: The current research comprised of a total of 130 individuals, all of whom were placed into one of three primary groups ; 50 (25 males and 25 females) diabetic patients without nephropathy; 50 (25 males and 25 females) diabetic patients with nephropathy as cases, and 30 (15 males and 15 females) persons in good health, who were of the same age as the patients being studied. In order to measure the concentrations of the parameters, the standard procedures and techniques were used.

Results: The mean value of serum TGF-β1was significantly higher in diabetic patients without nephropathy and diabetic patients with nephropathy (40.19 ± 3.56 ng/ml),(51.21 ± 5.20 ng/ml) respectively as compared to controls (24.80 ± 3.51 ng/ml) with a high significant difference (P < 0.01). The level of TGF β1 showed a positive correlation in the study population with fasting blood sugar (r = 0.273, P = 0.006), creatinine (r = 0.546, P = 0.004), and cystatin C (r = 0.597, P = 0.005). The main finding of the present study was that the mean value of serum TGF-β1 in diabetic patients without nephropathy was 40.19 ± 3.56, and for diabetic patients with nephropathy 51.21 ± 5.20, while for controls was 24.80 ± 3.51, a significant difference (P < 0.05). In addition, there was a statistically significant and positive link between the blood TGF 1 levels and the serum creatinine levels of the individuals who participated in the study (r = 0.611, P = 0.003). In addition, patients who had diabetes for a longer period of time (more than five years) had higher levels of TGF β1 than those who had just been diagnosed with the condition. This difference, however, was not statistically significant (P > 0.05), which may be due to the fact that the level of TGF 1 rises with increasing duration of the participant's cases.

Conclusion: There is no significant correlation between cystatin-C with fasting blood sugar. There was a statistically significant and positive link between the blood TGF-β1 levels and the serum creatinine and cystatin-C levels.

Key words: Type-2 Diabetes Mellitus; TGF-β1; Cystatin-C.

Citation: Jasem AH, Haddad NS, Yaseen NT. Association of TGF-β1 with cystatin-C in patients with diabetic nephropathy. Anaesth. pain intensive care 2024;28(1):151−154; DOI: 10.35975/apic.v28i1.2399
Received: October 26, 2023; Reviewed: December 11, 2023; Accepted: December 11, 2023  
 

1. INTRODUCTION

 

The prevalence of diabetes mellitus type 2 (T2DM), which now exceeds 9% globally, is estimated to impact around 463 million people, and is one of the top 10 main causes of mortality.1 This condition has been rising at an exponential rate. Therefore, one of the primary concerns of public health researchers and clinical health professionals2 is the development of methods to postpone the onset of type 2 diabetes mellitus and early treatment techniques to halt its progression. It has been shown that inflammation has a significant role in the development and progression of T2DM, including an increase in the levels of inflammatory biomarkers; hence, the treatment of T2DM should focus on lowering inflammation.3 Diabetes mellitus is a chronic metabolic illness that causes hyperglycemia. This hyperglycemia may be the consequence of a malfunction in insulin synthesis, insulin action, or both. Diabetes mellitus can raise the risk for the development of microvascular as well as macrovascular problems. An increase in oxidative stress and inflammation are two of the hallmarks of diabetes, which is recognized as a disorder in and of itself.

TGF-β is a member of the TGF-β subfamily, which is a part of the TGF-β superfamily.4,5 A wide variety of biological processes are controlled by the TGF-β subfamily, which is engaged in their regulation, such as the development of cells and differentiation, adhesion, proliferation, tissue repair, morphogenesis, and apoptosis.6,7 These are only some of the functions. Only TGF-β isoform 1-3 have been shown to be expressed in mammals,8 despite the fact that there have been five different TGF-β isoforms discovered in vertebrates belonging to this subfamily. The TGF-β1 isoform that is prominent in the kidney is the one that causes responses to be triggered by many signaling pathways. These pathways include SMAD (an acronym from the fusion of Caenorhabditis elegans Sma genes and the Drosophila Mad, Mothers against decapentaplegic), protein mitogen-activated kinases, extracellular regulatory kinase, p38, and Jun kinase9 were examples of proteins that play a role in signal transduction.  Through three mechanisms, renal fibrosis has been firmly linked to TGF-β1 activity in the setting of kidney disease:

(1) A pro-fibrotic phenotype is brought about in the kidneys as a result of an increase in the production of extracellular matrix (ECM) as well as epithelial-mesenchymal transition (EMT).

