Molecular aspects of renal tubular handling and regulation of inorganic sulfate

The renal proximal tubular reabsorption of sulfate plays an important role in the maintenance of sulfate homeostasis. Two different renal sulfate transport systems have been identified and characterized at the molecular level in the past few years: NaSi-1 and Sat-1. NaSi-1 belongs to a Na(+)-coupled transporter family comprising the Na(+)-dicarboxylate transporters and the recently characterized SUT1 sulfate transporter. NaSi-1 is a Na(+)-sulfate cotransporter located exclusively in the brush border membrane of renal proximal tubular and ileal cells. Recently, NaSi-1 was shown to be regulated at the protein and mRNA level by a number of factors, such as vitamin D, dietary sulfate, glucocorticoids and thyroid hormones, which are known to modulate sulfate reabsorption in vivo. The second member of renal sulfate transporters, denoted Sat-1, belongs to a family of Na+-independent sulfate transporter family comprising the DTDST, DRA and PDS genes. Sat-1 is a sulfate/bicarbonate-oxalate exchanger located at the basolateral membrane of proximal tubular epithelial cells and canalicular surface of hepatic cells. Contrary to NaSi-1, no physiological factor has been found to date to regulate Sat-1 gene expression. Both NaSi-1 and Sat-1 transporter activities are implicated in pathophysiological states such as heavy metal intoxication and chronic renal failure. This review focuses on recent developments in the molecular characterization of NaSi-1 and Sat-1 and the mechanisms involved in their regulation.     

Expression of bone type 1 PTH receptor in rats with chronic renal failure

Some researchers have speculated that a decrease in bone type 1 PTH receptor (PTH1R) may be among the causes of “skeletal resistance” in chronic renal failure (CRF). Indeed, the down-regulation of PTH1R mRNA has been identified in uremic bones. However, few studies have identified the patterns of PTH1R protein expression. In this article we compare the bone expression of PTH1R protein and mRNA under control and CRF conditions. Sprague–Dawley rats underwent 5/6 nephrectomies (Nx) or sham operations (control), and were killed 16 weeks later. Blood urea nitrogen (BUN), serum Cr, P, and parathyroid hormone (PTH) were higher in the Nx group than in the controls, while serum Ca and 1,25(OH)₂D₃ were lower in the Nx group. Immunohistochemical images of lumbar bone samples were analyzed by an image processing system. PTH1R was essentially identified in all osteoblasts. The expression of osteoblast PTH1R protein was quantified based on the gray value of PTH1R staining. The mean gray scale of osteoblasts was 25% lower in Nx rats than in control rats (P < 0.01), whereas osteoblast cell counts and cell sizes were not significantly different between the two groups. Thus, down-regulation of PTH1R protein expression under the CRF condition appeared likely. Total RNA extracted from the bone samples was reverse transcribed for real-time polymerase chain reaction (PCR). PTH1R mRNA expression was 33% lower in the Nx group than in the control group in the quantitative PCR analysis (P < 0.05). Our findings suggested that osteoblast PTH1R expression is down-regulated at both the protein and mRNA levels in the steady state of CRF.     

Adverse effects of hyperphosphatemia on myocardial hypertrophy, renal function, and bone in rats with renal failure

BACKGROUND: Hyperphosphatemia and disturbances in calcium or parathyroid hormone (PTH) metabolism contribute to the high incidence of cardiovascular disease and renal osteodystrophy in chronic renal failure (CRF). We evaluated the effect of hyperphosphatemia on the cardiovascular system, on renal function, and on bone in experimental uremia. METHODS: Wistar rats were submitted to parathyroidectomy (PTx) and 5/6 nephrectomy (Nx) with minipump implantation, delivering 1-34 rat PTH (physiologic rate), or were sham-operated and received vehicle. Only phosphorus content (low-phosphorus (LP) 0.2%; high-phosphorus (HP) 1.2%) differentiated diets. We divided the groups as follows: PTx +Nx +LP; sham + LP; PTx + Nx + HP; and sham + HP. Tail-cuff pressure and weight were measured weekly. After 2 months, biochemical, arterial, and myocardial histology and bone histomorphometry were analyzed. RESULTS: Heart weight normalized to body weight (heart weight/100 g body weight) was higher in PTx + Nx + HP rats (PTx + Nx + HP = 0.36 +/- 0.01 vs. sham + HP = 0.29 +/- 0.01, PTx + Nx + LP = 0.32 +/- 0.01, sham + LP = 0.28 +/- 0.01) (P < 0.05). Serum creatinine levels were higher in PTx + Nx + HP rats than in PTx + Nx + LP rats (1.09 +/- 0.13 vs. 0.59 +/- 0.03 mg/dL) (P < 0.05). Levels of PTH did not differ significantly between the groups. Myocardial and arterial histology detected no vascular calcification or fibrosis. Bone histomorphometry revealed an association, unrelated to uremia, between HP diets and decreased trabecular connectivity. CONCLUSION: Myocardial hypertrophy, impaired renal function, and adverse effects on bone remodeling were associated with hyperphosphatemia and were not corrected by PTH replacement. Although no vascular calcification was observed in this model, we cannot rule out an adverse effect of hyperphosphatemia on the vascular bed. Our finding underscores the importance of phosphorus control in reducing morbidity and mortality in CRF patients.     

