Long term of diabetes represents a threat to the patient’s kidney known as Diabetes nephropathy (DN), which is a major diabetic complication caused by hyperglycemia, which damages the nerves and blood vessels. DN is still the most noted explanation of end stage renal disease (ESRD) worldwide [28].
Kidney damage was obvious in the current study, where elevated creatinine and urea serum levels were recorded in untreated diabetic group. Greater protein catabolism is linked to a higher urea concentration in diabetic rats. The elevated serum levels of urea and creatinine were clear indicators of impaired kidney function, which lead to nephrotoxicity due to induction of STZ [29], as at least half of the kidney nephrons must be damaged for the serum creatinine level to increase. Also, Ceriello et al., [30] illustrated a favorable link between nephropathy advancement and hyperglycemia. As, it was recorded that diabetic rats' renal tubules accumulate glycogen in form of clusters by virtue of hyperglycemia [31] that was confirmed by our histopathological inspection of kidney tissue.
Contrarily, treated rats with DAPA displayed enhancement in renal function parameters compared to untreated diabetic animals, by reason of the prohibition of endoplasmic reticulum stress, inflammation, apoptosis and fibrosis, and due to reduction of lipids accumulation in the diabetic rats’ kidneys [32]. It was in harmony with Thomas [33], who denoted the possible anti-inflammatory and antioxidant effects of DAPA through decreasing the proximal tubule's reabsorption of glucose and sodium.
Furthermore, Diabetic rats treated with mulberry fruit and leaves extract and mix showed an improvement in serum glucose, urea and creatinine levels. Similarly, Abouzed et al., [20] stated that the black mulberry fruit extract ameliorated the renal function biomarkers in diabetic rats. Mulberry extract might advantageously affect diabetic confusions, like renal dysfunction, by keeping up with blood glucose homeostasis in diabetic rats. Rutin, as a major polyphenol in the MFE, has a hypoglycemic impact because it increases the action of the insulin-dependent receptor kinase, which activates the pathway for insulin signaling and causes an increase in glucose absorption and GLUT4 translocation [34]. Min et al., [35] demonstrated that increased antioxidant enzyme action in the pancreas and kidneys was linked to mulberry's antioxidant properties. Our results also revealed that rats treated with mulberry leaves showed significant improvement in renal function tests compared to mulberry fruits group because of the high chlorogenic acid content, which has anti-inflammatory and antioxidant characteristics and slows the development of DN by modifying the Nrf2/HO-1 and NF-κB pathways [36].
The trans-membrane protein, Kidney injury molecule (KIM-1) has been identified as a particular biomarker for the early detection of acute and chronic kidney impairment in laboratory animals [37]. Also, estimated glomerular filtration rate (eGFR) has been determined using beta-2-microglobulin (β2-MG) [38]. Serum KIM-1 and β2-MG levels showed notable rise in diabetic untreated animals related to control animals. This could be attributed to injection of STZ which increases the free radicals that damage the proximal tubule of the kidney. The present results are analogous to Humphreys et al., [39] who stated that mice's tubule-interstitial fibrosis was caused by chronic KIM-1 expression. It has been demonstrated that when renal function deteriorates, the serum concentration of β2-microglobulin increased before the concentration of serum creatinine. However, β2-MG level in serum has been recommended as a superior indicator of glomerular filtration rate than creatinine level in serum [40]. Where, serum globulin β2-MG is of low molecular weight, has high stability and can be filtered by the glomerular filtration membrane before being absorbed by the proximal convoluted tubule. Increased serum β2-MG levels in T2DM patients frequently signify that the patient's glomerular filtration function is impaired [41]. This is also in accord with Kim et al., and Kamal et al., [42] [43].
The existent findings revealed that diabetic animals treated with DAPA showed significant decrement in KIM-1 level. KIM-1 levels might be an indicator of novel treatments like SGLT2 inhibitors that protect the kidneys. Where, SGLT2 inhibitors can minimize proximal tubular damage in diabetic kidney disease by lowering the need for re-absorptive activity thus improving the tubular hypoxia [44]. Also, MFE, MLE and mix groups showed significant decline in KIM-1 level correlated to the untreated diabetic one. This reduction could be due to antioxidant activity of mulberry extract where numerous researches had proven the antioxidant effect of black mulberry with different methods, including DPPH assay [45–47] that came in the same line with the present results. Additionally, several flavonoids and phenolic compounds isolated from black mulberry extracts demonstrated potent anti-diabetic effects via α-glucosidase inhibition [48] that reduces glucose level in the blood which leads to reduction of KIM-1 level.
