肾上腺切除对5/6肾切除大鼠肾脏的保护作用

目的通过切除5／6肾切除大鼠的肾上腺,探讨醛固酮对慢性肾脏疾病发生及发展的作用。方法雄性Wister大鼠分成5组:(1)假手术组(SHAM组);(2)5／6肾切除组(SNX组); (3)SNX+双肾上腺切除组(ADX组);(4)ADX+地塞米松组(DXM组);(5)ADX+地塞米松+醛固酮组(ALDO组)。所有大鼠自由饮用生理盐水,于成模第8周测定大鼠收缩压、各项血尿指标及肾小球硬化程度。应用Western印迹和实时定量PCR检测大鼠肾皮质TGF-β1、醛固酮受体 (MR)及保护MR的酶11β-羟类固醇脱氢酶2(11β-HSD2)的mRNA表达水平。结果 SNX组大鼠表现为明显的白蛋白尿、高血压、肾小球硬化、肾皮质TGF-β1表达升高,血醛固酮水平是 SHAM组的4倍以上。与SNX组比较,ADX组大鼠血浆醛固酮水平明显下降,同时病变明显改善 [尿白蛋白(mg／24 h)19．7±2．0比31．7±1．7,P<0．01;收缩压(mmHg)173．8±4．3比210．4±4．1,P <0．01;肾小球硬化指数38．2±7．9比92．3±6．7,P<0．01;TGF-β1 3．8±0．6比10．3±1．2,P< 0．01]。ALDO组的血浆醛固酮水平为SHAM组的近2倍,与ADX组比较,以上病变又加重[尿白蛋白(mg／24 h)24．9±1．4,收缩压(mmHg)201．5±4．5,肾小球硬化指数88．1±7．2,TGF-β1 5．8± 0．6,P均<0．01]。肾脏皮质MR mRNA在SNX组的表达明显增加;在ADX组明显下降[SNX(复制数／百万GAPDH)39866．7±10579．0比SHAM 2366．7±446．3,P<0．05;比ADX 22100．0±4435．7, P<0．05]。然而,11β-HSD2 mRNA表达和MR相反,SNX组为9150．0±969．9,明显低于SHAM组 (48100．0±9315．2,P<0．05);而ADX组的表达比SNX组显著升高(30066．7±5150．2,P<0．05)。 4个实验组大鼠肾脏Ccr和肾重／体重无显著性差别。结论醛固酮参与慢性肾脏病变的进展, 其对肾小球损伤的作用除血流动力学效应外,还可能存在非血流动力学的直接致纤维化作用。     

FTY720 attenuates tubulointerstitial inflammation and fibrosis in subtotally nephrectomized rats

Abstract

Tubulointerstitial fibrosis is a common pathway that leads to kidney failure, and persistent tubulointerstitial inflammation is a key event in the development of tubulointerstitial fibrosis. The new immunosuppressive drug FTY720 modifies lymphocyte migration into injured tissues by sequestering lymphocytes within secondary lymphoid organs. However, its therapeutic effect on tubulointerstitial inflammation and fibrosis had not been well understood. This study was designed to explore the effect of FTY720 on tubulointerstitial inflammation and fibrosis in subtotally nephrectomized (SNX) rats. In total, 24 male Sprague–Dawley rats were used. Seven days after 5/6 nephrectomy, rats were randomized to FTY720 (1?mg/kg/d) and placebo-treated groups. Sham-operated rats served as controls. FTY720 significantly attenuated the rise in proteinuria, serum creatinine, urea nitrogen and N-acetyl-β-D-glucosaminidase activity in SNX rats, and reduced the count of peripheral white blood cells and lymphocytes in SNX rats. Morphological analysis revealed that there was severe tubulointerstitial inflammation and fibrosis in SNX group and much more tubulointerstitial infiltrating inflammatory cells with high expression of CD3, CD4, CD8, CD20, CD68, CD163 and CCR-7 in SNX group, as compared with the controls, but the lesions were attenuated significantly by treatment with FTY720. Furthermore, the expressions of proinflammatory molecules (IL-6, TNF-α and MCP-1), profibrotic molecule (TGF-β1) and production of extracellular matrix proteins such as fibronectin and types I and III collagens were upregulated in SNX rats. FTY720 administration significantly reduced these abnormalities. In summary, FTY720 exerts therapeutic effects on tubulointerstitial fibrosis in SNX rats by inhibiting the tubulointerstitial inflammatory response.     