Calcimimetic compound upregulates decreased calcium-sensing receptor expression level in parathyroid glands of rats with chronic renal insufficiency

The reduced expression level of the calcium-sensing receptor (CaR) is attributed to the hyposensitivity of parathyroid cells to extracellular calcium concentration [Ca2+]o, which plays a crucial role in the pathogenesis of secondary hyperparathyroidism (SHPT) in patients and rats with chronic renal insufficiency (CRI). Calcimimetic compounds have been demonstrated to improve the decreased sensitivity of CaR to extracellular calcium concentration and to suppress both parathyroid hormone (PTH) oversecretion and parathyroid cell proliferation. However, the effect of calcimimetics on the reduced CaR expression level in parathyroid cells in CRI remains unclarified. The aim of this investigation was to examine the effect of the calcimimetic compound NSP R-568 (R-568) on the CaR expression in the parathyroid cells of rats with experimental CRI. Subtotally nephrectomized rats were fed a high-phosphorus diet for 8 (n = 12; Nx-8 group) or 9 wk (n = 11; Nx-9 group) to induce severe SHPT. Another group of uremic rats were fed a high-phosphorus diet for 8 wk and then orally administered R-568 (100 micromol/kg body wt) once a day for 7 d (n = 11; Nx+R-568 group). Sham-operated rats that were fed a standard diet for 9 wk were used as controls (n = 8). R-568 treatment induced a significant reduction in plasma PTH level with significant decrease in serum calcium and without change in serum phosphorus concentration. Serum 1,25(OH)2D3 level was not affected by R-568 administration. CaR mRNA and protein levels in the Nx-8 and Nx-9 groups significantly decreased compared with those in the controls; however, no significant difference in these parameters was observed between the Nx-8 and Nx-9 groups. In the Nx+R-568 group, CaR mRNA and protein levels significantly increased compared with those in either the Nx-8 or Nx-9 group. R-568 was effective in reducing the number of proliferating cell nuclear antigen-positive cells along with parathyroid gland growth suppression in the Nx+R-568 group compared with that in the Nx-9 group. The results suggest that the calcimimetic compound R-568 upregulates decreased CaR expression, and the upregulation possibly has an enhancement effect on PTH secretion and parathyroid cell hyperplasia through the improved sensitivity of CaR to [Ca2+]o.     

低分子右旋糖酐铁和蔗糖铁对慢性肾衰竭大鼠氧化应激的影响

目的 评价小剂量反复多次低分子右旋糖酐铁和蔗糖铁静脉用药后对慢性肾衰竭大鼠氧化应激的影响。 方法 以5/6肾大部切除术建立慢性肾衰竭大鼠模型。右肾切除术后第4周,将实验大鼠分为4组：低分子右旋糖酐铁（糖酐铁）组、蔗糖铁组、对照组、假手术组。观察6周,检测各组大鼠体内氧化应激、铁代谢等指标。 结果 糖酐铁组和蔗糖铁组大鼠血红蛋白明显高于对照组（P < 0.05）,而两铁剂组间差异无统计学意义。对照组大鼠的血清铁、血清铁蛋白、转铁蛋白饱和度显著低于假手术组（P < 0.05）;两铁剂组大鼠上述指标均显著高于对照组（P < 0.05）,而两铁剂组间差异无统计学意义。糖酐铁组和蔗糖铁组血浆晚期氧化蛋白产物（AOPP）显著高于对照组[（127.84±21.19） μmol/L、（134.21±29.38） μmol/L比 （81.83±19.93） μmol/L,P < 0.05],而两铁剂组间差异无统计学意义。两铁剂组大鼠血浆丙二醛（MDA）高于对照组,而蔗糖铁组高于糖酐铁组[（6.06±0.73） nmol/L比（4.99±0.80） nmol/L, P < 0.05]。糖酐铁组、蔗糖铁组和对照组大鼠血清超氧化物歧化酶（SOD）和总抗氧化能力（TAOC）差异无统计学意义。模型组大鼠血浆谷胱甘肽过氧化物酶（GSH-Px）水平显著低于假手术组（P < 0.05）,而蔗糖铁组显著低于糖酐铁组和对照组[（2123.11±74.78） nmol&#8226;ml-1&#8226;min-1比（2352.84±163.90） nmol&#8226;ml-1&#8226;min-1、（2310.23±125.99） nmol&#8226;ml-1&#8226;min-1,P < 0.05]。 结论 静脉补铁可部分纠正5/6肾大部切除肾衰竭大鼠的贫血,改善铁代谢指标。反复静脉小剂量补铁对慢性肾衰竭大鼠氧化应激状态有不良影响,而低分子右旋糖酐铁的不良影响低于蔗糖铁。     

