Ameliorative Effects of Camel Milk and Its Exosomes on Diabetic Nephropathy in Rats

Contradictory results were obtained regarding the effects of extracellular vesicles such as exosomes (EXOs) on diabetes and diabetic nephropathy (DN). Some studies showed that EXOs, including milk EXOs, were involved in the pathogenesis of DN, whereas other studies revealed ameliorative effects. Compared to other animals, camel milk had unique components that lower blood glucose levels. However, little is known regarding the effect of camel milk and its EXOs on DN. Thus, the present study was conducted to evaluate this effect on a rat model of DN induced by streptozotocin. Treatment with camel milk and/or its EXOs ameliorated DN as evidenced by (1) reduced levels of kidney function parameters (urea, creatinine, retinol-binding protein (RBP), and urinary proteins), (2) restored redox balance (decreased lipid peroxide malondialdehyde (MDA) and increased the activity of antioxidants enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)), (3) downregulated expression of DN-related genes (transforming growth factor-beta 1 (TGFβ1), intercellular adhesion molecules 1 (ICAM1), and transformation specific 1 (ETS1), integrin subunit beta 2 (ITGβ2), tissue inhibitors of matrix metalloproteinase 2 (TIMP2), and kidney injury molecule-1 (KIM1)), and (4) decreased renal damage histological score. These results concluded that the treatment with camel milk and/or its EXOs could ameliorate DN with a better effect for the combined therapy.


Introduction
Persistent hyperglycemia in diabetic patients induces diabetic complications that negatively affect health outcomes [1]. Diabetic nephropathy (DN) is one of the most common microvascular problems in diabetic patients and is the main cause of end-stage kidney diseases and renal failure [2]. The key features of DN are prolonged proteinuria and glomerulosclerosis (thickened glomerular basement membrane). The main pathological lesions of DN involve abnormal mesangial cell proliferation, which initiates extracellular matrix aggregation and destroys podocytes, leading to glomerular sclerosis and tubulointerstitial fibrosis [3]. There is a close association between fibrosis/matrix remodeling and the progression of DN. Fibrosis-related genes (such as TGFβ1 and ICAM1), matrix remodeling-related genes (such as ETS1, ITGβ2, and TIMP2), and the kidney injury-related gene (KIM1) play important roles in DN pathogenesis [4][5][6]. Many treatment strategies in PBS. Exosomal protein concentration was determined by a BCA kit and aliquots of EXOs at a concentration less than 6 mg/mL were kept at −80 • C until use.

Characterization of Milk-Derived Exosomes
The diameter and shape of the EXOs were determined by using transmission electron microscopy (TEM). Before the examination, EXOs were fixed in 2.5% glutaraldehyde/2 h/room temperature, then 10 µL EXOs were mounted on a carbon-coated copper grid covered with nitrocellulose and examined by TEM (JEM2100, Joel Inc., Japan) at 80 kV. Size distribution of EXOs was measured by dynamic light scattering (DLS) by using a Nano Zeta Sizer System (Malvern Instruments) with a 633-nm laser wavelength and a 173 • scattering angle. EXOs were further characterized by the detection of specific exosomal proteins CD63 and CD81 by using flow cytometry. In brief, EXOs were incubated with either anti-CD63 (1:200, Santa Cruz, Germany) or anti-CD81 (1:200, Santa Cruz) for 1 h in the dark at room temperature. Data were detected by an Attune flow cytometer (Applied Biosystem, San Francisco, CA, USA), and a standard nanobead calibration kit containing beads (50 and 100 nm, Technologies Drive Fisher, Lenexa, KS, USA) was used to set the gate and count EXOs.

Chemical Induction of Diabetic Nephropathy
Rats were intraperitoneally (IP) injected with a single dose of STZ at a dose of 60 mg/kg body weight (BW) dissolved in 0.1 mol/L cold citrate buffer, pH 4.5 (Sigma-Aldrich Chemical Co). The volume of STZ injected was 0.25 mL/kg BW. Sucrose (15 g/L) was given to rats in drinking water for 2 days to decrease the death of rats from pancreatic β-cell damage induced by STZ. After 3 days of STZ injection, rats with fasting blood glucose of more than 250 mg/dL were considered diabetic and involved in the experiment. The desirable level of blood glucose (350 mg/dL) was maintained by subcutaneous injection of diabetic rats with long-lasting insulin (3 U/rat) to avoid ketonuria [47].

