Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive
Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Comparative Effects of Nigella sativa and Thymoquinone on Glucose Metabolism in a High Glucose-induced Renal Cell Line

A
A. Usta1,*
S
S. Çetin2
V
V. Yüksek3
S
S. Dede4
1Van Yuzuncu Yil University, Faculty of Science, Department of Chemistry, Van, Türkiye.
2Ankara Yildirim Beyazit University, Vocational School of Health Services, Department of Veterinary Medicine, Ankara, Türkiye.
3Van Yuzuncu Yil University, Ozalp Regional High School, Van, Türkiye.
4Van Yuzuncu Yil University, Faculty of Veterinary Medicine, Biochemistry Department, Van, Türkiye.

ABSTRACT

Background: A significant side effect of type 2 diabetes mellitus is diabetic nephropathy (DN), which is typified by poor glucose homeostasis and insulin resistance in renal tissues. Thymoquinone (TQ), the active ingredient in Nigella sativa (NS), has been shown to have antioxidant and antidiabetic properties. However, nothing is known about their relative effects on renal glucose metabolism in hyperglycemic situations. The objective of this work was to examine and contrast the regulatory effects of NS and TQ on insulin signaling pathways and glucose transporter expression in NRK-52E renal epithelial cells grown in high-glucose environments.

Methods: TQ (10 µM) or NS extract (0.5 µg/ml) were applied to NRK-52E cells after they were subjected to high glucose (246 mM). RT-qPCR was used to measure the mRNA expression levels of insulin signaling components (Ir, Irs1, Pi3k, Ampk, Gsk3β) and glucose transporters (Glut1, Glut2, Glut3, Glut5, Sglt2). Western blot was used to measure the protein levels of Glut2, Glut3, Sglt2 and Irs1. The Duncan test (p<0.05) and one-way ANOVA were used to determine statistical significance.

Result: Ir, Irs1, Pi3k and Ampk levels were downregulated and Glut1, Glut2, Glut3 and Sglt2 expression was markedly elevated by high glucose exposure. These changes were reversed by TQ and NS therapy, with TQ having a stronger effect. TQ increased the levels of Irs1 and Pi3k while more successfully suppressing the expression of Sglt2 and Glut2. The transcriptional alterations were confirmed by Western blot results. TQ and NS improved insulin signaling pathways and modulated glucose transporter expression to protect renal cells. These results confirm that TQ and NS are safe to use as possible therapeutic agents to treat diabetic nephropathy.

INTRODUCTION

Diabetic nephropathy is an important side effect of diabetes that can lead to chronic kidney failure and significantly reduce a patient’s quality of life. Vascular injury in intraglomerular arterioles is the main way that persistent hyperglycemia causes renal failure. It is thought to affect about 15% of patients with type 2 diabetes and 35% of those with type 1 diabetes (Eraslan Farisoğlu et al., 2026; Ozcan and Kaya, 2025; Raj and Rani, 2024).
       
Diabetes raises the risk of organ failure and persistent tissue damage due to impaired glucose regulation (Keembiyehetty et al., 2006). Cellular function depends on the movement of glucose across cell membranes, which is accomplished by either active or assisted diffusion. Tissue-specific expression of sodium-glucose cotransporters (Sglts) and glucose transporters (Gluts) occurs in the kidney. Sglt2, which is found on the apical membrane of proximal tubular cells, is principally in charge of sodium-dependent glucose reabsorption, which uses basolateral Glut transporters to move glucose into the bloodstream (Norton et al., 2017; Bailey et al., 2010).
       
Gluts comprise 14 isoforms that are categorized into three classes and are widely expressed in mammalian cells (Augustin, 2010; Mueckler et al., 2013). Hormonal, metabolic and oxidative stress-related factors impact their expression, which varies according to tissue type and pathological situations such diabetes, ischemia and cancer (Rizzo et al., 2013; Medina and Owen, 2002).
       
Insulin primarily uses the Pi3k/Akt and MAPK signaling pathways to control glucose homeostasis by activating the insulin receptor (Ir) and insulin receptor substrate (Irs) (Cardoso, 2009). Irs1 phosphorylation is essential for Pi3k activation, which increases glucose absorption through Glut translocation, especially Glut4 (Lee et al., 2012). Additionally, Ampk is a key target for treatment in type 2 diabetes and plays a crucial role in glucose metabolism (Bijland et al., 2013; Manna et al., 2017; Park et al., 2018).
       
Glut2-mediated glucose absorption in pancreatic β-cells raises intracellular ATP levels, which in turn cause membrane depolarization and calcium influx, which in turn stimulate insulin production (Champe and Harvey 2007). Furthermore, a crucial regulator of glycogen metabolism, glycogen synthase kinase 3 beta (Gsk3β), is still active in diabetes, which inhibits glycogen synthesis and lowers cellular glucose absorption.
       
In diabetic patients, Nigella sativa (NS) has been shown to enhance inflammatory indicators, oxidative stress, insulin resistance, lipid profiles and glycemic management. Additionally, it improves antioxidant capacity and β-cell activity (Shan et al., 2011; O’Neill, 2013; Hamdan et al., 2019; Kaatabi et al., 2015; Chatterjee et al., 2025; Heshmati et al., 2015). The primary bioactive component of NS, thymoquinone (TQ), has shown protective properties in diabetic mice by lowering inflammation, apoptosis and oxidative stress, in part by modifying the Pi3k/Akt pathway (Liu et al., 2016; He et al., 2023; Usta and Dede, 2017).
       
Despite these results, the effects of NS and TQ on renal glucose metabolism are still unknown and changes in glucose transporters under metabolic diseases are not completely understood. Therefore, by examining the mRNA and protein expression levels of important genes involved in glucose metabolism, such as Glut1, Glut2, Glut3, Glut5, Sglt2, Ir, Irs1, Pi3k, Ampk and Gsk3β, this study sought to determine how NS and TQ affected renal proximal tubular cells (NRK-52E) cultured under high-glucose conditions.

MATERIALS AND METHODS

Reagents and chemicals
 
Thymoquinone (TQ) and D-glucose were acquired from Sigma-Aldrich (USA). Western blot kits and antibodies were purchased from Affinity Biosciences (USA). The ABT 2x SYBR Green Master Mix (Türkiye) was used for RT-qPCR studies.
 
Cell culture
 
NRK-52E (ATCC® CRL-1571TM), a rat kidney epithelial cell line, was grown in RPMI-1640 media supplemented with 1% L-glutamine, 1% penicillin/streptomycin and 10% fetal bovine serum (Capricorn, Germany). The cells were kept in a humidified environment with 5% CO2 at 37oC (Esco CelCulture CO2, Singapore). The Van Yuzuncu Yil University Scientific Research Projects Coordination Unit project TYD-2020-8582 provided funding for all of the experiments, which were carried out in 2022 at the Cell Culture and Molecular  Biology Laboratory in the Biochemistry Department of the Faculty of Veterinary Medicine at Van Yuzuncu Yil University.
 
Preparation of solutions
 
In order to make Nigella sativa (NS) extract, 70% of the powdered seeds were extracted using methanol, filtered, the solvent was evaporated, the mixture was lyophilized and it was then stored at -20°C (Meziti et al., 2012). TQ and NS stock solutions were made in DMSO (Panreac Applichem, Spain) (<0.05%) and then diluted in culture medium. As previously stated, amounts of glucose (0-400 mM), NS (0-200 µg/ml) and TQ (0-100 µM) were prepared (Gümüş et al., 2018; Celik, 2013; Usta et al., 2024a).

MTT assay
 
Following the seeding of 1×104 cells per well in 96-well plates, the cells were incubated overnight. The MTT assay was used to evaluate cell viability following treatment. The formazan crystals made by metabolically active cells were dissolved and the absorbance at 570 nm was determined (Biochrom, Anthos Zenyth 200). The viable cell percentage was contrasted with the control (Usta et al., 2024b; Meerloo et al., 2011).
 
Experimental design
 
Treatment groups were established based on different concentrations of glucose, TQ and NS, as indicated in Table 1.

Table 1: Study groups based on the administration of glucose, TQ and NS to NRK-52E cells.


 
RNA isolation and cDNA synthesis
 
Each group received a 24-hour treatment after being planted with about 8×105 cells. Total RNA was isolated using Trizol reagent (GeneAll, Korea) and its purity was assessed by spectrophotometric analysis (Chomczynski and Mackey, 1995). The manufacturer’s instructions were followed for cDNA synthesis using a commercial kit (ABT High Capacity, Türkiye).
 
RT-qPCR analysis
 
The findings of RT-qPCR investigation of Sglt2, Glut1, Glut2, Glut3, Glut5, Ir, Irs1, Gsk3β, Pi3k and Ampk gene expression levels are displayed in Table 2 (Qiagen, Germany).

Table 2: Target gene primers’ base sequence.


       
Using β-actin as the internal reference, relative expression levels were determined using the 2- ΔΔCt approach (Livak and Schmittgen, 2001) (Table 3).

Table 3: RT-qPCR reaction conditions.


 
Western blot analysis
 
After the proteins were extracted (GeneAll, Korea) and quantified using the Bradford procedure (ABT Biosciences, Türkiye), they were separated using SDS-PAGE. After being moved to nitrocellulose membranes, samples were incubated with primary and secondary antibodies. Protein bands were seen using a chemiluminescent substrate and analysis was done using ImageJ software.
 
Statistical analysis
 
SPSS 22.0 was used for statistical analysis (IBM, USA). The Duncan test was used after one-way ANOVA and Kruskal-Wallis to assess group differences. Mean±standard deviation was used to express the data and p<0.05 was regarded as statistically significant.

RESULTS AND DISCUSSION

Effects of TQ, NS and glucose on cell viability
 
TQ, NS and glucose concentrations that were cytotoxic and proliferative in NRK-52E cells were measured using the MTT test (Fig 1). The best proliferative doses were determined to be TQ (10 μM) and NS (0.5 μg/ml), which both markedly improved cell viability (p<0.05). In the experiments that followed, the cytotoxic concentration of glucose was established to be 246 mM based on its IC50 value.