血管紧张素Ⅱ受体拮抗剂对慢性肾衰竭大鼠肾小管上皮细胞增殖和转分化的影响

目的：探讨血管紧张素Ⅱ受体拮抗剂（ARB）losartan对慢性肾衰竭（CRF）大鼠肾小管上皮细胞增殖和转分化的影响。方法：Wistar大鼠随机分为假手术（Sham）组、模型（Nx）组、ARB小剂量（ARB15,losartan 15mg·kg^-1·d^-1灌胃）组、ARB大剂量（ARB150,losartan 150mg·kg^-1·d^-1灌胃）组。5/6肾切除制作CRF模型。12周后,观察各组肾功能、蛋白尿、肾重/体重（BW）、心脏重量/BW、肾脏病理变化,免疫组化观察肾脏胰岛素样生长因子-1（IGF-1）、α-平滑肌肌动蛋白（α-SMA）、Vimentin表达变化及增殖细胞核抗原（PCNA）阳性细胞数,TUNEL法观察细胞凋亡。结果：（1）Nx组大鼠Scr、BUN、24h尿蛋白定量、肾重/BW、心脏重量/BW均明显高于Sham组（P〈0.05）;ARB治疗后肾功能明显改善、蛋白尿减少、肾重/BW、心脏重量/BW降低（P〈0.05）。（2）Nx组大鼠肾组织有明显的肾小球硬化及肾小管间质损伤,肾小球硬化指数（GSI）及肾小管间质损伤指数（TII）均较Sham组明显增高;ARB治疗后GSI及TII均较Nx组降低（P〈0.05）。（3）与Sham组比较,Nx组肾间质IGF-1表达明显增加（P〈0.05）,ARB可明显减少IGF-1的表达（P〈0.05）。（4）Sham组肾组织可见α-SMA表达,肾间质无Vimentin表达;Nx组肾间质Vimentin及α-SMA表达增加（均P〈0.05）,ARB治疗后较Nx组两者表达减少（P〈0.05）。（5）Nx组TUNEL阳性细胞和PCNA阳性细胞均明显增加,ARB治疗对TUNEL阳性细胞无明显影响,但可明显减少PCNA阳性细胞数（P〈0.05）。结论：ARB可明显减轻CRF大鼠的肾损伤,该作用与抑制局部IGF-1表达和肾小管上皮细胞转分化以及改变细胞凋亡与增生的平衡有关。     

Focal glomerulosclerosis in the remnant kidney model--an inflammatory disease mediated by cytokines

BACKGROUND: The mechanism of progression of established renal disease remains unclear. While a low protein diet slows this progression, the role of cytokines in this process has been little investigated. METHODS: We investigated cytokine expression by Northern blot and immunohistochemistry in two groups of 5/6 nephrectomized rats (5/6 Nx) fed a normal (24%) or low (6%) protein diet and compared them with sham operated controls. RESULTS: The rats on 6% protein diet had significantly less focal glomerulosclerosis (FGS) (17.4 +/- 4.4 vs 27.4 +/- 8.8%, P < 0.05) and global sclerosis (GGS) after 7 weeks (0.4 +/- 0.8 vs 3.5 +/- 2.1% of glomeruli P < 0.05). Both experimental groups showed three times control levels of MCP-I expression after 2 weeks. However in the 5/6 Nx 6% protein group the expression decreased at 4 weeks (1.5 times controls) and reached control levels after 7 weeks. In contrast, the 5/6 Nx 24% protein group exhibited a further marked increase after 4 weeks (5.6 times controls) and was still two-fold higher after 7 weeks. TGF-beta expression was modestly but consistently increased at all time points (120-160% of controls), with no difference between the two study groups. Neither IL-1 beta or TNF-alpha was detectable at any time. Immunohistochemistry demonstrated TGF-beta intracellularly in distal tubular cells in both experimental and control animals, while MCP-1 protein was found in the area of FGS and in the apical pole of distal tubular cells in both experimental groups. Glomerular and interstitial ED1 positive cells were significantly increased after four weeks in the 5/6 Nx 24% protein group (P < 0.05). CONCLUSIONS: A 'mechanical' injury to the kidney clearly results in an inflammatory response associated with the upregulation of MCP-1. A low protein diet modulates the expression of MCP-1 and improves the morphological sequelae seen after renal ablation.      

Mechanisms of PKC-dependent Na+ K+ ATPase phosphorylation in the rat kidney with chronic renal failure