(2) Increased type I and IV collagen, fibronectin, and laminin synthesis.

(3) The matrix breakdown may be reduced by increasing the expression of matrix metallo-protease inhibitors.10,11,12.
This study aimed to explore correlation between cystatin-C with fasting blood sugar, and a link between the blood TGF-β1 levels and the serum creatinine and cystatin-C levels.

 

2. METHODOLOGY

 

This case-control research was conducted using data obtained at the Faiha Specialised Diabetes, Endocrinology and Metabolism Centre (FDEMC) located in Basrah governorate between November 2022 to May 2023. The study included 100 patients (50 males and 50 females) with T2DM, and 30 controls apparently healthy subjects (15 males and 15 females).

Statistical analysis
The data were represented as means with the standard deviation. The statistical studies were conducted with SPSS, version 26, developed by IBM, United States. A significance level of P < 0.05 was deemed to be statistically significant.

 

3. RESULTS

 

There were 30 healthy controls to compare with the group containing known patients of T2DM, consisting of equal number of male and female patients (Table 1).

The data regarding BMI, FBS, HbA1c, creatinine, cystatin-C and TGF β1 are given in Table 1. BMI was insignificantly higher in the diabetics as compared to the healthy volunteers. The mean value of HbA1c% for diabetic patients was 8.83 ± 1.99. Concerning the serum TGF β1, the mean value of diabetic patients without nephropathy was 40.19 ± 3.56, and for diabetic patients with nephropathy 51.21 ± 5.20, while for controls was 24.80 ± 3.51. A statistically significant and positive association between the levels of blood TGF β1 and serum creatinine (Table 2).

 

4. DISCUSSION

 

Type 2 diabetes mellitus is the prevailing form of diabetes, characterized by a complex interplay of several factors and a persistent metabolic dysfunction. Its incidence has been progressively rising on a worldwide scale. Almost more than 95% worldwide of an individual with DM have T2DM.5
The objective of this research was to assess the use of TGF β1 as a biomarker for diabetic nephropathy in individuals with T2DM. This study linked the association between serum TGF β1, creatinine, cystatin c, blood glucose, HbA1c, and anthropometric measures in patients with T2DM. The mean value of HbA1c for diabetic patients was was lower than the results of other study.6 More than 61% of the patients in the present study had poor glycemic control and only 18%, and 21% of them achieved good and fair glycemic control respectively. Concerning the serum TGF β1, a statistically significant difference was observed (P < 0.05). A similar result was obtained by Shaker et al, who found that there is association between serum TGF β1 to T2DM.7 Moreover, The present investigation revealed a statistically significant and positive association between the levels of blood TGF β1 and serum creatinine within the study cohort (r = 0.546, P = 0.004), (r = 0.611, P = 0.003) respectively, Similar results were revealed by Shaker et al.7 The findings of the current investigation demonstrated that the average value for TGF β1 levels in females was higher but not significant when compared to male participants, as well as a higher significant difference in both males and females when compared with control (P < 0.05), Furthermore, it is worth noting that a statistically insignificant positive connection was seen between TGF β1 levels and gender (r = 0.043, P = 0.674) (Table 3).