Cigarette smoke condensate aggravates renal injury in the renal ablation model

BACKGROUND: Cigarette smoking increases the risk of progression of diabetic and non-diabetic renal diseases. The mechanisms underlying the adverse effects of smoking are largely unknown. We examined the subtotally nephrectomized rat (i) to investigate whether components of cigarette smoke dissolved in acetone (cigarette smoke condensate) aggravate structural renal damage and (ii) to establish whether this provides an animal model that can be used to investigate potential pathomechanisms of cigarette smoke-induced renal damage. Since nicotine activates the sympathetic nerve system in humans, we investigated whether interference with this system modulates the effects of cigarette smoke condensate on the damaged kidney. METHODS: One group of Sprague-Dawley rats was subtotally nephrectomized (SNX). Acetone (SNX + solvent) or cigarette smoke condensate (SNX + cigarette) was applied daily to the oral mucosa. Another group of Sprague-Dawley rats was sham-operated and received the same treatments (sham + solvent, sham + cigarette). To investigate whether increased activity of the sympathetic nerve system is involved, the remnant kidney was denervated by microsurgical technique in one SNX + cigarette group. The control group for this intervention was a solvent-treated SNX group with denervated remnant kidney. Blood pressure (BP) was measured weekly by tail plethysmography. The experiment was terminated after 12 weeks. Structural renal damage was assessed by morphometric techniques (indices of glomerulosclerosis, tubulointerstitial and vascular damage) and urinary albumin and endothelin-1 excretion were measured. RESULTS: Indices of structural renal damage were increased in all SNX-groups. Treatment with cigarette smoke condensate further increased the indices of glomerulosclerosis and tubulointerstitial damage in SNX, but not sham-operated rats. This increase was completely prevented by renal denervation. No differences in systemic blood pressure were observed in the different SNX groups. Urinary albumin excretion went in parallel with the indices of glomerulosclerosis and tubulointerstitial damage and urinary endothelin-1 excretion was significantly increased in SNX + cigarette animals. CONCLUSION: These findings document that acetone soluble components in cigarette smoke aggravate glomerulosclerosis and tubulointerstitial damage in the renal ablation model. Renal injury induced by cigarette smoke condensate in this model is reversed by renal denervation. We conclude that cigarette smoke-induced renal damage is due, at least in part, to activation of the sympathetic nerve system.     

Tranilast attenuates structural and functional aspects of renal injury in the remnant kidney model

Pathologic fibrosis is a key feature of progressive renal disease that correlates closely with kidney dysfunction and in which the prosclerotic growth factor TGF-beta has been consistently implicated. Tranilast (n-[3,4-dimethoxycinnamoyl] anthranilic acid), an antifibrotic agent that is used to treat hypertrophic scars and scleroderma, has also been shown to inhibit TGF-beta-induced extracellular matrix synthesis in a range of cell types, including those of renal origin. Therefore, the effects of tranilast on kidney fibrosis and dysfunction were examined in the subtotal nephrectomy model of progressive renal injury. Subtotal nephrectomy led to proteinuria and renal dysfunction in association with glomerulosclerosis, tubulointerstitial fibrosis, and macrophage accumulation. Despite persistent hypertension, treatment with tranilast led to a reduction in albuminuria (61.7 (x)/(/) 1.2 versus 20.5 (x)/(/) 1.3 mg/d; P < 0.01) and plasma creatinine (0.16 versus 0.08 mmol/L; P < 0.01) in subtotally nephrectomized rats. In addition, features suggestive of TGF-beta activation, including glomerulosclerosis, tubulointerstitial fibrosis, tubular atrophy, and macrophage accumulation, all were significantly attenuated by tranilast in association with evidence of reduced TGF-beta signaling in vivo. In the context of a recent pilot study in humans, the findings of the present report suggest that tranilast may provide a novel strategy for the treatment of progressive kidney disease characterized by fibrotic scarring.     