The present work was designed to study Na+ K+ ATPase alpha1-subunit phosphorylation in rats with chronic renal failure (CRF) in comparison with normal rats. Na+ K+ ATPase alpha1-subunit phosphorylation degree was measured by binding the McK-1 antibody to dephosphorylated Ser-23 in microdissected medullary thick ascending limb of Henle (mTAL) segments. In addition, the total Na+ K+ ATPase alpha1-subunit expression and activity were also measured in the outer renal medulla homogenates and membranes. CRF rats showed a higher Na+ K+ ATPase activity, as compared with control rats (18.95 +/- 2.4 vs. 11.21 +/- 1.5 micromol Pi/mg prot/h, p < 0.05), accompanied by a higher total Na+ K+ ATPase expression (0.54 +/- 0.04 vs. 0.27 +/- 0.02 normalized arbitrary units (NU), p < 0.05). When McK-1 antibody was used, a higher immunosignal in mTAL of CRF rats was observed, as compared with controls (6.3 +/- 0.35 vs. 4.1 +/- 0.33 NU, p < 0.05). The ratio Na+ K+ ATPase alpha1-subunit phosphorylation/total Na+ K+ ATPase alpha1-subunit expression per microg protein showed a non-significant difference between CRF and control rats in microdissected mTAL segments (2.11 +/- 0.12 vs. 2.26 +/- 0.18 NU, p = NS). The PKC inhibitor RO-318220 10(-6) M increased immunosignal (lower phosphorylation degree) in mTAL of CRF rats to 128.43 +/- 7.08% (p < 0.05) but did not alter McK1 binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10(-6) M and dopamine 10(-6) M decreased immunosignal in CRF rats, corresponding to a higher Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23 (55.26 +/- 11.17% and 53.27 +/- 7.12% compared with basal, p < 0.05). In mTAL of CRF rats, the calcineurin inhibitor FK-506 10(-6) M did not modify phosphorylation degree at Ser-23 of Na+ K+ ATPase alpha1-subunit (100.21 +/- 3.00% compared with basal CRF). In control rats, FK 506 10(-6) M decreased the immunosignal, which corresponds to a higher Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.     

Systemic and vascular inflammation is elevated in early IgA and type 1 diabetic nephropathies and relates to vascular disease risk factors and renal function.

BACKGROUND: Inflammation is implicated in cardiovascular disease (CVD) and mortality in end-stage renal failure (ESRF). Its importance in early renal disease is yet to be defined. METHODS: Serum levels of systemic and vascular inflammatory markers in early IgA nephropathy (IgAN) and control subjects were measured and related to renal function and vascular risk factors. A parallel study in type 1 diabetes mellitus subjects with (T1DM Nx) and without nephropathy (T1DM No Nx) was performed. RESULTS: Fifty-one IgAN patients aged 46+/-2 years (mean+/-SEM), calculated creatinine clearance (CrCl) 88+/-5 ml/min, were compared with 51 matched control subjects. Forty-six T1DM Nx patients aged 40+/-2 years, CrCl 84+/-5 ml/min, and 73 T1DM No Nx patients aged 38+/-2 years were also compared. High sensitivity C-reactive protein (hsCRP) was elevated in IgAN, T1DM Nx and T1DM No Nx patients compared with controls [4.2+/-0.6 (P < 0.001), 4.1+/-0.6 (P < 0.001), 2.6+/-0.4 (P < 0.05) vs 1.6+/-0.3 mg/l]. Levels in T1DM Nx patients were higher than in T1DM No Nx patients (P < 0.05). Inflammation and vascular dysfunction as measured by pulse pressure (PP) were related. HsCRP correlated with PP in IgAN and T1DM Nx (r = 0.47, P = 0.001; r = 0.40, P < 0.05). PP was the strongest independent predictor of hsCRP in IgAN (T = 2.45, P < 0.001), while body mass index (T = 7.83, P < 0.001) was the strongest predictor in T1DM Nx. Endothelial cell adhesion molecules were increased in T1DM Nx > IgAN > T1DM No Nx vs controls: soluble vascular adhesion molecule-1 (sVCAM-1) 760+/-30 (P < 0.001) > 663+/-34 (P = 0.001) > 601+/-21 (P < 0.05) vs 536+/-15 ng/ml; soluble intracellular adhesion molecule-1 (sICAM-1) 320+/-8 (P < 0.001) > 313+/-13 (P < 0.001) > 307+/-8 (P < 0.001) vs 244+/-6 ng/ml. sVCAM-1 levels were higher in T1DM Nx than in T1DM No Nx, P < 0.001. In IgAN and T1DM Nx, hsCRP correlated with sICAM-1 (r = 0.33, P = 0.017; r = 0.37; P = 0.017). sVCAM-1 was related to renal function in IgAN and T1DM Nx: serum cystatin C (r = 0.63, P < 0.001: r = 0.425, P = 0.002), and urine protein:creatinine ratio in IgAN (r = 0.48; P = 0.001). CONCLUSIONS: Systemic and vascular markers of inflammation are increased in early renal disease and relate to renal dysfunction and cardiovascular risk factors. Inflammation may be a common process in various renal diseases and may link and accelerate renal dysfunction and CVD.     