Experimental Design
Experimental protocols were approved by the Ethical Committee at the Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt with a license number of 32971/273. A total number of 60 male albino rats were enrolled in this study. Animals were kept in optimal environmental conditions (25 • C ± 2, 12-h light/dark cycle) and received a basal diet and water ad libitum. They were acclimatized to laboratory conditions for 2 weeks before the experiment. Rats were randomly divided into 5 groups. In the control (Cnt) group (n = 15), rats were IP injected with a single dose of 0.25 mL/kg body weight (BW) sodium citrate buffer as a vehicle. In the DN group (n = 15), rats were IP injected with a single dose of STZ (0.25 mL/kg BW) and left for 12 weeks without treatment. At the seventh week, 5 rats from this group and 5 from the Cnt group were euthanized by exsanguination and blood samples were collected and kidneys were dissected to confirm the occurrence of DN as revealed by significant increases in proteinuria and glomerular sclerosis relative to the Cnt group. In the CM group (n = 10), rats were injected with STZ as in the DN group and at the eighth week, they were orally administered CM (10 mL/rat/day) for one month, until the twelfth week [44]. In the EXO group (n = 10), rats were injected with STZ as in the DN group and at the eighth week, they were orally administered 1 mL camel milk EXOs (1.25 mg/kg BW) once per week for 1 month, until the twelfth week [32]. In the CM + EXO group (n = 10), rats were injected with STZ as in the DN group and at the eighth week, they were orally administered CM and EXOs as previously mentioned.

Sampling
At the end of the twelfth week, urine samples were collected from rats placed in metabolic cages. Blood samples were collected from the medial canthus of the eyes by capillary tubes and some samples were centrifuged at 3000 rpm for 5 min to get serum for the detection of kidney function parameters, and other whole blood samples were used to estimate fasting blood glucose. Rats were euthanized by exsanguination and kidneys were quickly excised, and some renal tissue specimens were homogenized, centrifuged at 12,000× g for 15 min at 4 • C, and the supernatant was used for the biochemical assay. Other kidney specimens were snap-frozen in liquid nitrogen for real time PCR or preserved in 10% formalin for histological examination.

Biochemical Parameters
The level of fasting blood glucose was determined by using a commercially available kit (Biodiagnostic, Egypt). Serum levels of creatinine and urea were estimated calorimetrically by using commercially available kits (Bio-Med, Egypt). Retinol-binding protein (RBP) concentration in serum was measured by an ELISA kit (LSBio, Inc., Seattle, WA, USA, # LS-F28065) according to the standard protocol of the manufacturer. The concentration of proteins in the urine was estimated based on the manufacturer's instructions (Fortress Diagnostics Ltd., Antrim, UK). The levels of lipid peroxidation biomarker malondialdehyde (MDA) and the activity of the antioxidant enzymes SOD, CAT, and GPx were measured in kidney homogenates by using commercially available kits (Biodiagnostics, Egypt) as previously detailed [48,49].

Real-Time PCR
The renal expression of fibrosis-related genes (TGFβ1 and ICAM1), matrix remodeling genes (ETS1, ITGβ2, and TIMP2), and the kidney injury-related gene (KIM1) following treatment with CM and/or its EXOs were detected by real-time PCR. First, total RNA was extracted from kidney specimens by using a Trizol reagent (Invitrogen, USA, Cat# 15596026). RNA concentration and purity were determined by using a Nanodrop (Q5000, Quawell, USA). Secondly, cDNA was synthesized from RNA by using the RevertAid H Minus Reverse (Thermo Scientific, #EP04 51). Finally, a PCR mix containing cDNA, 2XMaster Mix (QuantiTect SYBR Green), and primers (Table 1) was run in the Step One Plus thermal cycler (Applied Biosystem, USA) at the following conditions: one cycle initial denaturation at 94 • C for 4 min followed by 40 cycles of each denaturation at 94 • C for 40 s, annealing at 60 • C for 30 s, and extension at 72 • C for 30 s. The housekeeping gene β actin was utilized as an internal control. The results of qPCR were presented as fold change mean ± standard error of the mean (SEM) by using the Livak method (2 −∆∆Ct ) and as previously described [50].

