| Part one: Inhalation of hydrogen gas ameliorates glyoxylate-induced calcium oxalate deposition and renal injury in miceObjective: The aim of this study is to evaluate the protective effect of inhalation of high dose hydrogen gas(H2) to glyoxylate induced renal calcium oxalate(Ca Ox) crystal deposition and renal injury in mice.Methods: Rodent renal CaOx crystal deposition model was introduced by intra-abdominal injection of glyoxylate(100 mg/kg/d) for 5 days. The mixed gas consisting of 67% H2 and 33% O2 was produced by hydrogen oxygen nebulizer. Twenty-four male C57BL/6 mice were randomly divided into the following four groups of 6 mice each:(1) Nitrogen-oxygen gas(NO) group: The animals inhaled mixed gas consisting of 67% N2 and 33% O2(vol/vol) for 30 minutes daily for 7 consecutive days in addition to daily intra-abdominal injections with a normal volume of saline from day 3.(2) Hydrogen-oxygen gas(HO) group: The animals inhaled mixed gas consisting of 67% H2 and 33% O2(vol/vol) for 30 minutes daily for 7 consecutive days in addition to daily intra-abdominal injections with a normal volume of saline from day 3.(3) Calcium oxalate(Ca Ox) group: The animals inhaled mixed gas consisting of 67% N2 and 33% O2(vol/vol) for 30 minutes daily for 7 days in addition to daily intra-abdominal injections with glyoxylate from day 3.(4) Hydrogen-oxygen gas plus calcium oxalate(HO plus Ca Ox) group: The animals inhaled mixed gas consisting of 67% H2 and 33% O2(vol/vol) for 30 minutes daily for 7 days in addition to daily intra-abdominal injections with glyoxylate from day 3. By the end of study, the serum, urine and renal tissue were collected for biochemical and pathological assay. We measured the level of serum excretion of kidney injury molecule-1(KIM-1) to evaluate renal injury, the ratio of calcium and creatinine in urine to evaluate the excretion of calcium, the content of calcium in renal tissue and von Kossa staining to evaluate crystal deposition. The levels of renal malondialdehyde(MDA), 8-hydroxydeoxyguanosine(8-OHd G), myeloperoxidase(MPO) and the activities of superoxide dismutase(SOD), glutathione(GSH) and catalase(CAT) were measured to evaluate the redox state. The renal expressions of osteopontin(OPN), CD44, monocyte chemoattractant protein-1(MCP-1) and interleukin-10(IL-10) were measured to evaluate the inflammation state. To further evaluate the potential protective effect of H2, renal cell apoptosis was assessed using a terminal deoxytransferase uridine triphosphate nick end labeling(TUNEL) assay.Results:(1) The level of serum excretion of KIM-1 increased significantly(P<0.05) in Ca Ox group compared with NO group, and the effect was reversed in HO plus Ca Ox group. Meanwhile, there was no effect on the level of serum excretion of KIM-1 in HO group.(2) The ratio of calcium and creatinine in urine significantly increased in the Ca Ox group compared with the NO group(P<0.05) and decreased in the HO plus Ca Ox group(P<0.05). Additionally, the ratio of phosphate and creatinine increased in the Ca Ox group(P<0.05) and decreased in the HO plus Ca Ox group(P<0.05). Additionally, it showed no effect on the ratio of calcium, phosphate and creatinine in HO group compared with NO group.(3) Von Kossa staining showed that the histological examination of kidneys in the Ca Ox and HO plus Ca Ox groups, crystals were observed in the lumen(x400 magnification) and were primarily located at the border between the renal cortex and medulla(x40 magnification). Compared with the Ca Ox group, histological examination of kidneys in the HO plus Ca Ox group showed fewer crystal depositions in the kidney, and no crystals were observed in the NO or HO groups. The concentration of calcium in renal tissue increased significantly(P<0.05) after the mice were administered glyoxylate and decreased significantly(P<0.05) in the HO plus Ca Ox group compared with the Ca Ox group. H2 pretreatment alone showed no effect on the renal calcium level.