沉默信息调节因子1对缺氧条件下肠上皮屏障功能的保护作用及机制

目的观察沉默信息调节因子1（SIRT1）对肠缺氧上皮Caco-2细胞屏障功能的影响并探讨其分子机制。方法对Caco-2细胞分别进行1％O2浓度缺氧6h（缺氧组）和常规处理（常规组）及加浓度为40μmol／L Resveratrol（SIRT1激动剂）预处理6h后再缺氧6h（观察组）,分别检测3组肠上皮细胞跨上皮电阻（TER）,PCR及Western blot分别检测3组SIRT1及紧密连接蛋白ZO—1、Occludin和Claudin-1的mRNA与蛋白表达。结果缺氧组SIRT1mRNA及其蛋白的相对表达量均明显低于常规组（分别为0．40±0．02比0．70＋0．07,P=0．001;0．37±0．03比0．76±0．03,P=0．001）,也明显低于观察组（0．50±0．02,P=0．026;0．54±0．02,P=0．011）。缺氧组3种紧密连接蛋白mRNA表达均低于常规组（P〈0．05）,也低于观察组（P〈0．05）,观察组的ZO-1及Claudin-1表达与常规组比较,差异无统计学意义（P〉0．05）。3种紧密连接蛋白的表达水平常规组最高,观察组次之,缺氧组最低,两两比较,差异均具有统计学意义（均P〈0．05）。常规组、缺氧组和观察组的TER分别为（142±7）Ohm／cm^2、（94±3）Ohm／cm^2和（119±7）Ohm／cm^2;两两比较,差异均具有统计学意义（均P〈0．05）。结论缺氧条件下SIRT1水平降低,而SIRT1的激活可通过增加肠道紧密连接蛋白ZO-1、Occludin和Claudin-1mRNA与蛋白的表达,从而减轻缺氧对肠上皮屏障功能的损伤。     

Administration of ghrelin to young uraemic rats increases food intake transiently, stimulates growth hormone secretion and does not improve longitudinal growth.

BACKGROUND: Ghrelin administration stimulates appetite and growth hormone (GH) secretion. Whether these effects are preserved in young individuals with chronic renal failure (CRF) and their potential benefit on growth is questioned. METHODS: Three experiments were performed in subtotally nephrectomized young rats (Nx). (i) Food intake was monitored in CRF rats receiving saline intraperitoneally or a ghrelin dose (30 nmol) shown to increase food intake over 2 and 24 h in rats with normal renal function. (ii) Plasma levels of GH were measured after a dose of intravenous ghrelin (3 nmol) was given to three groups of five rats each: Nx, sham-operated fed ad libitum (SAL) and sham-operated pair-fed with Nx (SPF). (iii) Growth of Nx rats treated with intraperitoneal ghrelin (3 nmol) for 7 days (Nx-Ghr) was compared with that of SAL and Nx groups receiving saline (n=8-10 per group). RESULTS: In CRF rats, the dose of 30 nmol of ghrelin increased food consumption for 2 h (1.3+/-0.2 g vs 0.5+/-0.2 g, P<0.05) but not 24-h food intake (12.5+/-0.6 g vs 12.2+/-0.5 g). Ghrelin (3 nmol) increased plasma levels of GH, which were maximal 10 min after injection, no differences being observed among groups (SAL: 666.2+/-104.6 ng/ml; Nx: 691.6+/-90.7 ng/ml; SPF: 577.8+/-125.4 ng/ml). Return to basal GH levels was delayed in Nx. Ghrelin did not improve body length and weight gains, longitudinal bone growth rate or food intake in the Nx-Ghr group. CONCLUSIONS: In young uraemic rats, ghrelin increases appetite but not 24-h food intake, stimulates GH secretion and does not improve growth.     

慢性肾功能衰竭大鼠主动脉核因子κB活化及泛素通路的调节作用

目的　探讨慢性肾功能衰竭大鼠主动脉泛素（Ub）-NF-κB通路的表达变化规律。 方法　将大鼠随机分为假手术对照组（CON）、肾功能衰竭组（CRF）、肾功能衰竭加 MG-132干预组（CRF+M），观察6个月。采用部分肾动脉结扎加对侧肾切除法复制慢性肾功能衰竭大鼠模型。ELISA法测定血液中炎性因子白细胞介素1β（IL-1β）和肿瘤坏死因子α (TNF-α)的含量。RT-PCR半定量测定主动脉中NF-κB及Ub mRNA表达。免疫组织化学方法检测主动脉中NF-κB及Ub蛋白表达。Western印迹法测定主动脉泛素化蛋白的含量。凝胶电泳迁移率（EMSA）法测定动脉中NF-κB的活化情况。 结果　与CON组比较，CRF组大鼠术后4～6个月时血清中IL-1β[ (9.02±1.29)比(2.74±0.96) mg/L]和TNF-α[(50.02±9.52)比(14.04±1.29) mg/L]水平显著升高（P < 0.01）；主动脉NF-κB、Ub的 mRNA表达升高1.38倍和1.29倍（P < 0.01）；NF-κB、Ub的蛋白质表达升高 3.75倍和20.5倍（P < 0.01）；NF-κB活性增强1.82倍（P < 0.01），术后6个月各指标均有进一步上升趋势。与CRF组比较，CRF+M组大鼠干预治疗4～6个月后，大鼠血清中IL-1β[(2.94±0.33) mg/L]和TNF-α[(12.80±2.12) mg/L]含量显著下降（P < 0.01）；NF-κB 、Ub的mRNA及蛋白质表达显著减少（P < 0.01）；NF-κB的活性显著降低（P < 0.01），与CON组比较，差异已无统计学意义，而主动脉中泛素化蛋白含量显著升高（P < 0.01）。 结论 慢性肾功能衰竭大鼠主动脉泛素-NF-κB炎性反应信号明显活化，抑制蛋白酶体活性可能是降低主动脉NF-κB活化的重要药物靶点。     