Statistical Analysis
Data were checked for normal distribution before performing statistical analysis. Oneway ANOVA followed by Tukey's honestly significant difference as a post-doc test was applied by using GraphPad Prism 8 software to determine significant differences among the experimental groups. Data were presented as mean per group ± standard error of the mean (SEM), and the significance was declared at p < 0.05.

Characterization of CM-Derived EXOs
As examined under TEM, camel milk EXOs appeared as nanospheres with various sizes ranging from 50 to 100 nm ( Figure 1A). Size distribution, as detected by DLS, showed EXOs with different sizes ranging from 35 to 130 nm with a mean diameter of 87.3 ± 7.20 nm ( Figure 1B). The flow cytometry analyses ( Figure 1C) exhibited the presence of a high percentage of specific exosomal marker proteins CD63 (82.4%) and CD81 (80.2%). These results implied the effective isolation of EXOs from the camel milk.

Effects of CM and/or Its EXOs on Kidney Damage Parameters
Levels of kidney damage parameters (urea, creatinine, retinol-binding protein (RBP), and urinary proteins) and fasting blood glucose were significantly higher in STZ-injected rats (DN group) than in the control (Cnt) group ( Table 2). The levels of these parameters were significantly decreased in the three treated groups, with lowest levels in the cotreated (CM + EXO) group, compared to the DN group. The EXO group also showed significantly decreased urea and RBP levels relative to the CM group. However, the levels of

Effects of CM and/or Its EXOs on Kidney Damage Parameters
Levels of kidney damage parameters (urea, creatinine, retinol-binding protein (RBP), and urinary proteins) and fasting blood glucose were significantly higher in STZ-injected rats (DN group) than in the control (Cnt) group ( Table 2). The levels of these parameters were significantly decreased in the three treated groups, with lowest levels in the co-treated (CM + EXO) group, compared to the DN group. The EXO group also showed significantly decreased urea and RBP levels relative to the CM group. However, the levels of all parameters in the treated groups remained higher than in the control group. These results inferred that treatment with CM and/or its EXOs attenuated the elevated levels of kidney damage parameters and blood glucose induced by STZ in DN rats.

Effects of CM and/or Its EXOs on Oxidative Stress and Antioxidants Parameters
Rats in the DN group had significantly higher renal levels of the lipid peroxidation biomarker MDA and significantly lower renal levels of the antioxidant enzymes (SOD, CAT, and GPx) than the control group ( Figure 2). Treatments with CM and its EXOs each alone or in combination (CM and EXO) partially normalized the redox balance as indicated by the reduction of MDA and elevation of the antioxidant enzymes relative to the DN group. The co-treated group (CM and EXO) significantly achieved the best improvement followed by the EXO group compared to the CM group. However, none of the treatments returned the levels of these oxidative and antioxidant markers to normal levels. These results implied that CM and/or its EXOs restored oxidative stress associated with DN with the best effect when given together.

Effect of CM and/or Its EXOs on Fibrosis and Kidney Damage-Related Genes
The changes in relative renal expression of the fibrosis-related genes (TGFβ1 and ICAM1) and the kidney injury-related gene (KIM1) were monitored following treatment with STZ, CM, or EXO by using real-time PCR and the results were presented in Figure 3. The expression of these genes was significantly higher in the DN group than in the control group. However, DN rats treated with CM, EXO, or CM + EXO showed significantly reduced expression, with lowest levels in the co-treated group, compared to rats in the DN group. Again, none of these treatments restored the expression to normal levels. These results indicated that the administration of CM and its EXO could ameliorate fibrosis and renal injury caused by STZ in the rat model of DN with superior effects for the combined therapy. cated by the reduction of MDA and elevation of the antioxidant enzymes relative to the DN group. The co-treated group (CM and EXO) significantly achieved the best improvement followed by the EXO group compared to the CM group. However, none of the treatments returned the levels of these oxidative and antioxidant markers to normal levels. These results implied that CM and/or its EXOs restored oxidative stress associated with DN with the best effect when given together.  Values were expressed as mean ± SEM (n = 6/group). Columns carrying different letters (a (the highest value)-e (the lowest value)) are significantly different at p < 0.05. All groups were compared to each other. Each data point represents an average of three independent experiments with three replicates for each. Cnt, control group; DN, diabetic nephropathy group; CM, camel milk-treated group; EXO, camel milk exosomes-treated group; CM + EXO, cotreated group with camel milk and its exosomes.