(4) The levels of MDA and 8-OHd G of renal tissue increased significantly(P<0.05), and the activities of SOD and CAT and the GSH level of renal tissue decreased significantly(P<0.05) in the Ca Ox group. In the HO plus Ca Ox group, a significant reduction of MDA and 8-OHd G levels(P<0.05) was observed, and a significant increase in SOD and CAT activities and GSH levels(P<0.05) was observed. However, the level of MPO did not change significantly. There was no effect on the level of MDA, 8-OHd G and MPO and the activities of SOD, CAT and GSH by H2 pretreatment alone.(5) Immunostaining was used to determine the expression of OPN, CD44, MCP-1 and IL-10 in the kidneys. Staining for OPN, CD44 and MCP-1 increased significantly in Ca Ox group compared with NO group, and demonstrated a marked reduction in the kidneys of HO plus Ca Ox group. Compared with the NO group, the expression of IL-10 increased in the Ca Ox group, and a markedly increased expression was clearly observed in the HO plus Ca Ox group.(6) The number of TUNEL-positive cells was dramatically increased in Ca Ox group, to 8.7%, but this increase was dramatically blunted in HO plus Ca Ox group, to 3.1%. There was no obvious TUNEL-positive cells in NO and HOgroups.Conclusions: Inhalation of high dose H2 ameliorates Ca Ox crystal induced renal tubule injury, and reduces the excretion of calcium in urine and the concentration of calcium in renal tissue, renal crystallization, renal oxidative injury and inflammation and the tubule apoptosis. It may be a candidate agent with few adverse effects for prevention of nephrolithiasis.Part two: Inhalation of hydrogen gas ameliorates calcium oxalate crystal-induced renal injury based on serum metabonomics approach in miceObjective: The therapeutic effect of hydrogen gas on calcium oxalate crystal-induced renal injury is explored by analyzing serum metabolic profiles in mice, with ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry(UPLC/Q-TOF-MS).Methods: Eighteen male C57BL/6 mice were randomly divided into the following three groups of 6 mice each:(1) Nitrogen-oxygen gas(NO) group: The animals inhaled mixed gas consisting of 67% N2 and 33% O2(vol/vol) for 30 minutes daily for 7 consecutive days in addition to daily intra-abdominal injections with a normal volume of saline from day 3.(2) Calcium oxalate(Ca Ox) group: The animals inhaled mixed gas consisting of 67% N2 and 33% O2(vol/vol) for 30 minutes daily for 7 days in addition to daily intra-abdominal injections with glyoxylate from day 3.(3) Hydrogen-oxygen gas plus calcium oxalate(HO plus Ca Ox) group: The animals inhaled mixed gas consisting of 67% H2 and 33% O2(vol/vol) for 30 minutes daily for 7 days in addition to daily intra-abdominal injections with glyoxylate from day 3. By the end of study, the serum was collected for metabonomics analysis.Results: Analyzing the serum metabolic profiles showed that the metabolic profiles could be distinguished in NO, Ca Ox and HO plus Ca Ox groups. Compared with the NO group, there were 26 metabolites significantly difference in Ca Ox group. They were potential markers of Ca Ox crystals induced renal injury in mice. Inhaled of high dose hydrogen gas, 21 metabolites were reversed significantly(P<0.05) in HO plus Ca Ox group compared to Ca Ox group, and 5 metabolites were reversed but not significantly(P>0.05). These metabolites mainly included fatty acid metabolism, phospholipid metabolism and amino acid metabolism, the most of them were canitine metabolites.Conclusions: Inhaled of high dose hydrogen gas could effectively regulate the fatty acid metabolism, phospholipid metabolism and amino acid metabolism for Ca Ox crystals induced renal injury in mice. |
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