Fig 1: After administering different TQ, NS and glucose concentrations to the NRK-52E cell line for 24 hours.


 
Effects on glucose transporter gene expression
 
High glucose considerably increased the expression of Glut1, Glut2, Glut3 and Sglt2 in comparison to the control, according to RT-qPCR study (Fig 2). These increases were reversed by TQ and NS treatment, bringing expression levels back to nearly control levels. Nonetheless, the treated groups’ Glut2 expression was still lower than the control. All experimental groups had lower levels of Glut5 expression than the control.

Fig 2: (a-e) Glut1, Glut2, Glut3, Glut5 and Sglt2 mRNA transcription levels in NRK-52E cells after 24 hours.


 
Effects on Ir and Irs1 expression
 
Ir and Irs1 were downregulated in comparison to the control in high glucose circumstances (Fig 3). Both genes’ expression was elevated by TQ and NS treatments in comparison to the glucose group. Irs1 levels were brought back to almost control levels in treatment groups, but Ir expression remained below control.

Fig 3: (a-b) Irs1 and Ir mRNA transcription levels in NRK-52E cells after 24 hours.



Effects on Pi3k, Gsk3β and Ampk expression
 
Under high glucose circumstances, Pi3k and Ampk expression levels decreased; however, after TQ and NS treatment, they either increased or were recovered (Fig 4). While there was no discernible difference between the treatment groups and the control, Gsk3β expression was increased in the glucose group. On the other hand, Gsk3β expression was decreased by TQ and NS alone.

Fig 4: (a-c) Pi3k, Gsk3â and Ampk mRNA transcription levels at 24 hours in NRK-52E cells.


 
Protein expression analysis
 
The glucose-treated group had higher amounts of Glut2 protein than the control group, according to Western blot analysis and these levels dropped after TQ and NS treatment (Fig 5). In general, all groups had lower levels of other target proteins than the control. These results most likely represent early transcriptional responses at 24 hours, since longer exposure times may be necessary for protein-level alterations (48-72 h).

Fig 5: (a-d) Glut2, Glut3, Sglt2 and Irs1 protein translation levels in the NRK-52E cell line after 24 hours.


       
In this investigation, NRK-52E cells subjected to rising glucose concentrations for a full day demonstrated a dose-dependent decline in viability, with an IC50 of 246 mM, simulating advanced hyperglycemic stress in line with other findings. (Liu et al., 2016; Gholamnezhad et al., 2016). This method allowed for the assessment of renal tubular cells’ impaired insulin signaling and glucose-induced cytotoxicity.
       
Thymoquinone (TQ) is one of the main bioactives found in Nigella sativa (NS) extract. Both have been shown to have anti-inflammatory, anti-apoptotic, antioxidant and antidiabetic properties (Sangi et al., 2015; Ali and Blunden, 2003).
       
Sglt2 reabsorbs approximately 90% of filtered glucose in proximal tubules, while Glut1/Glut2 facilitates translocation to the circulation. Sglt and Glut transporters control glucose uptake (Bell et al., 1990; Iancu et al., 2022; Watson and Pessin, 2001; Umino et al., 2018; Girard, 2017; Marks et al., 2003; Mather and Pollock, 2011). Hyperglycemia enhances transporter expression and renal glucose reabsorption, which exacerbates glucose toxicity (Abdul-Ghani et al., 2011; Holman, 2020; Vestri et al., 2001; Rahmoune et al., 2005; Defronzo, 2009; Liu et al., 2012). High glucose increased Glut1, Glut2, Glut3 and Sglt2 in our investigation, which is consistent with the literature. However, TQ and NS therapy brought these levels down to almost control levels. All groups showed low levels of Glut5, which is consistent with its restricted role in the kidneys (Adeshara et al., 2017; Gnudi et al., 2007;  Brosius and Heilig, 2005; Vallon et al., 2011; Sugawara-Yokoo et al., 1999; Rand et al., 1993; Inukai et al., 1993).
       
Insulin signaling via the downstream Pi3k/Akt pathway and Ir/Irs1 is essential for maintaining glucose homeostasis. Elevated glucose inhibited the production of Ir and Irs1, which hindered insulin signaling and decreased the absorption of glucose. The expression of Pi3k/Akt and Irs1 was restored by TQ and NS, indicating improved glycogen production and insulin signaling (Tiwari et al., 2013; Peng and He, 2018; Mokashi et al., 2017; Linnemann et al., 2014; Gatica et al., 2013; Lay et al., 2024; Mima et al., 2011; Mima et al., 2023; Lay and Coward, 2018; Chen et al., 2022).
       
Under high glucose, AMPK, a crucial regulator of energy balance, was inhibited, which increased oxidative stress and insulin resistance (Coughlan et al., 2014; Soetikno et al., 2013; Han et al., 2021; Welsh et al., 2010). TQ and NS improved glucose utilization and reversed insulin resistance via restoring AMPK expression. In a similar vein, Gsk3β, which rises in diabetes and prevents the synthesis of glycogen, was increased in high glucose but repressed by TQ and NS, promoting better glycogen metabolism and decreased oxidative stress (Liang et al., 2020; Rayasam et al., 2009; Paeng et al., 2014).
       
Together, our results show that TQ and NS influence several pathways related to glucose metabolism, such as glucose transporters, Ir/Irs1, Pi3k/Akt, AMPK and Gsk3β (Fig 6). Their promise as treatment agents for diabetic nephropathy is highlighted by these effects, which improve insulin sensitivity and reduce hyperglycemia. To validate these protective effects, further extensive in vivo and clinical research is necessary.

Fig 6: Impact of TQ and NS supplementation on the downstream pathways of glucose metabolism in high-glucose environments.

CONCLUSION

In NRK-52E renal proximal tubular cells, high glucose exposure reduces cell survival, interferes with insulin signaling, and modifies glucose transporter expression. By lowering the expression of Glut1, Glut2, Glut3, and Sglt2, increasing Ir/Irs1, Pi3k/Akt, and AMPK signaling, and suppressing Gsk3β activity, treatment with Nigella sativa (NS) extract and thymoquinone (TQ) reduced these effects and promoted glycogen synthesis and glucose utilization.
       
TQ’s potential as a therapeutic agent for avoiding or lessening diabetic nephropathy was highlighted by its better effectiveness than NS in repairing impaired glucose transporter expression and metabolic signaling. These results shed light on the mechanisms behind NS and TQ’s renoprotective and antidiabetic actions. To confirm and expand these protective benefits, more in vivo and clinical research is necessary.

ACKNOWLEDGEMENT

We would like to thank Van Yuzuncu Yil University Scientific Research Projects Coordination Unit (TYD-2020-8582) for their contributions.

CONFLICT OF INTEREST

The authors declares no conflicts of interest to report regarding the present study.

REFERENCES


  1. Abdul-Ghani, M.A., Norton, L. and Defronzo, R.A. (2011). Role of sodium- glucose cotransporter 2 (Sglt 2) inhibitors in the treatment of type 2 diabetes. Endocr Rev. 32(4): 515-531. https://doi.org/10.1210/er.2010-0029.

  2. Adeshara, K.A., Diwan, A.G., Jagtap, T.R., Advani, K., Siddiqui, A. and Tupe, R.S. (2017). Relationship between plasma glycation with membrane modification, oxidative stress and expression of glucose transporter-1 in type 2 diabetes patients with vascular complications. J. Diabetes Complications. 31(2): 439-448. https://doi.org/10.1016/j.jdiacomp.2016.10.012.

  3. Ali, B.H. and Blunden, G. (2003). Pharmacological and toxicological properties of Nigella sativa. Phytother Res. 17: 299-305. https://doi.org/10.1002/ptr.1309.

  4. Augustin, R. (2010). The protein family of glucose transport facilitators: It’s not only about glucose after all. IUBMB Life. 62(5): 315-333. https://doi.org/10.1002/iub.315.

  5. Bailey, C.J., Gross, J.L., Pieters, A., Bastien, A. and List, J.F. (2010). Effect of dapagliflozin in patients with type 2 diabetes who have inadequate glycaemic control with metformin: A randomised, double-blind, placebocontrolled trial. Lancet. 375(9733): 2223-2233. https://doi.org/10.1016/S0140-6736(10)60407-2.

  6. Bell, G.I., Kayano, T., Buse, J.B., Burant, C.F., Takeda, J., Lin, D.,  Fukumoto, H and Seino, S. (1990). Molecular biology of mammalian glucose transporters. Diabetes Care. 13(3): 198-208. https://doi.org/10.2337/diacare.13.3.198.

  7. Bijland, S., Mancini, S.J. and Salt, I.P. (2013). Role of AMP-activated protein kinase in adipose tissue metabolism and inflammation. Clin Sci. 124(8): 491-507. https://doi.org/10.1042/CS20120536.

  8. Brosius, F.C. and Heilig, C.W. (2005). Glucose transporters in diabetic nephropathy. Pediatr Nephrol. 20(4): 447-451. https:// doi.org/10.1007/s00467-004-1748-x.

  9. Cardoso, S., Correia, S., Santos, R.X. et al. (2009). Insulin is a two-edged knife on the brain. J. Alzheimers Dis. 18(3): 483-507. https://doi.org/10.3233/JAD-2009-11.

  10. Champe, P.C. and Harvey, R.A. (2007). Lippincott’s Illustrated Reviews Series.

  11. Chatterjee, G., Saha, A.K., Khurshid, S. and Saha, A.A. (2025). Comprehensive Review of the antioxidant, antimicrobial and therapeutic efficacies of black cumin (Nigella sativa L.) seed oil and its thymoquinone. J. Med Food. 28(4): 325-339. https://doi.org/10.1089/jmf.2024.k.0149.