Effect of CM and/or Its EXOs on Matrix Remodeling-Related Genes
The renal expression of matrix remodeling-related genes (ETS1, ITGβ2, and TIMP2) was significantly upregulated in the DN group compared to the control group (Figure 4). Individual and combined treatment with CM and its EXOs significantly downregulated ETS1, ITGβ2, and TIMP2 expression, with lowest expression in the CM + EXO group, compared to the DN group. Moreover, the expression of ETS1 and ITGβ2 was significantly lower in the EXO group than in the CM group. However, no significant difference was noticed in TIMP2 between the two groups. Interestingly, cotreatment with CM and EXO restored the normal mRNA levels of ITGβ2 and TIMP2. This signified that the administration of CM and its EXO had the potential to inhibit the expression of matrix remodeling genes induced by DN with best improvement when they were given together.

Effect of CM and/or Its EXOs on Matrix Remodeling-Related Genes
The renal expression of matrix remodeling-related genes (ETS1, ITGβ2, and TIMP2) was significantly upregulated in the DN group compared to the control group (Figure 4) Individual and combined treatment with CM and its EXOs significantly downregulated Figure 3. Effect of camel milk and/or its EXOs on renal expression of fibrosis-related genes (TGFβ1 and ICAM1) and the kidney injury-related gene (KIM1) as detected by real-time PCR. Values were expressed as fold change mean ± SEM (n = 6/group). Columns carrying different letters (a (the highest value)-e (the lowest value)) are significantly different at p < 0.05. All groups were compared to each other. Each data point represents an average of three independent experiments with three replicates for each. Cnt, control group; DN, diabetic nephropathy group; CM, camel milk-treated group; EXO, camel milk exosomes-treated group; CM + EXO, co-treated group with camel milk and its exosomes. lower in the EXO group than in the CM group. However, no significant difference w noticed in TIMP2 between the two groups. Interestingly, cotreatment with CM and EX restored the normal mRNA levels of ITGβ2 and TIMP2. This signified that the administr tion of CM and its EXO had the potential to inhibit the expression of matrix remodelin genes induced by DN with best improvement when they were given together. Values were expressed as fold change mean ± SE (n = 6/group). Columns carrying different letters (a (the highest value)-e (the lowest value)) a significantly different at p < 0.05. All groups were compared to each other. Each data point repr sents an average of three independent experiments with three replicates for each. Cnt, control grou DN, diabetic nephropathy group; CM, camel milk-treated group; EXO, camel milk exosome treated group; CM + EXO, co-treated group with camel milk and its exosomes. Figure 5 shows the histopathological changes in the renal cortex of rats followin treatment with STZ, CM, or its EXOs. The control (Cnt) group showed intact renal co puscle (arrow), containing normal renal glomeruli surrounded by Bowman's capsule wi Figure 4. Effect of camel milk and/or its EXOs on renal expression of matrix remodeling genes (ETS1, ITGβ2, and TIMP2) as detected by real-time PCR. Values were expressed as fold change mean ± SEM (n = 6/group). Columns carrying different letters (a (the highest value)-e (the lowest value)) are significantly different at p < 0.05. All groups were compared to each other. Each data point represents an average of three independent experiments with three replicates for each. Cnt, control group; DN, diabetic nephropathy group; CM, camel milk-treated group; EXO, camel milk exosomes-treated group; CM + EXO, co-treated group with camel milk and its exosomes. Figure 5 shows the histopathological changes in the renal cortex of rats following treatment with STZ, CM, or its EXOs. The control (Cnt) group showed intact renal corpuscle (arrow), containing normal renal glomeruli surrounded by Bowman's capsule with normal size capsular space in addition to normal proximal (black arrowheads) and distal (white arrowheads) convoluted tubules. In contrast, kidneys of rats in the DN group had notable histopathological alterations including degeneration in renal glomeruli with glomerular sclerosis and mesangial hyperplasia (thick arrow), vacuolar degeneration (thin arrows), and desquamation of tubular epithelium (black arrowheads), in addition to marked inflammatory cells infiltration (white arrowheads). Treatment with CM and its EXOs each alone or in combination (CM + EXO) exhibited slight, moderate, and markable improvement in renal histology, respectively, with the absence of glomerular sclerosis. In the CM group, some renal corpuscles and tubules appeared normal, but others showed some pathological lesions, including mild mesangial hyperplasia (white arrowhead), mild enlargement of capsular space (thick arrow), moderate desquamation and degeneration in renal tubules (black arrowheads), and moderate inflammatory cells infiltration (thin arrows). The EXO group exhibited mild degeneration in some renal glomeruli (thick arrow), mild vacuolar degeneration in some renal tubules (thin arrows), and focal infiltration of inflammatory cells (arrowhead). The CM + EXO group showed notable regenerative changes such as intact renal corpuscles (arrows) but with mild desquamation and degeneration of some renal tubules (arrowheads). As shown in Figure 5, the kidney damage score revealed significantly higher renal damage in the DN group than in the control group. In contrast, the levels of this score were significantly lower in the three treated groups, with the lowest level in the CM + EXO group, rather than in the DN group.