氟伐他汀对肾衰大鼠肾脏纤维化病变进展影响及机制探讨

目的：探讨丙戊二酸辅酶A（HMG－CoA）还原酶抑制剂－氟伐他汀（Fluvastatin）在慢性进展性肾小球硬化和肾小管间质纤维化过程中圣贤有血管损伤、肾脏细胞外基质产生和降解过程中的影响。方法：采用5／6肾切除SD大鼠制备肾小球硬化及慢性肾衰实验动物模型,予氟伐他汀治疗后分别于治疗开始、治疗后第4周、第8周时间点观察大鼠血脂、尿蛋白、肾功能及血压变化,并评价该药对肾小球及小管间质组织病理学变化的干预作用。结果：氟伐他汀可降血压、减少蛋白尿、降低血脂、改善肾功能、防止和逆转肾血管硬化、狭窄。并通过影响PAI－1uPA等因子的表达而阻抑肾脏细胞外基质的产生,促进其降解。结论：提示氟伐他汀具有防治或改善慢性进行性肾小球硬化及间质纤维化的作用。     

Tacrolimus restores podocyte injury and stabilizes the expression of Cabin1 in 5/6 nephrectomized rats

Podocyte injury is a vital factor, which induces massive proteinuria. Studies have shown that tacrolimus (TAC) protected podocyte via stabilizing cytoskeleton. Our latest study indicates that calcineurin binding protein 1 (Cabin1) undergoes nuclear translocation during podocytes injury. Whether TAC targets on Cabin1 during podocyte injury is still not clear. This study establishes non-immunological proteinuric model. To observe the effect of the treatment of TAC on Cabin1 expression in 5/6 nephrectomized rats. Sprague–Dawley rats were injected with TAC (0.2?mg/kg/day) for 4–8 weeks after 5/6 nephrectomy. Then, rats were sacrificed in the eighth week after operation, renal tissues were processed for morphological studies under light and electrical microscope. Cabin1 expression and distribution were detected by western blot and indirect immunofluorescence staining. In 5/6 nephrectomized rats, urinary protein excretion reached 90.2?±?30.1?mg/24?h, glomerular sclerosis index and tubulointerstitial fibrosis score were significantly increased, and widespread of podocyte foot processes fusion was found. Moreover, Cabin1 protein expression was markedly increased, and its distribution became much more obviously in podocytes nuclei. In TAC treated rats, urinary protein excretion significantly decreased (44.9?±?22.5?mg/24?h), glomerular sclerosis and tubulointerstitial fibrosis were alleviated, and podocyte foot processes fusion was inhibited. Furthermore, TAC alleviated the increased protein expression and abnormal distribution of Cabin1. In conclusion, TAC restores podocyte injury and stabilizes the expression of Cabin1. Cabin1 may become a new target to demonstrate the mechanism of TAC in podocyte injury.     

Effects of ionizing radiation on progressive experimental renal disease: a hemodynamic approach

In order to evaluate the progression of renal disease, Munich-Wistar rats were submitted to 5/6 nephrectomy and given whole-body x- or gamma-irradiation with or without remnant kidney protection or were submitted only to remnant kidney irradiation. All groups received a single 6-Gy dose immediately after surgery. Whole-kidney function, glomerular hemodynamics, 24-hour proteinuria and histopathology were assessed 60 days after surgery and irradiation. The irradiated nephrectomized animals presented whole-kidney function parameters comparable to those of normal rats. In addition, they were less hypertensive and had higher hematocrit. They showed glomerular hyperfiltration and hypertension even greater than their respective nephrectomized controls. However, the interrelations among the glomerular filtration determinants were somewhat different in irradiated animals. Their 24-hour proteinuria was significantly lower and the sclerosis index and tubulointerstitial injury score were markedly smaller. Among irradiated animals, the worst sclerosis index was observed in those with a shielded remnant kidney and the best in those without protection of the remnant kidney. This led us to speculate about a possible influence of resident mesangial cells on the early events following renal mass ablation and on the maintenance of subsequent physiopathologic changes. Therefore, radiation undoubtedly provoked a beneficial change in the course of renal disease when the renal mass ablation model was employed. Many factors could have contributed to this favorable feature including lower levels of systemic arterial pressure, less increment in DeltaP, diminished proteinuria, and maintenance of tubulointerstitial space integrity. Our data also suggest that development of glomerulosclerosis seems to be determined by events occurring immediately after injury.     