Serum levels of AGEs, TGF-β1 and TNF-α were substantially raised in untreated diabetic group in comparison with the control one. AGEs are glycated proteins or lipids by the action of high glucose level [49]. One of the harmful impacts that non-enzymatic alteration of plasma proteins may have, is the formation of oxygen free radicals [50]. Glycation may alter the cell function, where the target protein and lipid get denaturated and lose their functionality, organopathy results from the tissue buildup of AGEs, cell receptor-mediated signalling pathways are activated, and oxidative and carbonyl stresses are produced [51]. It was previously recorded that, DN patients have higher levels of serum TNF-α and TNF receptors, which are indicators of renal deterioration and the likelihood of ESRD [52]. TNF-α is a pleiotropic cytokine that promotes pro-inflammatory mediators, apoptosis, and necroptosis via activating TNF receptors [53]. On the contrary, diabetic rats received DAPA showed significant decline in serum AGEs, TNF-α and TGF-β1 in contrast to rats in diabetic group. As previously demonstrated by Moses et al., [54] that type 2 diabetes patients may have raised SGLT-2 proteins levels, which raise the glucose renal threshold and aggravate hyperglycemia giving rise to renal damage and diabetic nephropathy. Therefore, oral administration of SGLT-2 inhibitors prevent the glucose reabsorption in order to manage blood sugar levels [55] and reduce renal tubule fibrosis and inflammatory response [56]. Over and above, Yuan et al., [57] observed that IL-1, TNF-α and IL-6 were reduced by dapagliflozin, which in turn reduced kidney fibrosis and inflammation. Mulberry leaves demonstrated anti-inflammatory effects by means of IL-6, TNF-α and IL-1β prohibition which in turn suppress NF-κB, which is implicated in inflammation brought on by macrophage activation [58].
Transforming growth factor β1 (TGF-β1) is a versatile cytokine that regulates a variety of biological processes, including apoptosis [59], cell division [60] and immunity [61], plus patients with diabetes mellitus have higher levels of TGF-β1 mRNA and protein expression in their kidneys [62]. As a result of TGF-β1 promoting extracellular matrix formation and mesangium cell hypertrophy, the rate of glomerular filtration is reduced, and chronic renal failure results. As well known that, diabetes decreases the glucose availability, the metabolic switch from glycolysis to oxidative phosphorylation takes place by utilizing more fatty acids as a source of energy [63]. Therefore, the mitochondrial electron transport chain stimulates the three fundamental hyperglycemic deterioration pathways - generation of AGEs, polyol pathway motivation and protein C kinase stimulation - and also boosts superoxide production [64]. Additionally, excessive hyperglycemia triggers reactive oxygen species (ROS) through NADPH oxidase, mitochondrial electron transport chain and protein C kinase [65].
It has been documented that, the primary ROS generator in the vasculature is NADPH oxidase [66]. As well, through NADPH oxidase, AGEs can exactly motivate the ROS genesis [67].
Our results declared that, untreated diabetic group was considerably greater than the diabetic treated groups and the control one regarding renal tissue IL-6, NADPH-oxidase and NF-κB. It has been discovered that a high glucose level led to the provocation of NF- κB and the stimulation of pro-inflammatory cytokines [68]. AGEs ligated to particular receptors (RAGEs) can also trigger NF-κB stimulation [69]. Yet, a NF-κB-dependent pathway increased IL-6 expression in podocytes subjected to raised levels of glucose [70].