  12. Chen, X., Wang, J., Lin, Y., Liu, Y. and Zhou, T. (2022). Signaling pathways of podocyte injury in diabetic kidney disease and the effect of sodium-glucose cotransporter 2 inhibitors. Cells. 11(23): 3913. https://doi.org/10.3390/cells11233913.

  13. Chomczynski, P. and Mackey, K. (1995). Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide-and proteoglycan-rich sources. Biotechniques. 19(6): 942-945. https://pubmed.ncbi.nlm.nih.gov/8747660/.

  14. Coughlan, K.A., Valentine, R.J., Ruderman, N.B. and Saha, A.K. (2014). AMPK activation: A therapeutic target for type 2 diabetes? Diabetes Metab Syndr Obes. 7(1): 241-253. https://doi.org/10.2147/DMSO.S43731.

  15. Celik, H. (2013). Investigation of the effects of garlic (Allium sativum) and black cumin seed (Nigella sativa) extracts against aluminum-induced cytotoxicity and genotoxicity in human lymphocyte cell culture medium. Gaziantep University Health Sciences Institute. PhD thesis. 

  16. Defronzo, R.A. (2009). From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes. 58(1): 773-795. https://doi.org/10.2337/db09-9028.

  17. Eraslan Farisoğlu, Ü., Özcem, B., Balcıoğlu, Ö., Ülker, S.N., Koçer, G. (2026). The diabetic parabiosis model: Unlocking new therapeutic pathways in diabetes research. Indian Journal of Animal Research. 59(SI): 177-183. doi: 10.18805/IJAR.BF-2066.

  18. Gatica, R., Bertinat, R., Silva, P., Carpio, D., Ramírez, M.J., Slebe, J.C. and Martín, R.S. (2013). Altered expression and localization of insulin receptor in proximal tubule cells from human and rat diabetic kidney. J. Cell Biochem. 114(3): 639- 649. https://doi.org/10.1002/jcb.24406.

  19. Gholamnezhad, Z., Havakhah, S. and Boskabady, M.H. (2016). Preclinical  and clinical effects of Nigella sativa and its constituent, thymoquinone: A review. J. Ethnopharmacol. 190(1): 372-386. https://doi.org/10.1016/j.jep.2016.06.061.

  20. Girard, J. (2017). Role of the kidneys in glucose homeostasis. Implication of sodium-glucose cotransporter 2 (Sglt2) in diabetes mellitus treatment. Nephrol Ther. 13(l): 35-41. https://doi.org/10.1016/j.nephro.2017.01.006.

  21. Gnudi, L., Thomas, S.M. and Viberti, G. (2007). Mechanical forces in diabetic kidney disease: A trigger for impaired glucose metabolism. J. Am Soc Nephrol. 18(8): 2226-2232. https://doi.org/10.1681/ASN.2006121362.

  22. Gümüş, A.F., Dede, S., Yüksek, V., Çetin, S. and Taşpınar, M. (2018). In vitro evaluation of thymoquinone on apoptosis and oxidative DNA damage in high glucose condition. Cell Mol Biol. 64(1): 79-83. https://doi.org/10.14715/cmb/2018.64.14.13.

  23. Hamdan, A., Idrus. R.H. and Mokhtar, M.H. (2019). Effects of Nigella sativa on Type-2 Diabetes Mellitus: A systematic review. Int J. Environ Res Public Health. 16(24): 4911: 1-12. https://doi.org/10.3390/ijerph16244911.

  24. Han, Y.C., Tang, S.Q., Liu, Y.T., Li, A.M., Zhan, M., Ming Yang, M., and Song, N. (2021). AMPK agonist alleviate renal tubulointerstitial fibrosis via activating mitophagy in high fat and streptozotocin induced diabetic mice. Cell Death Dis. 12(10): 925. https://doi.org/10.1038/s41419-021-04184-8.

  25. He, P., He, Y., Ma, J., Liu, Y., Liu, C., Baoping, Y. and Dong, W. (2023). Thymoquinone induces apoptosis and protective autophagy in gastric cancer cells by inhibiting the PI3K/Akt/mTOR pathway. Phytother Res. 37(8): 3467-3480. https://doi.org/10.1002/ptr.7820.

  26. Heshmati, J., Namazi, N., Memarzadeh, M.R., Taghizadeh, M. and Kolahdooz, F. (2015). Nigella sativa oil affects glucose metabolism and lipid concentrations in patients with type 2 diabetes: A randomized, double-blind, placebo-controlled trial. Food Res Int. 70(1): 87-93. https://doi.org/10.1016/j.foodres.2015.01.030.

  27. Holman, G.D. (2020). Structure, function and regulation of mammalian glucose transporters of the SLC2 family. Pflugers Arch. 472(9): 1155-1175. https://doi.org/10.1007/s00424-020-02411-3.

  28. Iancu, C.V., Bocci, G., Ishtikhar, M., Khamrai, M., Oreb, M., Oprea, T. and Jun-Yong, C. (2022). Glut3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems. Sci Rep. 12(1): 1429. https://doi.org/10.1038/s41598-022-05383-9.

  29. Inukai, K., Asano, T., Katagiri, H., Ishihara, H., Anai, M.,  Fukushima, Y., Tsukuda, K., Kikuchi, M., Yazaki, Y. and Oka, Y. (1993). Cloning and increased expression with fructose feeding of rat jejunal Glut5. Endocrinology. 13(1993): 2009-2014. https://doi.org/10.1210/endo.133.5.8404647.

  30. Kaatabi, H., Bamosa, A.O., Badar, A., Al-Elq, A., Abou-Hozaifa, B., Lebda, F., Al-Khadra, A. and Al-Almaie, S. (2015). Nigella sativa improves glycemic control and ameliortes oxidative stress in patients with type 2 diabetes mellitus: Placebo controlled participant blinded clinical trial. PLos One. 10(2): e0113486. https://doi.org/10.1371/journal.pone.0113486.eCollection 2015.

  31. Keembiyehetty, C., Augustin, R., Carayannopoulos, M.O., Sarah, S., Manolescu, A., Cheeseman,  C.I. and  Moley, K.H. (2006). Mouse glucose transporter 9 splice variants are expressed in adult liver and kidney and are up-regulated in diabetes. Mol Endocrinol. 20(3): 686-697. https://doi.org/10.1210/me.2005-0010.

  32. Lay, A.C. and Coward, R.J.M. (2018). The evolving importance of insulin signaling in podocyte health and disease. Front Endocrinol (Lausanne). 9: 693. https://doi.org/10.3389/fendo.2018.00693.

  33. Lay, A.C., Tran, V.D.T., Nair, V., Betin, V., Hurcombe, J.A., Barrington, A.F. and Pope, R.J. (2024). Profiling of insulin-resistant kidney models and human biopsies reveals common and cell-type-specific mechanisms underpinning Diabetic Kidney Disease. Nature Communications. 15: 10018. https://doi.org/10.1038/s41467-024-54089-1.

  34. Linnemann, A.K., Baan, M. and Davis, D.B. (2014). Pancreatic β-cell proliferation in obesity. Adv Nutr. 5(1): 278-288. https:// doi.org/10.3945/an.113.005488.

  35. Lee, A.S., Lee, Y.J., Lee, S.M., Yoon, J.J., Kim, J.S., Kang, D.J. and Lee, H.S. (2012). Portulaca oleracea ameliorates diabetic vascular inflammation and endothelial dysfunction in db/ db mice. Evid Based Complement Alternat Med. 741824: 1-9. https://doi.org/10.1155/2012/741824

  36. Liang, X., Wang, P., Chen, B., Ge, Y., Athena, Y., Flickinger, B. and Malhotra, D.K. (2020). Glycogen synthase kinase 3β hyperactivity in urinary exfoliated cells predicts progression of diabetic kidney disease. Kidney Int. 97(1): 175-192. https://doi.org/10.1016/j.kint.2019.08.036.

  37. Liu, H., Liu, H.Y., Jiang, Y.N. and Li, N. (2016). Protective effect of thymoquinone improves cardiovascular function and attenuates oxidativestress, inflammation and apoptosis by mediating the PI3K/Akt pathway in diabetic rats. Mol Med Rep. 13(3): 2836-2842. https://doi.org/10.3892/mmr.2016.4823.

  38. Liu, J.J., Lee, T. and DeFronzo, R.A. (2012). Why do Sglt2 inhibitors inhibit only 30-50% of renal glucose reabsorption in humans? Diabetes. 61(1): 2199-2204. https://doi.org/10.2337/db12-0052.

  39. Livak, K.J. and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods. 25(4): 402-408. https://doi.org/10.1006/meth.2001.1262.

  40. Manna, P., Achari, A.E. and Jain, S.K. (2017). Vitamin D supplementation inhibits oxidative stress and upregulate Sirt1/Ampk/Glut4 cascade in high glucose-treated 3T3L1 adipocytes and in adipose tissue of high fat diet-fed diabetic mice. Arch Biochem Biophys. 615(1): 22-34. https://doi.org/10.1016/j.abb.2017.01.002.

  41. Marks, J., Carvou, N.J., Debnam, E.S., Srai, S.K. and Unwin, R.J. (2003). Diabetes increases facilitative glucose uptake and Glut2 expression at the rat proximal tubule brush border membrane. J. Physiol. 553(1): 137-145. https://doi.org/10.1113/jphysiol.2003.046268.

  42. Mather, A. and Pollock, C. (2011). Glucose handling by the kidney. Kidney Int Suppl. 79(120): S1-6. https://doi.org/10.1038/ki.2010.509.

  43. Medina, R.A. and Owen, G.I. (2002). Glucose transporters: Expression, regulation and cancer. Biol Res. 35(1): 9-26. https:// doi.org/10.4067/s0716-97602002000100004.