Nilotinib attenuates renal injury and prolongs survival in chronic kidney disease

The tyrosine kinase inhibitor imatinib is beneficial in experimental renal diseases, but the effect of the new tyrosine kinase inhibitor nilotinib on the progression of renal failure is unknown. We administered either nilotinib or vehicle to Sprague-Dawley rats beginning 2 weeks after 5/6 nephrectomy (Nx) or laparotomy and continuing for 8 weeks. Serum creatinine levels were significantly lower in the nilotinib group after 6 and 8 weeks of treatment. Furthermore, nilotinib-treated rats had less proteinuria, attenuated glomerulosclerosis and tubulointerstitial damage, and reduced macrophage infiltration into the tubulointerstitium. Treatment with nilotinib also significantly decreased renal cortical expression of profibrogenic genes, such as IL-1β and monocyte chemotactic protein-1, which correlated closely with the tubulointerstitial damage score and ED1-positive macrophages score. In addition, nilotinib treatment significantly prolonged survival. Taken together, these results suggest that nilotinib may limit the progression of chronic kidney disease.     

Angiotensin-converting enzyme inhibition attenuates renal platelet-derived growth factor gene expression and cell proliferation in subtotal nephrectomy

Cell proliferation, matrix accumulation and cell infiltration are characteristic features of progressive glomerulosclerosis and tubulointerstitial fibrosis. Platelet‐derived growth factor (PDGF), a cytokine which has proliferative, prosclerotic and chemokine properties, has been shown to be upregulated in the rat remnant kidney model. Inhibition of the renin–angiotensin system by angiotensin‐converting enzyme (ACE) inhibitors has a beneficial effect on renal function and morphology, but the effect of ACE inhibition on PDGF gene expression and PDGF‐mediated cellular proliferation in subtotal nephrectomy has not been studied in detail. Twelve rats were subtotally nephrectomized (STNx) and received either the ACE inhibitor perindopril or a placebo for 12 weeks. Five sham‐operated rats served as controls. Subtotal nephrectomy was associated with hypertension, proteinuria, elevated plasma creatinine and increased kidney weight. After 12 weeks, PDGF B‐chain mRNA was significantly upregulated in the glomeruli and tubulointerstitium of subtotally nephrectomized rats. ACE inhibition attenuated PDGF mRNA expression in association with a reduction in tubular and glomerular proliferation, as assessed by staining for proliferating cell nuclear antigen. In the context of the known in vitro and in vivo effects of PDGF, it is postulated that the renoprotective action of ACE inhibitors may be partially related to PDGF‐mediated antiproliferative mechanisms.     

Blood pressure-independent additive effects of pharmacologic blockade of the renin-angiotensin and endothelin systems on progression in a low-renin model of renal damage.

Pharmacologic blockade of the renin and endothelin (ET) systems is an established strategy to interfere with progression of renal failure. In the Heyman nephritis model, additive benefits of decreases in BP with the combination of angiotensin-converting enzyme inhibitors (ACE-i) and ET(A) receptor antagonists (ET-RA) were demonstrated. To further investigate these findings and to exclude confounding effects of BP decreases, this issue was reassessed in a low-renin model of subtotal kidney resection. Subtotally nephrectomized (SNX) and sham-operated rats were left untreated or received an ACE-i, an angiotensin II subtype 1 receptor antagonist (AT1-RA), an ET-RA, or combinations thereof (ACE-i plus ET-RA or AT1-RA plus ET-RA). The parameters studied were the glomerulosclerosis index (GSI), tubulointerstitial index, vascular damage index, glomerular geometry, and albumin excretion. After 12 wk, BP values were comparable. Urinary albumin excretion rates were significantly higher for untreated SNX rats (24.3 +/- 31.3 mg/24 h), compared with untreated sham-operated rats (0.71 +/- 0.40 mg/24 h). Rates were significantly lower for all treated, compared with untreated, SNX groups. GSI values were significantly higher for untreated SNX rats than for untreated sham-operated rats. ACE-i caused significantly lower GSI in SNX rats (0.46 +/- 0.06), compared with AT1-RA (0.60 +/- 0.10) or ET-RA (0.65 +/- 0.10). GSI values were significantly decreased further with ACE-i plus ET-RA (0.29 +/- 0.09) or AT1-RA plus ET-RA (23 +/- 0.05) treatment. Changes in the tubulointerstitial index and vascular damage index proceeded in parallel. The results document BP-independent effects of the ACE-i and AT1-RA on the GSI and urinary albumin excretion and an effect of the ET-RA on the GSI. The contrasting results suggest different pathogenetic pathways for glomerulosclerosis and albuminuria. The combination of treatments provided superior effects on the GSI and tubulointerstitial index but not on urinary albumin excretion.     