 

Table 1: The research sample was distributed based on the biochemical characteristics:
Parameters Controls
(n = 30) 		Cases (n = 100) P-value*
(between groups)
DM DN
Gender Male 15 (11.5) 50 (38.5) -
Female 15 (11.5) 50 (38.5) -
BMI (kg/m2) 26.87 ± 1.92 29.77 ± 5.15 > 0.05⁂
FBS (mg/dl) 98.23 ± 11.42 211.10 ± 68.70 244 ± 92.27 < 0.01
HbA1c (%) 4.91 ± 0.58 8.78 ± 2.22 8.87 ± 1.76 < 0.01
Creatinine (mg/dl) 0.68 ± 0.11 0.81 ± 0.25 3.54 ± 2.44 < 0.01
Cystatin-C (ng/ml) 43.05 ± 8.92 77.02 ± 4.41 90.55 ± 8.06 < 0.01
TGF β1 24.8 ± 3.51 40.19 ± 3.56 51.21 ± 5.20 < 0.01
Data presented as mean ± SD or n (%); P < 0.05 considered as significant
 

Table 2: The use of receiver-operating characteristics (ROC) curve analysis in the diagnosis of Type 2 Diabetes Mellitus (T2DM).
Variables Area under the
ROC curve (AUC)		 Best cut-off Sensitivity (%) Specificity (%)
Cystatin-C 0.970 82.35 88.0 86.0
Serum TGF-β1 0.972 44.45 92.0 94.0
Creatinine 0.998 1.6 94 88.0
 

Table 3: Correlations of serum TGF-β1 levels with biochemical and clinical characteristics.
Variables r-value P-value
Age (y) 0.179 0.264
Gender 0.043 0.674
BMI (kg/m2) 0.202* 0.044
Duration of DM 0.045 0.654
FBG (mg/dL) 0.273** 0.006
HbA1c (%) 0.064 0.527
Cystatin-C 0.597** 0.005
Creatinine 0.546** 0.004
 

The findings of this research indicate that individuals diagnosed with T2DM have notably elevated levels of cystatin C in comparison to the control group (83.79 ± 9.38 vs. 43.05 ± 8.92 ng/ ml). Similar results were revealed by Jeon YL et al.8 They stated that the higher level of cystatin C is most likely due to that the level of cystatin C was strongly correlated with increment in the creatinine level and nephropathy. Jeon YL et al.8 found significantly higher levels of cystatin C in diabetic patients than in controls. The observed increase in the parameter is hypothesized to be attributable to the occurrence of the tubular phase prior to the onset of glomerular symptoms. This observation implies a potential association between serum cystatin C levels and subclinical tubular dysfunction.

Patients with a duration of diabetes exceeding five years exhibited elevated levels of TGF β1 compared to individuals who had recently been diagnosed. Nevertheless, this disparity did not reach statistical significance (P > 0.05), potentially due to the observed trend of TGF β1 levels increasing with the progression of the participants' cases.9
Although the present study revealed a non-significant difference in the level of TGF β1 with respect to diabetic treatment (P > 0.05), as well as the TGF β1 levels were higher in patients who used oral antihyperglycemic drugs (46.72 ± 7.18) and lower in patients who were kept on dietary control (40.55 ± 4.63). These findings were consistent with the results obtained by Shaker et al,7 who demonstrated that the TGF β1 levels had significant association with serum glucose. They suggested that the alteration in TGF β1 levels might be associated with nephropathy. Regarding HbA1c the research investigation revealed that there existed no statistically significant link between the levels of HbA1c and TGF β1 within the examined sample (r = 0.064, P = 0.527). These findings were consistent with the outcomes obtained by Shaker et al.7
The study revealed that serum TGF-β1 correlates positively and significantly with creatinine and cystatin-C. This study also shows that there is no significant correlation between cystatin-C with FBS. These results are consistent with the findings of the research carried out by Shaker et al.7
The current study demonstrated, that there was a significant positive correlation between serum TGF-β1 with creatinine, and cystatin-C (r = 0.546, P = 0.004) and (r = 0.597, P = 0.005) respectively. An increased level of serum TGF-β1 in type 2 diabetic patients may be associated with nephropathy.

 

5. LIMITATIONS

 

We studied a moderate sample size due to strict inclusion criteria, which may have some bearing on the results of the study. A larger sample and inclusion of multiple centers may yield similar results, but these would be more credible.