  44. Meerloo, J.V., Kaspers. G.J.L. and Cloos, J. (2011).  Cell sensitivity assays: The MTT assay. Methods Mol Biol. 731(1): 237-245. https://doi.org/10.1007/978-1-61779-080-5_20.

  45. Meziti, A., Meziti, H., Boudiaf, K., Mustapha, B. and Bouriche, H. (2012). Polyphenolic profile and antioxidant activities of Nigella sativa seed extracts in vitro and in vivo. World Acad Sci Eng Technol. 64(1): 24-32. https://doi.org/10.5281/zenodo.1329809.

  46. Mima, A., Murakami, A., Lee, R. and Lee, S. (2023). Predictive significance of glomerular insulin receptor substrate-1 in patients with diabetic kidney disease. Metabol Open. 18: 100240. https://doi.org/10.1016/j.metop.2023.100240.

  47. Mima, A., Ohshiro, Y., Kitada, M., Matsumoto, M., Geraldes, P., Li, C. and Li, Q. (2011). PKCβ-induced Selective Irs1 dysfunction and insulin resistance in renal glomeruli of rodent models of diabetes and obesity. Kidney Int. 79(8): 883-896. https://doi.org/10.1038/ki.2010.526.

  48. Mokashi, P., Bhatt, L.K., Khanna, A. and Pandita, N. (2017). Swertisin rich fraction from Enicostema littorale ameliorates hyper- glycemia and hyperlipidemia in high-fat fed diet and low dose streptozotacin induced type 2 diabetes mellitus in rats. Biomed Pharmacother. 96(1): 1427-1437. https://doi.org/10.1016/j.biopha.2017.09.153.

  49. Mueckler, M. and Thorens, B. (2013). The Slc2 (Glut) family of membrane transporters. Mol Aspects Med. 34(2-3): 121-138. https:// doi.org/10.1016/j.mam.2012.07.001.

  50. Norton, L., Shannon, C.E., Fourcaudot, M., Hu, C., Wang, N. and Ren, W. (2017). Sodium-glucose co-transporter (Sglt) and glucose transporter (Glut) expression in the kidney of type 2 diabetic subjects. Diabetesp Obes Metab. 19(9): 1322-1326. https://doi.org/10.1111/dom.13003.

  51. O’Neill, H.M. (2013). AMPK and Exercise: Glucose uptake and insulin sensitivity. Diabetes Metab. 37(1): 1-21. https://doi.org/ 10.4093/dmj.2013.37.1.1.

  52. Ozcan, B.G. and Kaya, H. (2025). Effects of varying ratios of hawthorn fruit extract on nesfatin-1, Glp-1 and ctni response in streptozotocin-induced diabetic rats. Indian Journal of Animal Research. 59(12): 2133-2137. doi: 10.18805/IJAR.BF-2045.

  53. Peng, J. and He, L. (2018). Irs posttranslational modifications in regulating insulin signaling. J. Mol Endocrinol. 60(1): R1-R8. https:// doi.org/10.1530/JME-17-0151.

  54. Paeng, J., Chang, J.H., Lee, S.H., Nam, B.Y., Hye-Young, K. and Kim, S. (2014).  Enhanced glycogen synthase kinase- 3â activity mediates podocyte apoptosis under diabetic conditions. Apoptosis. 19(12): 1678-90. https://doi.org/10.1007/s10495-014-1037-5.

  55. Park, J.E., Lee, J.S., Lee, H.A. and Han, J.S. (2018). Portulaca oleracea L. extract enhances glucose uptake by stimulating Glut4 translocation to the plasma membrane in 3T3-L1 adipocytes. J. Med Food. 12(3): 183-190. https://doi.org/10.1089/jmf.2017.4098.

  56. Rahmoune, H., Thompson, P.W., Ward, J.M., Smith, C.D., Hong, G., and Brown, J. (2005). Glucose transporters in human renal proximal tubular cells isolated from the urine of patients with non-insulin-dependent diabetes. Diabetes. 54(12): 3427-34. https://doi.org/10.2337/diabetes.54.12.3427.

  57. Raj, A. and Rani, S. (2024). Evaluating the efficacy of metformin and camel milk on streptozotocin-nicotinamide induced diabetic albino rats. Indian Journal of Animal Research. 1-8. doi: 10.18805/ajdfr.DR-2213.

  58. Rand, E.B., Depaoli, A.M., Davidson, N.O., Bell, G. and Burant, C.F. (1993). Sequence, tissue distribution and functional characteri- zation of the rat fructose transporter Glut5. Am J. Physiol. 264(6 Pt 1): G1169-76. https://doi.org/10.1152/ajpgi.1993.264.6.G1169.

  59. Rayasam, G.V., Tulasi, V.K., Sodhi, R., Davis, J.A. and Ray, A. (2009). Glycogen synthase kinase 3: More than a namesake. Br J. Pharmacol. 156(6): 885-898. https://doi.org/10.1111/j.1476-5381.2008.00085.x.

  60. Rizzo, B., Zambonin, L., Angeloni, C., Krejpcio, Z. and Olejnik, A. (2013). Steviol glycosides modulate glucose transport in different cell types. Oxid Med Cell Longev.  348169: 1-11. https://doi.org/10.1155/2013/348169.

  61. Sangi, S.M., Sulaiman, M.I., El-Wahab, M.F., Ahmedani, E.I. and Ali, S.S. (2015). Antihyperglycemic effect of thymoquinone and oleuropein, on streptozotocin-induced diabetes mellitus in experimental animals. Pharmacogn Mag. 11(2): S251- 257. https://doi.org/10.4103/0973-1296.166017.

  62. Shan, W.F., Chen, B.Q., Zhu, S.J., Jiang, L. and Zhou, Y.F. (2011). Effects of Glut4 expression on insulin resistance in patients with advanced liver cirrhosis. J. Zhejiang Univ Sci B. 12(8): 677-682. https://doi.org/10.1631/jzus.B1100001.

  63. Soetikno, V., Sari, F.R., Sukumaran, V., Lakshmanan, A.P., Harima, M., Suzuki, K., Kawachi, H. and Watanabe, K. (2013). Curcumin decreases renal triglyceride accumulation through AMPK- SREBP signaling pathway in streptozotocin-induced type 1 diabetic rats.  J. Nutr Biochem. 24(5): 796-802. https://doi.org/10.1016/j.jnutbio.2012.04.013.

  64. Sugawara-Yokoo, M., Suzuki, T., Matsuzaki, T., Naruse, T. and Takata, K. (1999). Presence of fructose transporter Glut5 in the S3 proximal tubules in the rat kidney. Kidney Int. 56(3): 1022-1028. https://doi.org/10.1046/j.1523-1755.1999.00635.x.

  65. Tiwari, S., Singh, R.S., Li, L., Tsukerman, S., Godbole, M., Pandey, G. and Ecelbarger, C.M. (2013). Deletion of the insulin receptor in the proximal tubule promotes hyperglycemia. J. Am Soc Nephrol. 24(8): 1209-1214. https://doi.org/10.1681/ASN.2012 060628.

  66. Usta, A. and Dede, S. (2017). The effect of thymoquinone on nuclear factor kappa b levels and oxidative DNA damage on experimental diabetic rats. Pharmacogn Mag. 13(S3): 458-461. https://doi.org/10.4103/pm.pm_134_17.

  67. Umino, H., Hasegawa, K., Minakuchi, H., Muraoka, H., Kawaguchi, T., Kanda, T., Tokuyama, H., Wakino, S. and Itoh, H. (2018). High basolateral glucose increases sodium-glucose cotransporter 2 and Reduces Sirtuin-1 in Renal Tubules through Glucose Transporter-2 Detection. Sci Rep. 8(1): 6791: 1-13. https://doi.org/10.1038/s41598-018-25054-y.

  68. Usta, A., Dede, S., Yüksek, V. and Öner, A.C. (2024a). Determination of the effect of thymoquinone on DNA damage in kidney cells applied to high glucose depending on time and dosage by comet assay. TURJAF. 12(8): 1359-1365. https:// doi.org/10.24925/turjaf.v12i8.1359-1365.6573.

  69. Usta, A., Yüksek, V., Çetin, S. and Dede, S. (2024b). Lycopene prevents cell death in NRK-52E cells by inhibition of high glucose- activated DNA damage and apoptotic, autophagic and necrotic pathways. J. Biochem Mol Toxicol. 38(3): e23678: 1-13. https://doi.org/10.1002/jbt.23678.

  70. Vallon, V., Platt, K.A., Cunard, R., Schroth, J., Whaley, J., Thomson, S.C., Koepsell, H.  and  Rieg, T. (2011). Sglt2 mediates glucose reabsorption in the early proximal tubule. J. Am Soc Nephrol. 22(1): 104-12. https://doi.org/10.1681/ASN.2010030246.

  71. Vestri, S., Okamoto, M.M., de Freitas, H.S.,  Eichler, R.A.D.S., Nunes, M.T., Morimatsu, M., Heimann, J. and Machado, U.F.  (2001). Changes in sodium or glucose filtration rate modulate expression of glucose transporters in renal proximal tubular cells of rat. J. Membr Biol. 182(2): 105-112. https://doi.org/10.1007/s00232-001-0036-y.

  72. Watson, R.T. and Pessin, J.E. (2001). Intracellular organization of insulin signaling and Glut4 translocation. Recent Prog Horm Res. 56(1): 175-193. https://doi.org/10.1210/rp.56.1.175.