Beneficial effects of calcimimetics on progression of renal failure and cardiovascular risk factors

In renal failure, parathyroid hormone (PTH) is not only involved in the genesis of disturbed calcium/phosphate metabolism and ostitis fibrosa; it is also a permissive factor in the genesis of hypertension, cardiovascular damage, and dyslipidemia. The allosteric activator of the calcium sensing receptor NPSR-568 (R-568) has been shown to reduce the serum intact PTH (iPTH) concentration in uremic rats. It was the purpose of this study in subtotally nephrectomized (SNX) rats to compare pharmacologic abrogation of secondary hyperparathyroidism by R-568 with parathyroidectomy (PTX). The effects on progression of renal failure, BP, and lipid and structural parameters of kidney and heart were studied. Four groups of male SD-rats were studied: (1) sham-operated + vehicle-treated rats (controls); (2) SNX + vehicle-treated rats (SNX); (3) parathyroidectomized SNX + vehicle-treated rats (SNX+PTX); and (4) SNX + calcimimetic R-568-treated rats (SNX+R-568). R-568 (50 micro mol/kg per d) was administered by gavage. Eight weeks after SNX, serum creatinine concentration, urinary albumin excretion, BP, and serum LDL-cholesterol concentration were significantly lower in both R-568-treated and parathyroidectomized SNX compared with vehicle-treated SNX. In addition, structural abnormalities of the kidney (glomerulosclerosis, tubulointerstitial changes) and the heart (interstitial fibrosis, capillary length density, arteriolar wall thickness) were significantly less pronounced than in vehicle-treated SNX. It is concluded that in experimental renal failure abrogation of hyperparathyroidism by administration of a calcimimetic or PTX similarly attenuates progression of renal failure. Furthermore, it interferes with the development of cardiovascular risk factors and cardiac remodeling.     

Effect of candesartan cilexetil (TCV-116) in rats with chronic renal failure.

BACKGROUND: Inhibition of the renin-angiotensin system by both angiotensin II type 1 receptor antagonists (AT1As) and angiotensin I-converting enzyme inhibitors (ACEIs) shows renoprotective effects in rats with chronic renal failure when treatment is started in the early phase of renal injury. In this study, we examined the renal protective effects of candesartan cilexetil (TCV-116), an AT1A, and enalapril, an ACEI, in the progressive phase of renal injury in 5/6 nephrectomized rats. METHODS: Candesartan cilexetil (1 mg/kg/day) and enalapril (10 mg/kg/day) were orally administered once a day for 4 weeks (the short-term experiment) or 16 weeks (the long-term experiment) to 5/6 nephrectomized rats beginning 15 weeks after the nephrectomy, that is, after they had already showed marked proteinuria. RESULTS: In vehicle-treated rats, proteinuria, glomerulosclerosis, and interstitial fibrosis developed. Moreover, enhanced expression of transforming growth factor-beta1 (TGF-beta1) in the injured glomeruli was observed. These adverse changes progressed with time, and in the short-term experiment, both drugs inhibited them. In the long-term experiment, the progressive proteinuria and the elevation of blood pressure were similarly attenuated by both drugs. However, candesartan cilexetil significantly inhibited the progression of glomerulosclerosis, the expression of TGF-beta1, and interstitial fibrosis, whereas enalapril did not. CONCLUSION: These results indicate that candesartan cilexetil shows potent and long-term preventive effects against the progression of previously developed renal injury.     

Rapamycin ameliorates proteinuria-associated tubulointerstitial inflammation and fibrosis in experimental membranous nephropathy