Downregulation of intestinal cytochrome p450 in chronic renal failure

Chronic renal failure (CRF) is associated with a decrease in intestinal drug metabolism. The mechanisms remain poorly understood, but one hypothesis involves a reduction in cytochrome P450 levels. This study aimed to investigate the effects of CRF on intestinal cytochrome P450. Two groups of rats were defined, i.e., rats with CRF (induced by 5/6 nephrectomy) and control pair-fed rats. Total cytochrome P450 levels and protein and mRNA expression of cytochrome P450 isoforms, as well as in vitro N-demethylation of erythromycin (a probe for CYP3A activity) and 7-ethoxyresorufin o-deethylase activity (a probe for CYP1A), were assessed in intestinal microsomes. Body weights were similar in the two groups. Creatinine clearance was reduced by 77% (P < 0.001) in CRF rats, compared with control pair-fed animals. Total intestinal cytochrome P450 activity was reduced by 32% (P < 0.001) in CRF rats. CYP1A1 and CYP3A2 protein expression was considerably reduced (>40%, P < 0.001) in rats with CRF. CYP2B1, CYP2C6, and CYP2C11 levels were the same in the two groups. RT-PCR assays revealed marked downregulation of CYP1A1 and CYP3A2 gene expression in CRF rats (P < 0.001). Although intestinal cytochrome P450 levels were reduced in CRF, induction by dexamethasone was present. N-Demethylation of erythromycin and 7-ethoxyresorufin o-deethylase activity were decreased by 25% (P < 0.05) in CRF rats, compared with control rats. In conclusion, CRF in rats is associated with decreases in intestinal cytochrome P450 activity (mainly CYP1A1 and CYP3A2) secondary to reduced gene expression.     

Enalapril inhibits growth and proliferation of various tissues in rat normotensive four-sixths kidney ablation nephropathy

Most experimental studies on kidney proliferation and its attenuation by angiotensin-converting enzyme inhibitors were performed in the rat hypertensive remnant-kidney model with a five-sixths kidney ablation. The developing hypertension rose the objections on the hypertension and its treatment in control rats. A normotensive four-sixths remnant-kidney model (Nx) was elaborated, compared with sham-operated (S) animals, and a subantihypertensive dosage of enalapril (E) was administered for 4 weeks of intensive kidney tissue proliferation (NxE). The pair-fed groups increased their body weight and blood pressure comparably. Moderately increased plasma creatinine and urea concentrations were found in the Nx group; markedly increased levels in the NxE group. Nx increased proteinuria, and E attenuated its increase. The remnant-kidney weight (Nx 912+/-31 vs. S 1,111+/-36 mg, p<0.001) was still lower, but collagen (Col; Nx 164+/-2 vs. S 148+/-5 mg/100 g, p<0.05) and tubular protein/DNA ratio (Nx 26.2+/-10.8 vs. S 9.8+/-1. 0, p<0.05) increased markedly in the Nx group; E attenuated the kidney growth (NxE 719+/-31 vs. Nx 912+/-31 mg, p<0.01) and decreased the tubular protein/DNA ratio remarkably (NxE 15.3+/-10.5 vs. Nx 26.2 +/-10.8), but E did not inhibit the Col accumulation. Nx decreased the heart (Nx 1,002+/-28 vs. S 1,130+/-41 mg, p<0.05), but not liver weights and did not influence Col concentrations or protein/DNA ratios either in heart or liver. E potentiated the weight decrease of heart (NxE 862+/-20 vs. Nx 1,002+/-28 mg, p<0.01) and liver (NxE 8.3+/-0.44 vs. Nx 10.3+/-0.51 g, p<0.001) and Col accumulation (heart: NxE 113+/-6 vs. Nx 92+/-5 mg/100 g, p<0.01; liver: NxE 134+/-8 vs. Nx 101+/-9 mg/100 g, p<0.01). Nx did not influence either the soleus muscle weight or its Col accumulation, but it increased its protein/DNA ratio (Nx 66.3+/-4.7 vs. S 35.5+/-2. 8 mg/100 g, p<0.01). E increased the Col concentration in muscle (NxE 141+/-3 vs. Nx 110+/-5 mg/100 g, p<0.01), while it attenuated the increase in protein/DNA ratio (NxE 36.6+/-2.1 vs. Nx 66.3+/-4.7, p<0.01). In conclusion, kidney ablation nephropathy stimulating kidney proliferation evokes only minor changes in heart, liver and striated muscle. E inhibits markedly the kidney proliferation and functional recovery, but does not prevent the Col accumulation. E evokes antiproliferative changes also in the heart and surprisingly even in the liver. Alterations in soleus muscle are only borderline.     