  73. Welsh, G.I., Hale, L.J., Eremina, V., Jeansson, M., Maezawa, Y.,  Lennon, R. et al. (2010). Insulin signaling to the glomerular podocyte is critical for normal kidney function. Cell Metab. 12(1): 329-340. https://doi.org/10.1016/j.cmet.2010.08.015.
Published In
Indian Journal of Animal Research
Article Metrics
Metrics Icon
24
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Comparative Effects of Nigella sativa and Thymoquinone on Glucose Metabolism in a High Glucose-induced Renal Cell Line

    A
    A. Usta1,*
    S
    S. Çetin2
    V
    V. Yüksek3
    S
    S. Dede4
    1Van Yuzuncu Yil University, Faculty of Science, Department of Chemistry, Van, Türkiye.
    2Ankara Yildirim Beyazit University, Vocational School of Health Services, Department of Veterinary Medicine, Ankara, Türkiye.
    3Van Yuzuncu Yil University, Ozalp Regional High School, Van, Türkiye.
    4Van Yuzuncu Yil University, Faculty of Veterinary Medicine, Biochemistry Department, Van, Türkiye.

    ABSTRACT

    Background: A significant side effect of type 2 diabetes mellitus is diabetic nephropathy (DN), which is typified by poor glucose homeostasis and insulin resistance in renal tissues. Thymoquinone (TQ), the active ingredient in Nigella sativa (NS), has been shown to have antioxidant and antidiabetic properties. However, nothing is known about their relative effects on renal glucose metabolism in hyperglycemic situations. The objective of this work was to examine and contrast the regulatory effects of NS and TQ on insulin signaling pathways and glucose transporter expression in NRK-52E renal epithelial cells grown in high-glucose environments.

    Methods: TQ (10 µM) or NS extract (0.5 µg/ml) were applied to NRK-52E cells after they were subjected to high glucose (246 mM). RT-qPCR was used to measure the mRNA expression levels of insulin signaling components (Ir, Irs1, Pi3k, Ampk, Gsk3β) and glucose transporters (Glut1, Glut2, Glut3, Glut5, Sglt2). Western blot was used to measure the protein levels of Glut2, Glut3, Sglt2 and Irs1. The Duncan test (p<0.05) and one-way ANOVA were used to determine statistical significance.

    Result: Ir, Irs1, Pi3k and Ampk levels were downregulated and Glut1, Glut2, Glut3 and Sglt2 expression was markedly elevated by high glucose exposure. These changes were reversed by TQ and NS therapy, with TQ having a stronger effect. TQ increased the levels of Irs1 and Pi3k while more successfully suppressing the expression of Sglt2 and Glut2. The transcriptional alterations were confirmed by Western blot results. TQ and NS improved insulin signaling pathways and modulated glucose transporter expression to protect renal cells. These results confirm that TQ and NS are safe to use as possible therapeutic agents to treat diabetic nephropathy.

    INTRODUCTION

    Diabetic nephropathy is an important side effect of diabetes that can lead to chronic kidney failure and significantly reduce a patient’s quality of life. Vascular injury in intraglomerular arterioles is the main way that persistent hyperglycemia causes renal failure. It is thought to affect about 15% of patients with type 2 diabetes and 35% of those with type 1 diabetes (Eraslan Farisoğlu et al., 2026; Ozcan and Kaya, 2025; Raj and Rani, 2024).
           
    Diabetes raises the risk of organ failure and persistent tissue damage due to impaired glucose regulation (Keembiyehetty et al., 2006). Cellular function depends on the movement of glucose across cell membranes, which is accomplished by either active or assisted diffusion. Tissue-specific expression of sodium-glucose cotransporters (Sglts) and glucose transporters (Gluts) occurs in the kidney. Sglt2, which is found on the apical membrane of proximal tubular cells, is principally in charge of sodium-dependent glucose reabsorption, which uses basolateral Glut transporters to move glucose into the bloodstream (Norton et al., 2017; Bailey et al., 2010).
           
    Gluts comprise 14 isoforms that are categorized into three classes and are widely expressed in mammalian cells (Augustin, 2010; Mueckler et al., 2013). Hormonal, metabolic and oxidative stress-related factors impact their expression, which varies according to tissue type and pathological situations such diabetes, ischemia and cancer (Rizzo et al., 2013; Medina and Owen, 2002).
           
    Insulin primarily uses the Pi3k/Akt and MAPK signaling pathways to control glucose homeostasis by activating the insulin receptor (Ir) and insulin receptor substrate (Irs) (Cardoso, 2009). Irs1 phosphorylation is essential for Pi3k activation, which increases glucose absorption through Glut translocation, especially Glut4 (Lee et al., 2012). Additionally, Ampk is a key target for treatment in type 2 diabetes and plays a crucial role in glucose metabolism (Bijland et al., 2013; Manna et al., 2017; Park et al., 2018).
           
    Glut2-mediated glucose absorption in pancreatic β-cells raises intracellular ATP levels, which in turn cause membrane depolarization and calcium influx, which in turn stimulate insulin production (Champe and Harvey 2007). Furthermore, a crucial regulator of glycogen metabolism, glycogen synthase kinase 3 beta (Gsk3β), is still active in diabetes, which inhibits glycogen synthesis and lowers cellular glucose absorption.
           
    In diabetic patients, Nigella sativa (NS) has been shown to enhance inflammatory indicators, oxidative stress, insulin resistance, lipid profiles and glycemic management. Additionally, it improves antioxidant capacity and β-cell activity (Shan et al., 2011; O’Neill, 2013; Hamdan et al., 2019; Kaatabi et al., 2015; Chatterjee et al., 2025; Heshmati et al., 2015). The primary bioactive component of NS, thymoquinone (TQ), has shown protective properties in diabetic mice by lowering inflammation, apoptosis and oxidative stress, in part by modifying the Pi3k/Akt pathway (Liu et al., 2016; He et al., 2023; Usta and Dede, 2017).
           
    Despite these results, the effects of NS and TQ on renal glucose metabolism are still unknown and changes in glucose transporters under metabolic diseases are not completely understood. Therefore, by examining the mRNA and protein expression levels of important genes involved in glucose metabolism, such as Glut1, Glut2, Glut3, Glut5, Sglt2, Ir, Irs1, Pi3k, Ampk and Gsk3β, this study sought to determine how NS and TQ affected renal proximal tubular cells (NRK-52E) cultured under high-glucose conditions.

    MATERIALS AND METHODS

    Reagents and chemicals
     
    Thymoquinone (TQ) and D-glucose were acquired from Sigma-Aldrich (USA). Western blot kits and antibodies were purchased from Affinity Biosciences (USA). The ABT 2x SYBR Green Master Mix (Türkiye) was used for RT-qPCR studies.
     
    Cell culture
     
    NRK-52E (ATCC® CRL-1571TM), a rat kidney epithelial cell line, was grown in RPMI-1640 media supplemented with 1% L-glutamine, 1% penicillin/streptomycin and 10% fetal bovine serum (Capricorn, Germany). The cells were kept in a humidified environment with 5% CO2 at 37oC (Esco CelCulture CO2, Singapore). The Van Yuzuncu Yil University Scientific Research Projects Coordination Unit project TYD-2020-8582 provided funding for all of the experiments, which were carried out in 2022 at the Cell Culture and Molecular  Biology Laboratory in the Biochemistry Department of the Faculty of Veterinary Medicine at Van Yuzuncu Yil University.
     
    Preparation of solutions
     
    In order to make Nigella sativa (NS) extract, 70% of the powdered seeds were extracted using methanol, filtered, the solvent was evaporated, the mixture was lyophilized and it was then stored at -20°C (Meziti et al., 2012). TQ and NS stock solutions were made in DMSO (Panreac Applichem, Spain) (<0.05%) and then diluted in culture medium. As previously stated, amounts of glucose (0-400 mM), NS (0-200 µg/ml) and TQ (0-100 µM) were prepared (Gümüş et al., 2018; Celik, 2013; Usta et al., 2024a).

    MTT assay
     
    Following the seeding of 1×104 cells per well in 96-well plates, the cells were incubated overnight. The MTT assay was used to evaluate cell viability following treatment. The formazan crystals made by metabolically active cells were dissolved and the absorbance at 570 nm was determined (Biochrom, Anthos Zenyth 200). The viable cell percentage was contrasted with the control (Usta et al., 2024b; Meerloo et al., 2011).
     
    Experimental design
     
    Treatment groups were established based on different concentrations of glucose, TQ and NS, as indicated in Table 1.

    Table 1: Study groups based on the administration of glucose, TQ and NS to NRK-52E cells.


     
    RNA isolation and cDNA synthesis
     
    Each group received a 24-hour treatment after being planted with about 8×105 cells. Total RNA was isolated using Trizol reagent (GeneAll, Korea) and its purity was assessed by spectrophotometric analysis (Chomczynski and Mackey, 1995). The manufacturer’s instructions were followed for cDNA synthesis using a commercial kit (ABT High Capacity, Türkiye).
     
    RT-qPCR analysis
     
    The findings of RT-qPCR investigation of Sglt2, Glut1, Glut2, Glut3, Glut5, Ir, Irs1, Gsk3β, Pi3k and Ampk gene expression levels are displayed in Table 2 (Qiagen, Germany).

    Table 2: Target gene primers’ base sequence.


           
    Using β-actin as the internal reference, relative expression levels were determined using the 2- ΔΔCt approach (Livak and Schmittgen, 2001) (Table 3).

    Table 3: RT-qPCR reaction conditions.


     
    Western blot analysis
     
    After the proteins were extracted (GeneAll, Korea) and quantified using the Bradford procedure (ABT Biosciences, Türkiye), they were separated using SDS-PAGE. After being moved to nitrocellulose membranes, samples were incubated with primary and secondary antibodies. Protein bands were seen using a chemiluminescent substrate and analysis was done using ImageJ software.
     
    Statistical analysis
     
    SPSS 22.0 was used for statistical analysis (IBM, USA). The Duncan test was used after one-way ANOVA and Kruskal-Wallis to assess group differences. Mean±standard deviation was used to express the data and p<0.05 was regarded as statistically significant.

    RESULTS AND DISCUSSION

    Effects of TQ, NS and glucose on cell viability
     
    TQ, NS and glucose concentrations that were cytotoxic and proliferative in NRK-52E cells were measured using the MTT test (Fig 1). The best proliferative doses were determined to be TQ (10 μM) and NS (0.5 μg/ml), which both markedly improved cell viability (p<0.05). In the experiments that followed, the cytotoxic concentration of glucose was established to be 246 mM based on its IC50 value.