To the contrary, diabetic rats received dapagliflozin manifested significant reduction in renal NF-κB and IL-6 expression in correspondence to untreated diabetic rats. In addition to suppressing NF-κB DNA binding action induced by extreme glucose, the SGLT2 inhibitor reduced IL6 expression [71]. The current study noted DAPA’s useful impacts on inhibition of renal NF-κB in diabetic rats, with subsequent diminution of IL-6 generation. Besides, Yao et al., [55] studied the reno-protective impact of DAPA on human diabetic nephropathy via inhibiting NF-κB pathway.
Moreover, MFE, MLE and mix groups significantly diminished renal NF-κB and IL-6 related to the untreated diabetic group. These outcomes could be coming from the presence of flavonoids and chlorogenic acid, which regulate NF-κB expression and prevent inflammation-related oxidative stress. Additionally, they are crucial in the inflammatory cytokine signaling process [72]. Moreover, Chen et al., [45] said that the anti-inflammatory properties of total flavonoids of mulberry may be related to their antioxidant capacity.
Renal NADPH oxidase was actually diminished in DAPA group in correspondence to the untreated diabetic one. The conservative outcome of dapagliflozin has been related to glucose level normalization, loss of body weight, natriuresis and the fall of intra-glomerular pressure [73, 74]. It was similar to Terami et al., [10], who found that dapagliflozin may lessen oxidative strain by inhibiting the output of ROS in the kidneys of mice that are derived from Nox4. Moreover, Liu et al., [75] concluded that the inhibition of AGEs/RAGE and the prohibition of NADPH oxidase and NF-κB stimulation, are linked to the influence of pioglitazone on insulin resistance in rats consuming fructose.
Moreover, renal NADPH oxidase was significantly decreased in rats supplied with MFE, MLE and mix groups corresponded to the untreated diabetic one. It could be accounted for the existence of anthocyanins in the extract of mulberry fruit, which are outstanding antioxidants that are more capable for hunting free radicals [76]. Also, the presence of flavonoids and phenolic compounds which are the largest phytochemical molecules owning a strong antioxidant action [77, 78]. It is similar to Ji et al., [79] who demonstrated that mulberry leaves inhibited the activity of NADPH oxidase.
Both the development of chronic renal failure and normal renal physiology are significantly influenced by IL-10. In a healthy adult kidney, the primary local source of IL-10 is mesangial cells [80]. Mesangial cells that have been activated begin to secrete a lot of chemokines, cytokines and extracellular matrix proteins which affect the renal IL-10 expression. In turn, these factors have affect the mesangial cells and mediate interactions with blood inflammatory cells and endothelial and epithelial tubular cells [81]. So, in the existing study, the untreated diabetic group showed inhibition in renal IL-10 gene expression compared to control group. It was parallel to Summers et al., [82], who reported that the histological changes and decreased renal function are both achieved by inhibition of IL-10. But, DAPA and mix treated rats exhibited raised IL-10 renal expression which explained the anti-inflammatory effect of DAPA. Also, Yuan et al., [57] recorded elevated levels of IL-10 in diabetic mice.
The earliest glomerular hemodynamic disruption in DN are largely mediated by the renin-angiotensin system (RAS). The kidney's juxtaglomerular cells produce the proteolytic enzyme renin. Angiotensinogen is converted by renin into angiotensin I, that is transformed into angiotensin II (Ang II) by the action of angiotensin-converting enzyme (ACE) [83]. Angiotensin II is another important protein that controls the expression and synthesis of cytokines that are implicated in various processes like cell proliferation, inflammation, fibrosis, and death [84, 85]. In the existing study, renal renin protein expression was decreased in the untreated diabetic group. While, diabetic treated groups with DAPA, MFE, MLE and their mix showed enhancement in renal IL-10 and renin expression when compared to untreated diabetic group. Dapagliflozin increases renal IL-10 and renin expression as a result of histopathological changes in kidney which declared nearly normal renal glomeruli and renal tubules. Similar to Yang et al., [86], who found an improvement in renal histopathological findings in diabetic rats treated with dapagliflozin.
Moreover, renal tissue of diabetic rats supplied with mulberry extracts revealed regular proximal renal tubules with nuclei that were either round or oval and cuboidal cytoplasm with prominent brush borders that increase the expression of renal IL-10 and renin in the current study. It was similar to Zhang et al., [87] who confirmed the effect of mulberry components in renal tissue.