Proteinuria is a risk factor for progression of chronic renal failure. A model of proteinuria-associated tubulointerstitial injury was developed and was used to examine the therapeutic effect of rapamycin. Two studies were performed. In study A, proteinuric rats were given sheep anti-Fx1A to induce experimental membranous nephropathy; control rats received normal sheep serum. Four weeks later, groups were subdivided and underwent laparotomy alone (two kidneys), nephrectomy alone (one kidney), or nephrectomy with polectomy (0.6 kidney). Renal function and morphology were evaluated 4 wk later. Whereas control rats never developed proteinuria, anti-Fx1A induced severe proteinuria. Proteinuria was unaffected by renal mass reduction. Proteinuric rats developed tubulointerstitial disease that was most severe in rats with 0.6 kidneys. Renal function (GFR) was reduced by loss of renal mass and was reduced further in proteinuric rats with 0.6 kidneys. In study B, the effect of rapamycin on the expression of candidate proinflammatory and profibrotic genes and the progression of proteinuria-associated renal disease were examined. All rats received an injection of anti-Fx1A and were nephrectomized and then divided into groups to receive rapamycin or vehicle. Gene expression, renal morphology, and GFR were evaluated after 4, 8, and 12 wk. Rapamycin reduced expression of the proinflammatory and profibrotic genes (monocyte chemotactic protein-1, vascular endothelial growth factor, PDGF, TGF-beta(1), and type 1 collagen). Tubulointerstitial inflammation and progression of interstitial fibrosis that were present in vehicle-treated rats were ameliorated by rapamycin. Rapamycin also completely inhibited compensatory renal hypertrophy. In summary, rapamycin ameliorates the tubulointerstitial disease associated with chronic proteinuria and loss of renal mass.     

Effects of Sirt1 gene knockout on chronic kidney disease induced by 5/6 nephrectomy in mice and VEGF/Flk?1 signaling pathway

Objective To investigate the effect of Sirt1 gene knockout on chronic kidney disease induced by 5/6 nephrectomy in mice and vascular endothelial growth factor (VEGF)/fetal liver kinase-1 (Flk-1) signaling pathway. Methods Twenty four male Sirt1+/+ and Sirt1+/- mice were randomly divided into four groups: Sirt1+/+ mice with sham-operation (WT-Sham, n=6), Sirt1+/- mice with sham-operation (KO-Sham, n=6), Sirt1+/+ mice with 5/6 nephrectomy (WT-Nx, n=6) and Sirt1+/- mice with 5/6 nephrectomy (KO-Nx, n=6). Proteinuria was determined by urine collection from 8:00 to 8:00 the next day at 20 weeks. Serum creatinine (Scr), urea nitrogen (BUN) and the renal pathological changes were measured after 20 weeks. Expressions of Sirt1, collagenⅠ and transforming growth factor β (TGF-β) were used to analyze the changes of renal fibrosis by immunohistochemistry staining. Real-time PCR and Western blotting were used to measure the mRNA and protein expressions of Sirt1, fibronectin, collagenⅠ,VEGF and Flk-1 in kidney. Results Sirt1 expressed in glomerular endothelial cells, podocytes, mesangial cells and renal tubular epithelial cells in Sirt1+/+ mice, while Sirt1 expression intensity was significantly reduced in Sirt1+/- mice. Compared with the WT-Sham group, WT-Nx group had increased proteinuria, BUN, Scr, glomerular sclerosis index and tubulointerstitial fibrosis index at 12 weeks after operation (all P＜0.01), and KO-Nx group had exacerbated the above up-regulations (all P＜0.01). Compared with those in WT-Sham group, the expressions of fibronectin, collagenⅠ and TGF-β were up-regulated in WT-Nx group (all P＜0.01), and were significantly augmented in KO-Nx group (all P＜0.01). Compared with those in WT-Sham group, renal mRNA and protein expressions of VEGF and Flk-1 were decreased in WT-Nx group, and KO-Nx group aggravated their down-regulation (all P＜0.01). Conclusions Sirt1 gene knockout can increase proteinuria and Scr, and aggravate renal pathology and renal fibrosis in 5/6 nephrectomized mice, which is associated with the inhibition of VEGF/Flk-1 signaling pathway. It is suggested that Sirt1 may be a potential therapeutic target of chronic kidney disease.     

Inhibition of Na/H exchanger 1 by cariporide alleviates burn-induced multiple organ injury

Background

Severe burns initiate an inflammatory response characterized by the upregulation of proinflammatory cytokine, which contributes to multiple organ injury. Na⁺/H⁺ exchanger 1 (NHE1) plays a significant role in several inflammatory processes. This study was designed to investigate the role of NHE1 in burn-induced inflammation and multiple organ injury.

Materials and methods

Rats were subjected to a 30% total body surface area full-thickness burn. Cariporide was used to assess the function of NHE1 in burn-induced multiple organ injury by biochemical parameters, histologic changes, and inflammatory cytokine production.