    Fig 1: After administering different TQ, NS and glucose concentrations to the NRK-52E cell line for 24 hours.


     
    Effects on glucose transporter gene expression
     
    High glucose considerably increased the expression of Glut1, Glut2, Glut3 and Sglt2 in comparison to the control, according to RT-qPCR study (Fig 2). These increases were reversed by TQ and NS treatment, bringing expression levels back to nearly control levels. Nonetheless, the treated groups’ Glut2 expression was still lower than the control. All experimental groups had lower levels of Glut5 expression than the control.

    Fig 2: (a-e) Glut1, Glut2, Glut3, Glut5 and Sglt2 mRNA transcription levels in NRK-52E cells after 24 hours.


     
    Effects on Ir and Irs1 expression
     
    Ir and Irs1 were downregulated in comparison to the control in high glucose circumstances (Fig 3). Both genes’ expression was elevated by TQ and NS treatments in comparison to the glucose group. Irs1 levels were brought back to almost control levels in treatment groups, but Ir expression remained below control.

    Fig 3: (a-b) Irs1 and Ir mRNA transcription levels in NRK-52E cells after 24 hours.



    Effects on Pi3k, Gsk3β and Ampk expression
     
    Under high glucose circumstances, Pi3k and Ampk expression levels decreased; however, after TQ and NS treatment, they either increased or were recovered (Fig 4). While there was no discernible difference between the treatment groups and the control, Gsk3β expression was increased in the glucose group. On the other hand, Gsk3β expression was decreased by TQ and NS alone.

    Fig 4: (a-c) Pi3k, Gsk3â and Ampk mRNA transcription levels at 24 hours in NRK-52E cells.


     
    Protein expression analysis
     
    The glucose-treated group had higher amounts of Glut2 protein than the control group, according to Western blot analysis and these levels dropped after TQ and NS treatment (Fig 5). In general, all groups had lower levels of other target proteins than the control. These results most likely represent early transcriptional responses at 24 hours, since longer exposure times may be necessary for protein-level alterations (48-72 h).

    Fig 5: (a-d) Glut2, Glut3, Sglt2 and Irs1 protein translation levels in the NRK-52E cell line after 24 hours.


           
    In this investigation, NRK-52E cells subjected to rising glucose concentrations for a full day demonstrated a dose-dependent decline in viability, with an IC50 of 246 mM, simulating advanced hyperglycemic stress in line with other findings. (Liu et al., 2016; Gholamnezhad et al., 2016). This method allowed for the assessment of renal tubular cells’ impaired insulin signaling and glucose-induced cytotoxicity.
           
    Thymoquinone (TQ) is one of the main bioactives found in Nigella sativa (NS) extract. Both have been shown to have anti-inflammatory, anti-apoptotic, antioxidant and antidiabetic properties (Sangi et al., 2015; Ali and Blunden, 2003).
           
    Sglt2 reabsorbs approximately 90% of filtered glucose in proximal tubules, while Glut1/Glut2 facilitates translocation to the circulation. Sglt and Glut transporters control glucose uptake (Bell et al., 1990; Iancu et al., 2022; Watson and Pessin, 2001; Umino et al., 2018; Girard, 2017; Marks et al., 2003; Mather and Pollock, 2011). Hyperglycemia enhances transporter expression and renal glucose reabsorption, which exacerbates glucose toxicity (Abdul-Ghani et al., 2011; Holman, 2020; Vestri et al., 2001; Rahmoune et al., 2005; Defronzo, 2009; Liu et al., 2012). High glucose increased Glut1, Glut2, Glut3 and Sglt2 in our investigation, which is consistent with the literature. However, TQ and NS therapy brought these levels down to almost control levels. All groups showed low levels of Glut5, which is consistent with its restricted role in the kidneys (Adeshara et al., 2017; Gnudi et al., 2007;  Brosius and Heilig, 2005; Vallon et al., 2011; Sugawara-Yokoo et al., 1999; Rand et al., 1993; Inukai et al., 1993).
           
    Insulin signaling via the downstream Pi3k/Akt pathway and Ir/Irs1 is essential for maintaining glucose homeostasis. Elevated glucose inhibited the production of Ir and Irs1, which hindered insulin signaling and decreased the absorption of glucose. The expression of Pi3k/Akt and Irs1 was restored by TQ and NS, indicating improved glycogen production and insulin signaling (Tiwari et al., 2013; Peng and He, 2018; Mokashi et al., 2017; Linnemann et al., 2014; Gatica et al., 2013; Lay et al., 2024; Mima et al., 2011; Mima et al., 2023; Lay and Coward, 2018; Chen et al., 2022).
           
    Under high glucose, AMPK, a crucial regulator of energy balance, was inhibited, which increased oxidative stress and insulin resistance (Coughlan et al., 2014; Soetikno et al., 2013; Han et al., 2021; Welsh et al., 2010). TQ and NS improved glucose utilization and reversed insulin resistance via restoring AMPK expression. In a similar vein, Gsk3β, which rises in diabetes and prevents the synthesis of glycogen, was increased in high glucose but repressed by TQ and NS, promoting better glycogen metabolism and decreased oxidative stress (Liang et al., 2020; Rayasam et al., 2009; Paeng et al., 2014).
           
    Together, our results show that TQ and NS influence several pathways related to glucose metabolism, such as glucose transporters, Ir/Irs1, Pi3k/Akt, AMPK and Gsk3β (Fig 6). Their promise as treatment agents for diabetic nephropathy is highlighted by these effects, which improve insulin sensitivity and reduce hyperglycemia. To validate these protective effects, further extensive in vivo and clinical research is necessary.

    Fig 6: Impact of TQ and NS supplementation on the downstream pathways of glucose metabolism in high-glucose environments.

    CONCLUSION

    In NRK-52E renal proximal tubular cells, high glucose exposure reduces cell survival, interferes with insulin signaling, and modifies glucose transporter expression. By lowering the expression of Glut1, Glut2, Glut3, and Sglt2, increasing Ir/Irs1, Pi3k/Akt, and AMPK signaling, and suppressing Gsk3β activity, treatment with Nigella sativa (NS) extract and thymoquinone (TQ) reduced these effects and promoted glycogen synthesis and glucose utilization.
           
    TQ’s potential as a therapeutic agent for avoiding or lessening diabetic nephropathy was highlighted by its better effectiveness than NS in repairing impaired glucose transporter expression and metabolic signaling. These results shed light on the mechanisms behind NS and TQ’s renoprotective and antidiabetic actions. To confirm and expand these protective benefits, more in vivo and clinical research is necessary.

    ACKNOWLEDGEMENT

    We would like to thank Van Yuzuncu Yil University Scientific Research Projects Coordination Unit (TYD-2020-8582) for their contributions.

    CONFLICT OF INTEREST

    The authors declares no conflicts of interest to report regarding the present study.

    REFERENCES


    1. Abdul-Ghani, M.A., Norton, L. and Defronzo, R.A. (2011). Role of sodium- glucose cotransporter 2 (Sglt 2) inhibitors in the treatment of type 2 diabetes. Endocr Rev. 32(4): 515-531. https://doi.org/10.1210/er.2010-0029.

    2. Adeshara, K.A., Diwan, A.G., Jagtap, T.R., Advani, K., Siddiqui, A. and Tupe, R.S. (2017). Relationship between plasma glycation with membrane modification, oxidative stress and expression of glucose transporter-1 in type 2 diabetes patients with vascular complications. J. Diabetes Complications. 31(2): 439-448. https://doi.org/10.1016/j.jdiacomp.2016.10.012.

    3. Ali, B.H. and Blunden, G. (2003). Pharmacological and toxicological properties of Nigella sativa. Phytother Res. 17: 299-305. https://doi.org/10.1002/ptr.1309.

    4. Augustin, R. (2010). The protein family of glucose transport facilitators: It’s not only about glucose after all. IUBMB Life. 62(5): 315-333. https://doi.org/10.1002/iub.315.

    5. Bailey, C.J., Gross, J.L., Pieters, A., Bastien, A. and List, J.F. (2010). Effect of dapagliflozin in patients with type 2 diabetes who have inadequate glycaemic control with metformin: A randomised, double-blind, placebocontrolled trial. Lancet. 375(9733): 2223-2233. https://doi.org/10.1016/S0140-6736(10)60407-2.

    6. Bell, G.I., Kayano, T., Buse, J.B., Burant, C.F., Takeda, J., Lin, D.,  Fukumoto, H and Seino, S. (1990). Molecular biology of mammalian glucose transporters. Diabetes Care. 13(3): 198-208. https://doi.org/10.2337/diacare.13.3.198.

    7. Bijland, S., Mancini, S.J. and Salt, I.P. (2013). Role of AMP-activated protein kinase in adipose tissue metabolism and inflammation. Clin Sci. 124(8): 491-507. https://doi.org/10.1042/CS20120536.

    8. Brosius, F.C. and Heilig, C.W. (2005). Glucose transporters in diabetic nephropathy. Pediatr Nephrol. 20(4): 447-451. https:// doi.org/10.1007/s00467-004-1748-x.

    9. Cardoso, S., Correia, S., Santos, R.X. et al. (2009). Insulin is a two-edged knife on the brain. J. Alzheimers Dis. 18(3): 483-507. https://doi.org/10.3233/JAD-2009-11.

    10. Champe, P.C. and Harvey, R.A. (2007). Lippincott’s Illustrated Reviews Series.

    11. Chatterjee, G., Saha, A.K., Khurshid, S. and Saha, A.A. (2025). Comprehensive Review of the antioxidant, antimicrobial and therapeutic efficacies of black cumin (Nigella sativa L.) seed oil and its thymoquinone. J. Med Food. 28(4): 325-339. https://doi.org/10.1089/jmf.2024.k.0149.

