Effects of Hydrogen Sulphide, a Gaseous Signal Molecule, in Rat of Tubulointerstitial Fibrosis
|Keywords||hydrogen sulfide tubulointerstitial fibrosis angiotensin Ⅱ transforming growth factor betal type Ⅲ collagen kidney injury molecule-1 ectodermal dysplasia1 proliferating cell nuclear antigen|
Background Tubulointerstitial fibrosis (TIF) is the final common pathway to end-stage renal disease. Understanding the mechanisms of TIF is essential in establishing novel therapeutic strategies for the prevention or arrest of progressive kidney diseases. Endogenous hydrogen sulfide (H2S), a new gaseous signal molecule, is synthesized by L-cysteine under the action of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) in mammalian tissues. Endogenous H2S participates in a number of physiological and pathophysiological processes and exerts a protective effect in cardiovascular system, digestive system, endocrine system, nervous system, immune system and so on. The growing evidences demonstrated the renal protective effect of H2S against renal injury multiple factors induced such as diabetic nephopathy, hyperhomocysteinemia, hypertensive nephropathy, hyperhomocysteinaemia, renal ischemia/reperfusion injury and so on. But the role of H2S in TIF caused by chronic renal failure is unclear.Objectives Unilateral ureteral obstruction (UUO) rat model will be established to observe level of serum H2S, expressions of angiotensin II (Ang Ⅱ), transforming growth factors-β1(TGF-β1), type III collagen (Col-III), Kidney Injury Molecule-1(KIM-1)(ED-1), proliferating cell nuclear antigen (PCNA), and the effects of sodium hydrosulphide, a hydrogen sulphide donor, in TIF induced by UUO.Methods TIF was established in rat model induced by UUO.96Sprague-Dauley(SD) male rats were divided into four groups randomly:sham-operated group(n=24), UUO model group(n=24), sodium hydrosulphid low-dose group (low-dose group, n=24) and sodium hydrosulphid high-dose group(high-dose group, n=24). Rats were accepted intraperitoneally sodium hydrosulphid (7.0μmol/kg, twice a day), and sodium hydrosulphid (1.4μmol/kg, twice a day), respectively. Rats in sham-operated group and UUO model group were injected intraperitoneally with identical voluminal normal saline.8rats in each group were killed randomly at7d,14d and21d, respectively. The concentration of plasma H2S was measured. Gross appearance of obstructed kidneys in experimental rats were observed. Renal tubulo interstitial damage and interstitial fibrosis were evaluated with routine Hematoxylin and eosin (HE) staining and Masson staining under microscope. The expressions of AngⅡ, TGF-β1, Col-Ⅲ, KIM-1, ED-1, PCNA were measured with immunohistochemical staining.Results①The kidney structure in sham-operated group is normal. However, the obstructed kidneys in the model group were markly enlarged, pale. The boundary between renal cortical and medulla was indefinite. Renal parenchyma became thin, and dilatating collecting ducts were observed. The changes mentioned above were more severier with prolongation of UUO. While, compared the kidney with UUO, worsen degree of obstructed kidneys in sodium hydrosulphide-treatment group was lessen at three time point, respectively.②Renal pathological changes in sham-operated group were not predominant. However, HE staining showed inflammatory cells infiltrated in interstitial district and diffusing vacuolar degeneration in renal tubule in the renal tissue of UUO model group after UUO. Partial renal tubules display focal distend, atrophy, necrosis and interstitial fibrosis were more serious. These changes mentioned above got gradually severe with the time goes on. But, the glomerular was almost normal. These changes in sodium hydrosulphide-treated group were lighter than that in UUO model group (P<0.05). The renal tubulointerstitial damage was greater in model group than that in sodium hydrosulphide-treated group by pathologic image analysis (P<0.05). We found interstitial expand, fibrous tissues proliferation in obstructed kidnys, and the degree of TIF got gradually severe as the time goes. However, the glomeruluar was almost normal. These changes in sodium hydrosulphide-treated group were lighter than that in UUO model group. The interstitial fibrosis area expanded markedly in UUO model group by pathologic image analysis. The interstitial fibrosis area of sodium hydrosulphide-treated group was decreased markedly compared with UUO model group (P<0.05).③Expressions of AngⅡ, TGF-β1, Col-Ⅲ, KIM-1, PCNA were rather low in sham-operated group, while interstitial fibrosis and injury tubule were becoming aggravated in UUO model group. ED-1express most on14d and express lower on21d than14d.Meanwhile, compared with the UUO model group, expressions levels of AngⅡ, TGF-β1, Col-Ⅲ, KIM-1, ED-1, PCNA in high-dose group and low-dose group at different time point were less (P<0.05).④Positive correlation between expressions of AngⅡ and TGF-β1, Col-Ⅲ, KIM-1, PCNA, the tubulointerstitial score and relative area of interstitium were analysed (r=0.881,0.975,0.951,0.971,0.971,0.907, P<0.01) respectively. Positive correlation between expressions of TGF-β1and Col-Ⅲ, KIM-1, PCNA, the tubulointerstitial score and relative area of interstitium were present (r=0.872,0.949,0.982,0.937,0.935, P<0.01) respectively. Positive correlation between expressions of Col-Ⅲ and KIM-1, PCNA, the tubulointerstitial score and relative area of interstitium were present (r=0.880,0.975,0.951,0.911, P<0.01) respectively. Positive correlation between expressions of KIM-1and PCNA, the tubulointerstitial score and relative area of interstitium were present (r=0.832,0.972,0.865, P<0.01) respectively. Positive correlation between expressions of ED-1and the tubulointerstitial score, relative area of interstitium at all time points were present (r=0.757,0.853on7d. r=0.897,0.984on14d. r=0.833,0.964on21d, P<0.01) respectively. Positive correlation between expression of PCNA and the tubulointerstitial score, relative area of interstitium were present (r=0.892,0.864, P<0.01) respectively.⑤Compared with control group, the concentration of plasma H2S in UUO group decreased signifcantly on7d (P<0.001), and the concentration increased clearly with the time go on (P<0.001). We also found that the concentration in treated group was more than UUO model group, but still lower than sham-operated group.Conclusions①H2S attenuates renal injury and delay progression of TIF partly against infiltration of monocyte/macropage in interstitial area and lesion of macropage-TGF-β1-renal fibrosis axis;②H2S attenuates renal injury and delay progression of TIF partly through suppressing proliferation of fibroblasts, tubular epithelial cell(TECs), renal tubular interstitial cell transdifferentiation and decressing over accumulation of Col-III, a major component of extracellular matrix (ECM);③H2S attenuates renal injury and delay progression of TIF partly by affecting Ang Ⅱ-TGF-β1-ECM axis to inhibit over accumulation of ECM.