Results

We found that NHE1 expression was significantly increased after burn injury. Inhibition of NHE1 by cariporide attenuated burn-induced edema and tissue injury in heart, lung, kidney, and small intestine. Cariporide also inhibited plasma levels of tumor necrosis factor α, interleukin 6, and myeloperoxidase activity.

Conclusions

These results indicate that NHE1 inhibition prevents burn-induced multiple organ injury. The salutary effects afforded by NHE1 inhibition, at least in part, are mediated by attenuating systemic inflammatory response.     

Luminal osmolarity downregulates gene expression of Na+/H+ exchanger (NHE3) in rat colon mucosa

Water-coupled Na⁺ absorption in the colon is mediated principally by Na⁺/H⁺ exchange (isoforms NHE2 and NHE3). To determine whether luminal ion composition or osmolarity influences NHE expression in colon mucosa, two groups (n = 6 in each) of adult male Sprague-Dawley rats underwent sham laparotomy or loop ileostomy. In these studies, diversion did not markedly alter mRNA levels for NTHE2, NHE3, or Na⁺/K⁺, at 8 or 21 days, indicating that loss of luminal volume does not alter NHE gene expression. To evaluate the effects of specific luminal components, we infused equal volumes of half-normal (154 mOsm) or iso-osmolar (308 mOsm) solutions of saline and mannitol into the diverted colon. All solutions elicited significant (45% to 60%; P <0.05) decreases in mRNA levels for NHE3, with iso-osmolar mannitol eliciting the greatest changes. Decreases in NHE2 and Na⁺/K⁺ mRNA levels were observed following these infusions but were not as marked as the changes for NHE3. These findings suggest that (1) loss of luminal Na⁺ is not, in itself, a signal that regulates NHE expression and (2) infusion of any solute, including Na⁺ itself, provides a signal to downregulate expression of NHE3 in colon mucosa. Supported by the Brigham Surgical Group Foundation and National Institutes of Health Award RO1-DK44571 (D.I.S.).     

曲尼司特联合贝拉普利对5／6肾切除大鼠肾脏的保护作用

目的观察曲尼司特联合贝拉普利对5／6肾切除大鼠肾脏的保护作用,并探讨其机制。方法将50只雄性SD大鼠随机分为5组：假手术组（S组）、5／6肾切除模型组（M组）、曲尼司特干预组（T组）、贝拉普利干预组（B组）和曲尼司特联合贝拉普利干预组（C组）,每组10只。术后第12周观察各组大鼠体重、血压、尿蛋白量、血肌酐及肾组织病理改变,用逆转录聚合酶链反应（RT-PCR）法检测残肾组织转化生长因子β1（TGF-β1）mRNA的表达,Western blotting法检测残肾组织Ⅲ型胶原蛋白的表达。结果与S组相比,M组大鼠血压、尿蛋白量和血肌酐水平明显升高,肾小球硬化、肾间质纤维化程度加重,残肾组织TGFβ1 mRNA和Ⅲ型胶原蛋白的表达升高,差异均有统计学意义（P〈0．05）。与M组相比,T组和B组上述指标减轻,差异均有统计学意义（P〈0．05）。与T组和B组相比,C组均更明显地减轻上述指标,差异均有统计学意义（P〈0．01）。结论曲尼司特联合贝拉普利对5／6肾切除大鼠的肾脏有更佳的保护作用,其机制可能与其减轻TGF-β1引起的细胞外基质的沉积有关。     

Renal structure and function evaluation of rats from dams that received increased sodium intake during pregnancy and lactation submitted or not to 5/6 nephrectomy

Adult rats submitted to perinatal salt overload presented renin-angiotensin system (RAS) functional disturbances. The RAS contributes to the renal development and renal damage in a 5/6 nephrectomy model. The aim of the present study was to analyze the renal structure and function of offspring from dams that received a high-salt intake during pregnancy and lactation. We also evaluated the influence of the prenatal high-salt intake on the evolution of 5/6 nephrectomy in adult rats. A total of 111 sixty-day-old rat pups from dams that received saline or water during pregnancy and lactation were submitted to 5/6 nephrectomy (nephrectomized) or to a sham operation (sham). The animals were killed 120 days after surgery, and the kidneys were removed for immunohistochemical and histological analysis. Systolic blood pressure (SBP), albuminuria, and glomerular filtration rate (GFR) were evaluated. Increased SBP, albuminuria, and decreased GFR were observed in the rats from dams submitted to high-sodium intake before surgery. However, there was no difference in these parameters between the groups after the 5/6 nephrectomy. The scores for tubulointerstitial lesions and glomerulosclerosis were higher in the rats from the sham saline group compared to the same age control rats, but there was no difference in the histological findings between the groups of nephrectomized rats. In conclusion, our data showed that the high-salt intake during pregnancy and lactation in rats leads to structural changes in the kidney of adult offspring. However, the progression of the renal lesions after 5/6 nephrectomy was similar in both groups.     