Downregulation of neuronal nitric oxide synthase in the rat remnant kidney

Chronic renal failure is associated with disturbances in nitric oxide (NO) production. This study was conducted to determine the effect of 5/6 nephrectomy (5/6 Nx) on expression of intrarenal neuronal nitric oxide synthase (nNOS) in the rat. In normal rat kidney, nNOS protein was detected in the macula densa and in the cytoplasm and nuclei of cells of the inner medullary collecting duct by both immunofluorescence and electron microscopy. Western blot analysis revealed that 2 wk after 5/6 Nx, there were significant decreases in nNOS protein expression in renal cortex (sham: 95.42+/-15.60 versus 5/6 Nx: 47.55+/-12.78 arbitrary units, P<0.05, n = 4) and inner medulla (sham: 147.70+/-26.96 versus 5/6 Nx: 36.95+/-17.24 arbitrary units, P<0.005, n = 8). Losartan treatment was used to determine the role of angiotensin II (AngII) AT1 receptors in the inhibition of nNOS expression in 5/6 Nx. Losartan had no effect on the decreased expression of nNOS in the inner medulla, but partially increased nNOS protein expression in the cortex of 5/6 Nx rats. In contrast, in sham rats losartan significantly inhibited nNOS protein expression in the cortex (0.66+/-0.04-fold of sham values, P<0.05, n = 6) and inner medulla (0.74+/-0.12-fold of sham values, P<0.05, n = 6). nNOS mRNA was significantly decreased in cortex and inner medulla from 5/6 Nx rats, and the effects of losartan on nNOS mRNA paralleled those observed on nNOS protein expression. These data indicate that 5/6 Nx downregulates intrarenal nNOS mRNA and protein expression. In normal rats, AngII AT1 receptors exert a tonic stimulatory effect on expression of intrarenal nNOS. These findings suggest that the reduction in intrarenal nNOS expression in 5/6 Nx may play a role in contributing to hypertension and altered tubular transport responses in chronic renal failure.     

Expression of renal aquaporins 1, 2, and 3 in a rat model of cisplatin-induced polyuria

BACKGROUND: Cisplatin (CP)-induced polyuria in rats is attributed to decreased medullary hypertonicity and/or an end-organ resistance to vasopressin. However, the roles of renal aquaporins (AQPs) have not yet been explored. METHODS: Male Sprague-Dawley rats (230 to 245 g) received either a single injection of CP (5 mg/kg, N = 4) or saline (N = 4) intraperitoneally five days before sacrifice. Urine, blood, and kidney samples were analyzed. RESULTS: Platinum accumulated in the cortex and outer medulla of CP-treated rats (39.05 +/- 7.50 and 36.48 +/- 12.44 microg/g vs. 2.52 +/- 0.43 and 1.87 +/- 0.84 microg/g dry tissue in controls, respectively). Histologically, tubular damage and decreased AQP1 immunolabeling were detected in the S3 segment of proximal tubules. CP treatment caused 4.4- and 4.8-fold increases, respectively, in blood urea nitrogen and urine volume, and a 4. 4-fold decrease in urine osmolality. Immunoblots showed that AQP2 and AQP3 were significantly reduced to 33 +/- 10% (P < 0.001) and 69 +/- 11% (P < 0.05), respectively, in the inner medulla of CP-treated rats. Immunocytochemical analysis showed a decrease in AQP2 labeling in the inner medulla of CP-treated rats. Northern hybridization revealed a 33 +/- 11% (P < 0.002) decrease in AQP2 mRNA expression in the inner medulla of CP-treated rats. AQP1 protein expression levels were modestly (67 +/- 7%, P = 0.057) and significantly (53 +/- 13%, P < 0.007) decreased in outer and inner medullae, respectively, of CP-treated rats. CONCLUSIONS: CP-induced polyuria in rats is associated with a significant decrease in the expression of collecting duct (AQP2 and AQP3) and proximal nephron and microvascular (AQP1) water channels in the inner medulla.     

胰岛素样生长因子1对慢性肾功能衰竭大鼠骨骼肌蛋白质代谢的影响

目的 探讨胰岛素样生长因子1（IGF-1）在慢性肾功能衰竭（CRF）大鼠骨骼肌蛋白质代谢上的作用。方法 用放射免疫分析法测定CRF大鼠和配对喂养的假手术（SO）对照大鼠血清及骨骼肌中IGF-1的含量：计算骨骼肌蛋白质的基础合成率及分解率；评估重组人胰岛素样生长因子-1（rhIGF-1）对骨骼肌蛋白质合成与分解的影响。结果（1）CRF组血清和骨骼肌中IGF-1含量明显低于SO组；（2）CRF组骨骼肌基础蛋白合成率显著低于SO组，基础蛋白分解率显著高于SO组；（3）rhIGF-1对CRF大鼠骨骼肌蛋白质合成的促进作用仅为SO大鼠的25%～44%，对其分解的抑制作用仅为SO大鼠的15%～42%。结论CRF时，血清及骨骼肌的IGF-1含量的减少和骨骼肌对IGF-1促进蛋白质合成代谢作用的抵抗可能是白质合成减少、分解增强，导致营养不良、肌肉萎缩的主要原因。     

Effect of erythropoietin on urinary liver-type fatty-acid-binding protein in patients with chronic renal failure and anemia