 

6. CONCLUSION

 

The findings of the present research show that the mean levels of serum TGF-β1 were substantially greater for diabetic individuals suffering from nephropathy than in controls. These results also revealed that serum levels of serum TGF-β1were significantly higher with poorly controlled diabetes patients.

7. Conflict of interest
The authors declare no competing interests.

8. Authors’ contribution
AHJ : writing the manuscript

NSH: conduction of the study work and manuscript editing

NTY: conduction of the study work and manuscript editing

MMMA: Evaluation and sending the manuscript

 

9. REFERENCES

 
  1. Saeedi P, Petersohn I, Salpea P, Malanda B, Karuranga S, Unwin N, et al. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: results from the International Diabetes Federation Diabetes Atlas, 9th edition. Diabetes Res Clin Pract 2019; 157:107843.
  2. Teixeira-Lemos E, Nunes S, Teixeira F, Reis F. Regular physical exercise training assists in preventing type 2 diabetes develop ment: focus on its antioxidant and anti-inflammatory properties. Cardiovasc Diabetol 2011; 10:12.
  3. Berbudi A, Rahmadika N, Tjahjadi AI, Ruslami R. Type 2 diabetes and its impact on the immune system. Curr Diabetes Rev 2020; 16:442-9.
  4. Wrighton, K.H., X. Lin, and X.H. Feng, Phospho-control of TGF-beta superfamily signaling. Cell Res, 2009. 19(1): p. 8-20.
  5. WHO (2017), Diabetes fact sheet, updated 2017 July; htpp://www.who.media centre/factsheets/fs312.
  6. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res. 2010 Oct 29;107(9):1058-70. DOI: 10.1161/CIRCRESAHA.110.223545. PMID: 21030723; PMCID: PMC2996922.
  7. Shaker YM, Soliman HA, Ezzat E, Hussein NS, Ashour E, Donia A, Eweida SM. Serum and urinary transforming growth factor beta 1 as biochemical markers in diabetic nephropathy patients. Beni-Suef Univ J Basic Appl Sci. 2014;3(1)..
  8. Jeon YL, Kim MH, Lee WI, Kang SY. Cystatin C as an early marker of diabetic nephropathy in patients with type 2 diabetes. Clin Lab. 2013;59(11-12):1221-9. DOI: 10.7754/clin.lab.2013.120804. PMID: 24409655.
  9. Hills CE, Squires PE. The role of TGF-β and epithelial-to mesenchymal transition in diabetic nephropathy. Cytokine & Growth Factor Reviews, 2011.
  10. Santibañez, Juan F., M. Quintanilla, and C. Bernabeu, TGF-β/TGF-β receptor system and its role in physiological and pathological conditions. Clinical Science, 2011. 121(6): p. 233-251.
  11. Pelton, R.W., et al., Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J Cell Biol, 1991. 115(4): p. 1091-105.
  12. Hills, C.E. and P.E. Squires, TGF-beta1-induced epithelial-to-mesenchymal transition and therapeutic intervention in diabetic nephropathy. Am J Nephrol, 2010. 31(1): p. 68-74.
  13. Sharma, K. and F.N. Ziyadeh, Biochemical events and cytokine interactions linking glucose metabolism to the development of diabetic nephropathy. Semin Nephrol, 1997. 17(2): p. 80-92.
  14. Wang, W., V. Koka, and H.Y. Lan,Transforming growth factor-beta and Smad signalling in kidney diseases. Nephrology (Carlton), 2005. 10(1): p. 48-56.
  15. Sureshbabu A, Muhsin SA, Choi ME. TGF-β signaling in the kidney: profibrotic and protective effects. Am J Physiol Renal Physiol. 2016 Apr 1;310(7):F596-F606. DOI: 10.1152/ajprenal.00365.2015. Epub 2016 Jan 6. PMID: 26739888; PMCID: PMC4824143.