Involvements of Rho-kinase and TGF-beta pathways in aldosterone-induced renal injury

Recent studies have suggested a role for aldosterone in the pathogenesis of renal injury. This study investigated the potential contributions of Rho-kinase and TGF-beta pathways to aldosterone-induced renal injury. Rats were uninephrectomized and then treated for 5 wk with 1% NaCl in a drinking solution and one of the following: Vehicle (2% ethanol, subcutaneously; n = 9); aldosterone (0.75 microg/h, subcutaneously; n = 9); or aldosterone + fasudil, a specific Rho-kinase inhibitor (10 mg/kg per d, subcutaneously; n = 8). Phosphorylation of myosin phosphate target subunit-1 (MYPT1) and Smad2/3 in renal cortical tissue was measured by Western blotting with anti-phospho MYPT1 and Smad2/3 antibodies, respectively. Rats that received aldosterone infusion exhibited hypertension and severe renal injury characterized by proteinuria, glomerular sclerosis, and tubulointerstitial fibrosis with increases in alpha-smooth muscle actin staining and numbers of monocytes/macrophages in the interstitium. Renal cortical mRNA levels of types I and III collagen, TGF-beta, connective tissue growth factor, and monocyte chemoattractant protein-1 as well as Smad2/3 phosphorylation were significantly increased in rats that received aldosterone infusion. All of these changes were associated with an increase in renal tissue MYPT1 phosphorylation. Treatment with fasudil did not alter BP but significantly ameliorated proteinuria and renal injury in rats that received aldosterone infusion. Furthermore, fasudil prevented MYPT1 phosphorylation and markedly decreased alpha-smooth muscle actin staining, numbers of monocytes/macrophages, mRNA levels of types I and III collagen, TGF-beta, connective tissue growth factor and monocyte chemoattractant protein-1, and Smad2/3 activity in renal cortical tissues. These results provide evidence, for the first time, that Rho-kinase is substantially involved in aldosterone-induced renal injury through activation of a TGF-beta-dependent pathway.     

Cyclosporin A stimulates apical Na+/H+ exchange in LLC-PK1/PKE20 proximal tubular cells

Cyclosporin A (CyA) causes renal Na⁺ retention which may lead to arterial hypertension. The apical Na⁺/H⁺ exchanger (NHE3) is responsible for bulk proximal tubular Na⁺ reabsorption. The aim of this study was to investigate the effects of CyA on the NHE3 of polarized proximal tubular cells to evaluate cellular mechanisms of CyA-associated arterial hypertension. The change of the intracellular pH (Δ-[pH]_i/min) was determined as a measure of the activity of the NHE in LLC-PK₁/PKE₂₀ cells using 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The NHE activity was identified as the apical NHE3 since it could be inhibited by the inhibitor S3226, but not by inhibitors of the basolateral isoform (NHE1) amiloride or HOE 694. CyA stimulated the NHE3 activity dose dependently. The mean increase stimulated by relevant CyA concentrations was 61±11%. A 24-h application of CyA also stimulated an increase of NHE3 activity which did not seem to be mediated by an increase of NHE3 RNA expression. The less immunosuppressive derivatives cyclosporin H and cyclosporin G caused NHE3 activation as well. Carbachol and ATP, which both induce a Ca²⁺ release from internal Ca²⁺ stores, also increased the NHE3 activity. The Ca²⁺ chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,-N′,N′-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) abolished the CyA-associated NHE3 stimulation, whereas low extracellular Ca²⁺ had no effect. CyA-associated effects did not seem to be mediated via inhibition of protein kinase C (PKC). CyA had no additive effects on the angiotensin II-associated NHE3 stimulation. Concurrent application of losartan did not impair the CyA-induced NHE3 stimulation. In conclusion CyA stimulates the apical NHE3 in proximal tubular cells. This is mediated by Ca²⁺ release from intracellular stores but is independent of the action of angiotensin II or PKC.