Effects of CM and/or Its EXOs on Renal Histology
Membranes 2022, 12, x FOR PEER REVIEW 12 of 18 Figure 5. Effect of camel milk and/or its EXOs on renal cortex structure as revealed by histopathological examination of H&E-stained slides, scale bars = 50 µ m. All labels (arrows and arrowheads) were explained in the main text. Values of the kidney damage score were expressed as mean ± SEM (n = 6/group). Columns carrying different letters (a (the highest value)-e (the lowest value)) are significantly different at p < 0.05. All groups were compared to each other. Cnt, control group; DN, diabetic nephropathy group; CM, camel milk-treated group; EXO, camel milk exosomes-treated group; CM + EXO, co-treated group with camel milk and its exosomes.

Discussion
In diabetic patients, chronic hyperglycemia induces a strong inflammatory response, oxidative stress damage, and glomerular hemodynamics which all cause glomerulosclerosis and proteinuria, thereby leading to DN [52]. Because the progress to end-stage renal disease is irreversible, it is necessary to find methods to delay renal damage progress [53]. There are many contradictions regarding the effect of EXOs derived from MSCs, cow, and human milk on diabetes and DN. A previous study reported ameliorated effect [40], but several other studies showed participation in diabetes and DN progression [33][34][35]37]. Figure 5. Effect of camel milk and/or its EXOs on renal cortex structure as revealed by histopathological examination of H&E-stained slides, scale bars = 50 µm. All labels (arrows and arrowheads) were explained in the main text. Values of the kidney damage score were expressed as mean ± SEM (n = 6/group). Columns carrying different letters (a (the highest value)-e (the lowest value)) are significantly different at p < 0.05. All groups were compared to each other. Cnt, control group; DN, diabetic nephropathy group; CM, camel milk-treated group; EXO, camel milk exosomes-treated group; CM + EXO, co-treated group with camel milk and its exosomes.