    12. Chen, X., Wang, J., Lin, Y., Liu, Y. and Zhou, T. (2022). Signaling pathways of podocyte injury in diabetic kidney disease and the effect of sodium-glucose cotransporter 2 inhibitors. Cells. 11(23): 3913. https://doi.org/10.3390/cells11233913.

    13. Chomczynski, P. and Mackey, K. (1995). Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide-and proteoglycan-rich sources. Biotechniques. 19(6): 942-945. https://pubmed.ncbi.nlm.nih.gov/8747660/.

    14. Coughlan, K.A., Valentine, R.J., Ruderman, N.B. and Saha, A.K. (2014). AMPK activation: A therapeutic target for type 2 diabetes? Diabetes Metab Syndr Obes. 7(1): 241-253. https://doi.org/10.2147/DMSO.S43731.

    15. Celik, H. (2013). Investigation of the effects of garlic (Allium sativum) and black cumin seed (Nigella sativa) extracts against aluminum-induced cytotoxicity and genotoxicity in human lymphocyte cell culture medium. Gaziantep University Health Sciences Institute. PhD thesis. 

    16. Defronzo, R.A. (2009). From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes. 58(1): 773-795. https://doi.org/10.2337/db09-9028.

    17. Eraslan Farisoğlu, Ü., Özcem, B., Balcıoğlu, Ö., Ülker, S.N., Koçer, G. (2026). The diabetic parabiosis model: Unlocking new therapeutic pathways in diabetes research. Indian Journal of Animal Research. 59(SI): 177-183. doi: 10.18805/IJAR.BF-2066.

    18. Gatica, R., Bertinat, R., Silva, P., Carpio, D., Ramírez, M.J., Slebe, J.C. and Martín, R.S. (2013). Altered expression and localization of insulin receptor in proximal tubule cells from human and rat diabetic kidney. J. Cell Biochem. 114(3): 639- 649. https://doi.org/10.1002/jcb.24406.

    19. Gholamnezhad, Z., Havakhah, S. and Boskabady, M.H. (2016). Preclinical  and clinical effects of Nigella sativa and its constituent, thymoquinone: A review. J. Ethnopharmacol. 190(1): 372-386. https://doi.org/10.1016/j.jep.2016.06.061.

    20. Girard, J. (2017). Role of the kidneys in glucose homeostasis. Implication of sodium-glucose cotransporter 2 (Sglt2) in diabetes mellitus treatment. Nephrol Ther. 13(l): 35-41. https://doi.org/10.1016/j.nephro.2017.01.006.

    21. Gnudi, L., Thomas, S.M. and Viberti, G. (2007). Mechanical forces in diabetic kidney disease: A trigger for impaired glucose metabolism. J. Am Soc Nephrol. 18(8): 2226-2232. https://doi.org/10.1681/ASN.2006121362.

    22. Gümüş, A.F., Dede, S., Yüksek, V., Çetin, S. and Taşpınar, M. (2018). In vitro evaluation of thymoquinone on apoptosis and oxidative DNA damage in high glucose condition. Cell Mol Biol. 64(1): 79-83. https://doi.org/10.14715/cmb/2018.64.14.13.

    23. Hamdan, A., Idrus. R.H. and Mokhtar, M.H. (2019). Effects of Nigella sativa on Type-2 Diabetes Mellitus: A systematic review. Int J. Environ Res Public Health. 16(24): 4911: 1-12. https://doi.org/10.3390/ijerph16244911.

    24. Han, Y.C., Tang, S.Q., Liu, Y.T., Li, A.M., Zhan, M., Ming Yang, M., and Song, N. (2021). AMPK agonist alleviate renal tubulointerstitial fibrosis via activating mitophagy in high fat and streptozotocin induced diabetic mice. Cell Death Dis. 12(10): 925. https://doi.org/10.1038/s41419-021-04184-8.

    25. He, P., He, Y., Ma, J., Liu, Y., Liu, C., Baoping, Y. and Dong, W. (2023). Thymoquinone induces apoptosis and protective autophagy in gastric cancer cells by inhibiting the PI3K/Akt/mTOR pathway. Phytother Res. 37(8): 3467-3480. https://doi.org/10.1002/ptr.7820.

    26. Heshmati, J., Namazi, N., Memarzadeh, M.R., Taghizadeh, M. and Kolahdooz, F. (2015). Nigella sativa oil affects glucose metabolism and lipid concentrations in patients with type 2 diabetes: A randomized, double-blind, placebo-controlled trial. Food Res Int. 70(1): 87-93. https://doi.org/10.1016/j.foodres.2015.01.030.

    27. Holman, G.D. (2020). Structure, function and regulation of mammalian glucose transporters of the SLC2 family. Pflugers Arch. 472(9): 1155-1175. https://doi.org/10.1007/s00424-020-02411-3.

    28. Iancu, C.V., Bocci, G., Ishtikhar, M., Khamrai, M., Oreb, M., Oprea, T. and Jun-Yong, C. (2022). Glut3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems. Sci Rep. 12(1): 1429. https://doi.org/10.1038/s41598-022-05383-9.

    29. Inukai, K., Asano, T., Katagiri, H., Ishihara, H., Anai, M.,  Fukushima, Y., Tsukuda, K., Kikuchi, M., Yazaki, Y. and Oka, Y. (1993). Cloning and increased expression with fructose feeding of rat jejunal Glut5. Endocrinology. 13(1993): 2009-2014. https://doi.org/10.1210/endo.133.5.8404647.

    30. Kaatabi, H., Bamosa, A.O., Badar, A., Al-Elq, A., Abou-Hozaifa, B., Lebda, F., Al-Khadra, A. and Al-Almaie, S. (2015). Nigella sativa improves glycemic control and ameliortes oxidative stress in patients with type 2 diabetes mellitus: Placebo controlled participant blinded clinical trial. PLos One. 10(2): e0113486. https://doi.org/10.1371/journal.pone.0113486.eCollection 2015.

    31. Keembiyehetty, C., Augustin, R., Carayannopoulos, M.O., Sarah, S., Manolescu, A., Cheeseman,  C.I. and  Moley, K.H. (2006). Mouse glucose transporter 9 splice variants are expressed in adult liver and kidney and are up-regulated in diabetes. Mol Endocrinol. 20(3): 686-697. https://doi.org/10.1210/me.2005-0010.

    32. Lay, A.C. and Coward, R.J.M. (2018). The evolving importance of insulin signaling in podocyte health and disease. Front Endocrinol (Lausanne). 9: 693. https://doi.org/10.3389/fendo.2018.00693.

    33. Lay, A.C., Tran, V.D.T., Nair, V., Betin, V., Hurcombe, J.A., Barrington, A.F. and Pope, R.J. (2024). Profiling of insulin-resistant kidney models and human biopsies reveals common and cell-type-specific mechanisms underpinning Diabetic Kidney Disease. Nature Communications. 15: 10018. https://doi.org/10.1038/s41467-024-54089-1.

    34. Linnemann, A.K., Baan, M. and Davis, D.B. (2014). Pancreatic β-cell proliferation in obesity. Adv Nutr. 5(1): 278-288. https:// doi.org/10.3945/an.113.005488.

    35. Lee, A.S., Lee, Y.J., Lee, S.M., Yoon, J.J., Kim, J.S., Kang, D.J. and Lee, H.S. (2012). Portulaca oleracea ameliorates diabetic vascular inflammation and endothelial dysfunction in db/ db mice. Evid Based Complement Alternat Med. 741824: 1-9. https://doi.org/10.1155/2012/741824

    36. Liang, X., Wang, P., Chen, B., Ge, Y., Athena, Y., Flickinger, B. and Malhotra, D.K. (2020). Glycogen synthase kinase 3β hyperactivity in urinary exfoliated cells predicts progression of diabetic kidney disease. Kidney Int. 97(1): 175-192. https://doi.org/10.1016/j.kint.2019.08.036.

    37. Liu, H., Liu, H.Y., Jiang, Y.N. and Li, N. (2016). Protective effect of thymoquinone improves cardiovascular function and attenuates oxidativestress, inflammation and apoptosis by mediating the PI3K/Akt pathway in diabetic rats. Mol Med Rep. 13(3): 2836-2842. https://doi.org/10.3892/mmr.2016.4823.

    38. Liu, J.J., Lee, T. and DeFronzo, R.A. (2012). Why do Sglt2 inhibitors inhibit only 30-50% of renal glucose reabsorption in humans? Diabetes. 61(1): 2199-2204. https://doi.org/10.2337/db12-0052.

    39. Livak, K.J. and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods. 25(4): 402-408. https://doi.org/10.1006/meth.2001.1262.

    40. Manna, P., Achari, A.E. and Jain, S.K. (2017). Vitamin D supplementation inhibits oxidative stress and upregulate Sirt1/Ampk/Glut4 cascade in high glucose-treated 3T3L1 adipocytes and in adipose tissue of high fat diet-fed diabetic mice. Arch Biochem Biophys. 615(1): 22-34. https://doi.org/10.1016/j.abb.2017.01.002.

    41. Marks, J., Carvou, N.J., Debnam, E.S., Srai, S.K. and Unwin, R.J. (2003). Diabetes increases facilitative glucose uptake and Glut2 expression at the rat proximal tubule brush border membrane. J. Physiol. 553(1): 137-145. https://doi.org/10.1113/jphysiol.2003.046268.

    42. Mather, A. and Pollock, C. (2011). Glucose handling by the kidney. Kidney Int Suppl. 79(120): S1-6. https://doi.org/10.1038/ki.2010.509.

    43. Medina, R.A. and Owen, G.I. (2002). Glucose transporters: Expression, regulation and cancer. Biol Res. 35(1): 9-26. https:// doi.org/10.4067/s0716-97602002000100004.

