الفهرس 
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Abstract
The burden of diabetes mellitus (DM) is rapidly rising. Current projections estimate the global prevalence of diabetic individuals to rise from 6.4% in 2010 to 7.7% in 2030. The main problem with DM is its macro- and microvascular complications.
Diabetic nephropathy (DN) is a progressive kidney disease caused by damage to the renal glomerular capillaries. It is due to longstanding DM, and affects about 40% of type 1 and type 2 diabetic patients. It is classified as a microvascular complication of diabetes, and defined as increased urinary albumin excretion (UAE) in the absence of other renal diseases. It increases the risk of death mainly from cardiovascular causes. DN remains the most common cause of end stage renal disease (ESRD), and is considered a prime reason for dialysis worldwide.
The biological actions of hydrogen sulfide (H2S) are still expanding, and its role in both renal physiology and pathology is still not well-understood. Therefore, in a trial to assess the underlying mechanisms of the renoprotective effects of H2S in DN, as well as its cross-talk with the other gasotransmitters; CO and NO, the present study was conducted on of 60 adult male albino rats of average weight 190 g. The overall survival ratio was 70% (42/60), they were divided into 7 groups; each group consists of 6 rats as follows:
1. Control rats.
2. Rats treated with NaHS “H2S donor”.
3. Rats treated with STZ “to induce early diabetic nephropathy”.
4. Rats treated with STZ and NaHS.
5. Rats treated with STZ, L-NAME “NO inhibitor”, and NaHS.
6. Rats treated with STZ, ZnPP-IX “CO inhibitor”, and NaHS.
7. Rats treated with STZ, glibenclamide, and NaHS.
All drugs used were given once daily for 1 week, starting 3 weeks after STZ injection to ensure the recovery of the kidneys from the acute mild nephrotoxic effects of STZ.
One day before the end of the study, each rat was housed separately in a metabolic cage for collection of 24 h urine. After that, the rats were sacrificed by decapitation.
The blood samples were immediately collected from the jugular veins in 10 ml Eppendorf tubes, allowed to clot, and then centrifuged at 3000 rpm for 20 minutes. The serum samples were separated in 2 ml Eppendorf tubes, and stored at -20oC until used. The kidneys were also removed from each rat, and then immediately frozen and kept at -80oC until used.
(A) Serum parameters:
Serum samples were used to determine the renal injury markers (RIM); urea, creatinine, uric acid and K+ levels.
(B) Urinary parameters:
Urine samples were used to determine urinary albumin excretion (UAE) and urinary albumin/creatinine (A/C) ratio.
(C) Renal tissue parameters:
Renal tissue samples were used to determine total antioxidant capacity (T-AOC), as well as total NO, TNF-α, CRP and IL-10 levels.
The results obtained in this study clearly demonstrated that:
STZ-induced diabetic rats had developed DN indicated by elevated RIM, UAE, and urinary A/C ratio. The development of DN was explained by decreased renal tissue T-AOC, increased renal tissue total NO level, increased renal tissue pro-inflammatory cytokines (TNF-α, and CRP), as well as decreased renal tissue anti-inflammatory cytokine (IL-10).
NaHS treatment led to improvement of the renal functions in the diabetic group. This improvement was indicated by decreased RIM, UAE, and urinary A/C ratio. This improvement was explained by increased renal tissue T-AOC, decreased renal tissue total NO level, decreased renal tissue TNF-α and CRP levels, as well as increased renal tissue IL-10.
It is becoming increasingly clear that there are important interactions among the gasotransmitters: NO, CO, and H2S. Previous studies reported that there is a possible crosstalk between them. In order to investigate whether the renoprotective effects of H2S in case of DN are dependent on NO and CO pathways or not, we treated the diabetic rats with either; NaHS and L-NAME (NO inhibitor), or NaHS and ZnPP-IX (CO inhibitor).
Furthermore, to assess if K+ has a role in the mechanism by which H2S produced its renoprotective effects in the case of DN, glibenclamide (Gli), a potent and selective ATP-sensitive potassium channels (KATP) blocker, was given with NaHS to diabetic rats.
Regarding RIM, L- NAME combination produced the worst higher levels, while ZnPP combination produced the best lower levels. NaHS treatment alone produced the best improvement of kidney function parameters in diabetic rats. As regards urinary parameters, L- NAME combination produced the worst higher levels, while Gli combination produced the best lower levels in the diabetic rats.
Regarding renal tissue T-AOC, L- NAME and ZnPP combinations produced the worst lower levels, while Gli combination produced the best higher levels in the diabetic rats. As regards renal total NO level, L- NAME combination produced the lowest total NO level, while ZnPP produced the higher level.
Regarding renal tissue inflammatory cytokines, the improvement in the pro-inflammatory (TNF-α, and CRP) levels produced by NaHS was reduced by combination with either L-NAME or ZnPP but not Gli. L- NAME and ZnPP combination produced the worst higher levels, while NaHS treatment alone as well as Gli combination produced the best lower levels. On the other hand, the improvement in the anti-inflammatory (IL-10) level produced by NaHS was reduced by combination with ZnPP but not with either L-NAME or Gli. ZnPP combination produced the worst lower levels, while treatment with NaHS alone as well as L-NAME and Gli combinations produced the best higher levels
Conclusion:
In conclusion, the present study has shown that:
1- DN induced by induction of diabetes, and indicated by elevated RIM, UAE, and urinary A/C ratio.
2- DN was explained by decreasing renal tissue T-AOC and anti-inflammatory cytokine (IL-10), and by increasing renal tissue total NO level and pro-inflammatory cytokines (TNF-α, and CRP).
3- H2S is effective in ameliorating the development of DN through decreasing renal NO level, and through its anti-inflammatory and antioxidative stress effects.
4- The renoprotective effects of H2S are dependent partly on other gasotransmitters (NO and CO) and ATP-sensitive K+ channels; mainly NO pathways.
Limitations:
• The limitation of the present study is that it did not show any involvement of apoptotic markers in the sulfide’s renoprotective mechanisms and its link with endogenous CO or NO.
Recommendations:
• Since H2S has been provided in this study as a therapy for renal protection in diabetes mellitus, this can open the door for further future studies, in which multiple dosing groups can be established with involving distant organs.
• A similar study in human subjects is desirable to determine whether these results can be applied to human diabetic patients or not.