Discussion
In diabetic patients, chronic hyperglycemia induces a strong inflammatory response, oxidative stress damage, and glomerular hemodynamics which all cause glomerulosclerosis and proteinuria, thereby leading to DN [52]. Because the progress to end-stage renal disease is irreversible, it is necessary to find methods to delay renal damage progress [53]. There are many contradictions regarding the effect of EXOs derived from MSCs, cow, and human milk on diabetes and DN. A previous study reported ameliorated effect [40], but several other studies showed participation in diabetes and DN progression [33][34][35]37]. Unlike bovine and human milk, previous studies have reported beneficial effects of CM on diabetes and attributed this effect to its high levels of insulin [18,19]. Besides, CM-derived EXOs had a proliferative effect on normal pancreatic cells [42]. Therefore, this study aimed to evaluate whether CM and/or its EXOs could ameliorate DN in rats. To the best of our knowledge, this is the first study to report that CM-derived EXOs could attenuate the STZ-induced DN in rats as evidenced by restored kidney function and histology.
DN is characterized by raised urinary excretion of proteins and reduced renal function as indicated by an elevation in creatinine, urea, and RBP serum levels [40,44,52,54]. We also found significantly higher levels of these parameters in DN rats compared to the control group. DN is also associated with a disturbance of redox balance with induction of oxidative markers and inhibition of antioxidant enzyme activities [44,55]. Similarly, in the present study, DN rats exhibited significantly higher renal levels of the lipid peroxidation biomarker (MDA) and significantly lower activities of the antioxidant enzymes (SOD, CAT, and GPx) compared to the control group. Free radical overproduction induces the damage of various cellular components including DNA, proteins, and lipids. To protect these components from oxidative damage, the activities of antioxidant enzymes (such as SOD, GPx, and CAT) increase within the cells to get rid of reactive oxygen species (ROS) [56][57][58]. However, if this antioxidant defense system is interrupted by excessive release of ROS, the cellular components, particularly the cell membrane which is rich in phospholipid, will be damaged by lipid peroxidation [48,59,60].
At a molecular level, many genes such as fibrosis-related genes (TGFβ1 and ICAM1), matrix remodeling-related genes (ETS1, ITGβ2, and TIMP2), and the kidney injury-related gene KIM1 were involved in the pathogenesis of DN [4][5][6]61]. In support, we also found significantly higher expression of these genes in the renal tissue of DN rats relative to rats in the control group. Elevated free radical levels and the upregulation of TGFß1 expression are the main hallmarks of DN [55]. Excessive ROS can also induce overexpression of TGFß1 that exaggerating DN [62]. Upregulated expression of TGFß1 triggered renal fibrosis through the upregulation of extracellular matrix (ECM)-related genes such as ICAM1 [61]. Higher expression of ICAM1 attracted inflammatory cells, particularly macrophages, and T-cells which resulted in the over-release of inflammatory cytokines and chemokines resulting in renal fibrosis [63]. DN patients had higher serum levels of ICAM1 than nondiabetic individuals [64]. TGFß1 also induced the upregulation of the transcription factor ETS1 [65]. ETS1 plays an important role in the progression of DN through modulation of matrix metalloproteinase expression [66]. Indeed, upregulated expression of ETS1 is associated with TIMP2 expression and both genes participated in the progression of DN [5,6]. Moreover, ETS1 can induce DN in mice through upregulation of its downstream target ITGβ2 which is involved in matrix remodeling and the activation of neutrophil adherence in DN [6,18].
At the histological level, DN rats showed typical lesions of the nephropathy including degeneration in renal glomeruli with glomerular sclerosis and mesangial hyperplasia. Consistent with our findings, previous studies also reported similar pathological changes in DN animals [6,40,61]. As a result of these deleterious changes, nephrotoxicity caused by diabetes could induce the death of glomerular cells and vasodilatation of glomerular blood vessels [40,67]. Mechanical stretching of podocytes caused by increased blood flow to the kidneys may cause the podocytes foot process to become effaced and the cells to detach causing glomerular filtration abnormalities and proteinuria [68,69]. We also found marked infiltration of inflammatory cells, mainly macrophages. In agreement, Tesch [70] also reported excessive accumulation of macrophages in kidneys of DN rodent models and DN patients, and the degree of infiltration was positively associated with DN progression.