    44. Meerloo, J.V., Kaspers. G.J.L. and Cloos, J. (2011).  Cell sensitivity assays: The MTT assay. Methods Mol Biol. 731(1): 237-245. https://doi.org/10.1007/978-1-61779-080-5_20.

    45. Meziti, A., Meziti, H., Boudiaf, K., Mustapha, B. and Bouriche, H. (2012). Polyphenolic profile and antioxidant activities of Nigella sativa seed extracts in vitro and in vivo. World Acad Sci Eng Technol. 64(1): 24-32. https://doi.org/10.5281/zenodo.1329809.

    46. Mima, A., Murakami, A., Lee, R. and Lee, S. (2023). Predictive significance of glomerular insulin receptor substrate-1 in patients with diabetic kidney disease. Metabol Open. 18: 100240. https://doi.org/10.1016/j.metop.2023.100240.

    47. Mima, A., Ohshiro, Y., Kitada, M., Matsumoto, M., Geraldes, P., Li, C. and Li, Q. (2011). PKCβ-induced Selective Irs1 dysfunction and insulin resistance in renal glomeruli of rodent models of diabetes and obesity. Kidney Int. 79(8): 883-896. https://doi.org/10.1038/ki.2010.526.

    48. Mokashi, P., Bhatt, L.K., Khanna, A. and Pandita, N. (2017). Swertisin rich fraction from Enicostema littorale ameliorates hyper- glycemia and hyperlipidemia in high-fat fed diet and low dose streptozotacin induced type 2 diabetes mellitus in rats. Biomed Pharmacother. 96(1): 1427-1437. https://doi.org/10.1016/j.biopha.2017.09.153.

    49. Mueckler, M. and Thorens, B. (2013). The Slc2 (Glut) family of membrane transporters. Mol Aspects Med. 34(2-3): 121-138. https:// doi.org/10.1016/j.mam.2012.07.001.

    50. Norton, L., Shannon, C.E., Fourcaudot, M., Hu, C., Wang, N. and Ren, W. (2017). Sodium-glucose co-transporter (Sglt) and glucose transporter (Glut) expression in the kidney of type 2 diabetic subjects. Diabetesp Obes Metab. 19(9): 1322-1326. https://doi.org/10.1111/dom.13003.

    51. O’Neill, H.M. (2013). AMPK and Exercise: Glucose uptake and insulin sensitivity. Diabetes Metab. 37(1): 1-21. https://doi.org/ 10.4093/dmj.2013.37.1.1.

    52. Ozcan, B.G. and Kaya, H. (2025). Effects of varying ratios of hawthorn fruit extract on nesfatin-1, Glp-1 and ctni response in streptozotocin-induced diabetic rats. Indian Journal of Animal Research. 59(12): 2133-2137. doi: 10.18805/IJAR.BF-2045.

    53. Peng, J. and He, L. (2018). Irs posttranslational modifications in regulating insulin signaling. J. Mol Endocrinol. 60(1): R1-R8. https:// doi.org/10.1530/JME-17-0151.

    54. Paeng, J., Chang, J.H., Lee, S.H., Nam, B.Y., Hye-Young, K. and Kim, S. (2014).  Enhanced glycogen synthase kinase- 3â activity mediates podocyte apoptosis under diabetic conditions. Apoptosis. 19(12): 1678-90. https://doi.org/10.1007/s10495-014-1037-5.

    55. Park, J.E., Lee, J.S., Lee, H.A. and Han, J.S. (2018). Portulaca oleracea L. extract enhances glucose uptake by stimulating Glut4 translocation to the plasma membrane in 3T3-L1 adipocytes. J. Med Food. 12(3): 183-190. https://doi.org/10.1089/jmf.2017.4098.

    56. Rahmoune, H., Thompson, P.W., Ward, J.M., Smith, C.D., Hong, G., and Brown, J. (2005). Glucose transporters in human renal proximal tubular cells isolated from the urine of patients with non-insulin-dependent diabetes. Diabetes. 54(12): 3427-34. https://doi.org/10.2337/diabetes.54.12.3427.

    57. Raj, A. and Rani, S. (2024). Evaluating the efficacy of metformin and camel milk on streptozotocin-nicotinamide induced diabetic albino rats. Indian Journal of Animal Research. 1-8. doi: 10.18805/ajdfr.DR-2213.

    58. Rand, E.B., Depaoli, A.M., Davidson, N.O., Bell, G. and Burant, C.F. (1993). Sequence, tissue distribution and functional characteri- zation of the rat fructose transporter Glut5. Am J. Physiol. 264(6 Pt 1): G1169-76. https://doi.org/10.1152/ajpgi.1993.264.6.G1169.

    59. Rayasam, G.V., Tulasi, V.K., Sodhi, R., Davis, J.A. and Ray, A. (2009). Glycogen synthase kinase 3: More than a namesake. Br J. Pharmacol. 156(6): 885-898. https://doi.org/10.1111/j.1476-5381.2008.00085.x.

    60. Rizzo, B., Zambonin, L., Angeloni, C., Krejpcio, Z. and Olejnik, A. (2013). Steviol glycosides modulate glucose transport in different cell types. Oxid Med Cell Longev.  348169: 1-11. https://doi.org/10.1155/2013/348169.

    61. Sangi, S.M., Sulaiman, M.I., El-Wahab, M.F., Ahmedani, E.I. and Ali, S.S. (2015). Antihyperglycemic effect of thymoquinone and oleuropein, on streptozotocin-induced diabetes mellitus in experimental animals. Pharmacogn Mag. 11(2): S251- 257. https://doi.org/10.4103/0973-1296.166017.

    62. Shan, W.F., Chen, B.Q., Zhu, S.J., Jiang, L. and Zhou, Y.F. (2011). Effects of Glut4 expression on insulin resistance in patients with advanced liver cirrhosis. J. Zhejiang Univ Sci B. 12(8): 677-682. https://doi.org/10.1631/jzus.B1100001.

    63. Soetikno, V., Sari, F.R., Sukumaran, V., Lakshmanan, A.P., Harima, M., Suzuki, K., Kawachi, H. and Watanabe, K. (2013). Curcumin decreases renal triglyceride accumulation through AMPK- SREBP signaling pathway in streptozotocin-induced type 1 diabetic rats.  J. Nutr Biochem. 24(5): 796-802. https://doi.org/10.1016/j.jnutbio.2012.04.013.

    64. Sugawara-Yokoo, M., Suzuki, T., Matsuzaki, T., Naruse, T. and Takata, K. (1999). Presence of fructose transporter Glut5 in the S3 proximal tubules in the rat kidney. Kidney Int. 56(3): 1022-1028. https://doi.org/10.1046/j.1523-1755.1999.00635.x.

    65. Tiwari, S., Singh, R.S., Li, L., Tsukerman, S., Godbole, M., Pandey, G. and Ecelbarger, C.M. (2013). Deletion of the insulin receptor in the proximal tubule promotes hyperglycemia. J. Am Soc Nephrol. 24(8): 1209-1214. https://doi.org/10.1681/ASN.2012 060628.

    66. Usta, A. and Dede, S. (2017). The effect of thymoquinone on nuclear factor kappa b levels and oxidative DNA damage on experimental diabetic rats. Pharmacogn Mag. 13(S3): 458-461. https://doi.org/10.4103/pm.pm_134_17.

    67. Umino, H., Hasegawa, K., Minakuchi, H., Muraoka, H., Kawaguchi, T., Kanda, T., Tokuyama, H., Wakino, S. and Itoh, H. (2018). High basolateral glucose increases sodium-glucose cotransporter 2 and Reduces Sirtuin-1 in Renal Tubules through Glucose Transporter-2 Detection. Sci Rep. 8(1): 6791: 1-13. https://doi.org/10.1038/s41598-018-25054-y.

    68. Usta, A., Dede, S., Yüksek, V. and Öner, A.C. (2024a). Determination of the effect of thymoquinone on DNA damage in kidney cells applied to high glucose depending on time and dosage by comet assay. TURJAF. 12(8): 1359-1365. https:// doi.org/10.24925/turjaf.v12i8.1359-1365.6573.

    69. Usta, A., Yüksek, V., Çetin, S. and Dede, S. (2024b). Lycopene prevents cell death in NRK-52E cells by inhibition of high glucose- activated DNA damage and apoptotic, autophagic and necrotic pathways. J. Biochem Mol Toxicol. 38(3): e23678: 1-13. https://doi.org/10.1002/jbt.23678.

    70. Vallon, V., Platt, K.A., Cunard, R., Schroth, J., Whaley, J., Thomson, S.C., Koepsell, H.  and  Rieg, T. (2011). Sglt2 mediates glucose reabsorption in the early proximal tubule. J. Am Soc Nephrol. 22(1): 104-12. https://doi.org/10.1681/ASN.2010030246.

    71. Vestri, S., Okamoto, M.M., de Freitas, H.S.,  Eichler, R.A.D.S., Nunes, M.T., Morimatsu, M., Heimann, J. and Machado, U.F.  (2001). Changes in sodium or glucose filtration rate modulate expression of glucose transporters in renal proximal tubular cells of rat. J. Membr Biol. 182(2): 105-112. https://doi.org/10.1007/s00232-001-0036-y.

    72. Watson, R.T. and Pessin, J.E. (2001). Intracellular organization of insulin signaling and Glut4 translocation. Recent Prog Horm Res. 56(1): 175-193. https://doi.org/10.1210/rp.56.1.175.

    73. Welsh, G.I., Hale, L.J., Eremina, V., Jeansson, M., Maezawa, Y.,  Lennon, R. et al. (2010). Insulin signaling to the glomerular podocyte is critical for normal kidney function. Cell Metab. 12(1): 329-340. https://doi.org/10.1016/j.cmet.2010.08.015.
    In this Article
      Contact us
      Follow us
      Published In
      Indian Journal of Animal Research

      Editorial Board

      View all (0)