BACKGROUND/AIM: The urinary liver-type fatty-acid-binding protein (L-FABP) level reflects the clinical progression of chronic kidney disease. We conducted a study to determine whether administration of erythropoietin (EPO), which is produced in response to hypoxic stress, affects urinary protein excretion and L-FABP levels in patients with chronic renal failure (CRF) and anemia. METHODS: The study was an interventional trial that included 20 anemic CRF patients (median serum creatinine level 2.0 mg/dl, range 1.3-2.9 mg/dl; median hemoglobin concentration 9.2 g/dl, range 8.2-9.8 g/dl; median estimated glomerular filtration rate 20.5 ml/min, range 15.0-28.0 ml/min; group A). Recombinant EPO (12,000 U twice/month) was given to these patients for 6 months. Urinary protein, L-FABP, 8-hydroxy-2'-deoxyguanosine, and hemoglobin levels were measured before and 3 and 6 months after treatment. Twenty nonanemic CRF patients were enrolled as controls (group B). RESULTS: After 6 months, the hemoglobin level was increased as compared with the baseline level in group A treated with EPO (median 11.3 g/dl, range 9.3-13.8 g/dl, vs. median 9.2 g/dl, range 8.2-9.8 g/dl; p < 0.01) but not in the untreated group B (median 11.8 g/dl, range 10.2-13.0 g/dl, vs. median 12.1 g/dl, range 10.8-13.4 g/dl; not significant). The urinary protein excretion was decreased as compared with the baseline level in group A (median 1.2 g/day, range 0.6-1.9 g/day, vs. median 1.9 g/day, range 1.1-2.6 g/day; p < 0.01) but not in group B (median 1.4 g/day, range 0.7-2.2 g/day, vs. median 1.6 g/day, range 0.7-2.3 g/day; not significant). The urinary L-FABP level was also decreased as compared with the baseline level in group A (median 50.0 microg/g creatinine, range 7.5-90.0 microg/g creatinine, vs. median 115.0 microg/g creatinine, range 20.0-225.0 microg/g creatinine; p < 0.01) but not in group B (median 82.0 microg/g creatinine, range 15.5-158.0 microg/g creatinine, vs. median 76.0 microg/g creatinine, range 25.0-138.5 microg/g creatinine; not significant). The glomerular filtration rate changed little throughout the study period in either group. The urinary 8-hydroxy-2'-deoxyguanosine level was decreased as compared with the baseline level in group A (median 22.0 ng/mg creatinine, range 8.0-30.0 ng/mg creatinine, vs. median 38.5 ng/mg creatinine, range 14.0-68.0 ng/mg creatinine; p < 0.01) but not in group B (median 33.0 ng/mg creatinine, range 9.0-56.0 ng/mg creatinine, vs. median 30.0 ng/mg creatinine, range 10.0-54.0 ng/mg creatinine; not significant). CONCLUSION: EPO supplementation may ameliorate renal tubular damage, in part, due to a reduction of oxidative stress in CRF patients with anemia.     

The Receptor Activator of Nuclear Factor-κB Ligand Inhibitor Osteoprotegerin Is a Bone-Protective Agent in a Rat Model of Chronic Renal Insufficiency and Hyperparathyroidism

Osteoprotegerin (OPG) acts by neutralizing the receptor activator of nuclear factor-κB ligand (RANKL), the primary mediator of osteoclast differentiation, function, and survival. We examined whether OPG could affect the bone loss associated with chronic kidney disease (CKD) in a rodent model of CKD and secondary hyperparathyroidism (SHPT). SHPT was induced in rats by 5/6 nephrectomy (5/6 Nx) and a 1.2% P/0.6% Ca²⁺ diet. Starting 1 week after 5/6 Nx, rats were treated with vehicle (veh) or OPG-Fc (3 mg/kg, intravenously) every 2 weeks for 9 weeks. At baseline, 3, 6, and 9 weeks, blood was taken and bone mineral density (BMD) and bone mineral content (BMC) were assessed by dual-energy X-ray absorptiometry. Serum parathyroid hormone (sPTH) levels reached 912 pg/ml in 5/6 Nx rats vs. 97 pg/ml in shams at 9 weeks. OPG-Fc had no effect on sPTH or Ca²⁺ levels throughout the 9-week study, indicating that SHPT was a renal effect independent of bone changes. At 3 weeks, 5/6 Nx-veh rats had osteopenia compared with sham-veh rats and 5/6 Nx-OPG-Fc rats had significantly higher percent changes in whole-body BMC, leg BMD, and lumbar BMD versus 5/6 Nx-veh rats. By 6–9 weeks, elevated sPTH was associated with reversal of bone loss and osteitis fibrosa in the proximal tibial metaphysis. OPG-Fc decreased this sPTH-driven high bone turnover, resulting in augmented thickness of proximal tibial trabeculae in 5/6 Nx rats. Thus, RANKL inhibition with OPG-Fc can block the deleterious effects of continuously elevated sPTH on bone, suggesting that RANKL may be an important therapeutic target for protecting bone in patients with CKD and SHPT.