The main findings of the present study were the ameliorative effect of CM and its EXOs against STZ-induced DN as evidenced by restored kidney function (as indicated by the reduction of serum levels of urea, creatinine, and RBP) and redox balance (as revealed by decreasing MDA levels and increasing SOD, CAT, and GPx activities in the kidney), as well as the inhibited expression of TGFβ1, ICAM1, ETS1, ITGβ2, TIMP2, and KIM1 and the improved renal histology. A similar ameliorative effect for CM on DN induced by STZ was reported by Korish, Abdel Gader, Korashy, Al-Drees, Alhaider, and Arafah [44] who attributed this effect to CM hypoglycemic, antioxidant and anti-inflammatory properties [10][11][12]. CM is rich in insulin and insulin-like substances which are present within micelles preventing digestion in the stomach [18,19]. CM also had many components that possessed anti-oxidant and anti-inflammatory potential [71], which could also play a crucial role to relieve DN. Whey protein extracted from the CM inhibited the extended inflammation in diabetic rats by decreasing the release of pro-inflammatory cytokines such as TNFα, IL6, and IL1β [72]. CM also downregulated the expression of TNFα, IL1β, and NFκB in the kidney of 5-fluorouracil-treated rats [73].
To the best of our knowledge, this is the first study to report that CM-derived EXOs could attenuate the STZ-induced DN in rats. In agreement, EXOs derived from BM-MSCs also relieved the deleterious effects induced by STZ in the rat model of DN and improved renal function and structure [40]. Moreover, CM and MSCs-derived EXOs induced the proliferation of normal human pancreatic H6c7 cells [42] and pancreatic βcells [41], respectively. As most EXO actions are mediated through their cargo of miRNAs, the effect of CM-derived EXOs reported in our study could be attributed to the miRNAsmediated paracrine effect of these EXOs. Exosomal miRNAs could inhibit the expression of inflammation, fibrosis, and matrix remodeling genes and control the expression of antioxidant genes. Indeed, previous studies have reported antioxidant effects for CM-EXOs against oxidative effects induced by cyclophosphamide and breast cancer in rats [32,45]. Additionally, CM and its EXOs inhibited IL6, TNFα, and NFκB expression in spleens of immunocompromised rats and mammary tumor tissues [32,45]. In contrast, several other studies reported stimulatory effects for EXOs derived from cow/human milk, pancreatic β-cells or stem cells during DN pathogenesis. A previous study reported that EXOs derived from rats and human pancreatic β-cells participated effectively in the pathogenesis of type I diabetes through the activation of dendritic cells, which induced cytokine release and apoptosis of β-cells [33]. EXOs derived from MSCs resident in the pancreas of diabetic mice induce T cell-mediated destruction of β-cells [34]. Bone marrow (BM) MSC-derived EXOs loaded with miR-let7a-5p could induce DN [35]. Moreover, urine stem cell-derived EXOs loaded with miR-145 and miR-320c induced renal fibrosis through activation of TGFβ1 expression [36]. BM-MSCs-EXOs can also induce glomerular sclerosis by activating the PI3K/Akt/mTOR pathway [37]. Adipose tissue MSC-derived EXOs loaded with miR-486 triggered the overproduction of free radicals that modulate the PI3K/Akt/mTOR pathway leading to apoptosis of podocytes and DN progression [38]. The contradictory results regarding the effect of EXOs on DN may be attributed to the source of EXOs which could produce different exosomal miRNAs. Therefore, further studies are required to investigate the miRNAs content of camel milk EXOs and compare them with those derived from cow and human milk as well as MSCs. Further investigations are also required to perform proteomics, metabolomics, and RNA-seq of the exosomes from milk of different animals such as camel, goat, cow, and buffalo to compare their cargoes. As a bad side of EXOs, cancer cell-derived EXOs play a crucial role in the formation and progression of tumor. Recent studies used advanced tools such as localized surface plasmon resonance (LSPR), atomic force microscopy (AFM), and self-assembly gold nanoislands (SAM-AuNIs) to effectively detect these exosomal biomarkers [74][75][76][77][78][79][80]. It would be worth applying this advanced biosensing technology to biosense animal's health via milk-derived, exosomebased biomarkers.

Conclusions
Camel milk and its exosomes ameliorated the deleterious effects associated with STZ-induced diabetic nephropathy in rats through reduction of kidney damage scores, maintenance of redox balance, and inhibition of fibrosis and matrix remodeling-related genes. Thus, camel milk exosomes can be used as novel agents for the treatment strategies of diabetic nephropathy in the future. Further investigations are required to explain the exact molecular mechanisms underlying their effects with a special focus on miRNAs, proteins, and metabolites cargoes of exosomes.