Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation

The expression of the renoprotective antiaging gene Klotho is decreased in uremia. Recent studies suggest that Klotho may be a tumor suppressor, and its expression may be repressed by DNA hypermethylation in cancer cells. Here we investigated the effects and possible mechanisms by which Klotho expression is regulated during uremia in uninephrectomized B-6 mice given the uremic toxins indoxyl sulfate or p-cresyl sulfate. Cultured human renal tubular HK2 cells treated with these toxins were used as an in vitro model. Injections of indoxyl sulfate or p-cresyl sulfate increased their serum concentrations, kidney fibrosis, CpG hypermethylation of the Klotho gene, and decreased Klotho expression in renal tubules of these mice. The expression of DNA methyltransferases 1, 3a, and 3b isoforms in HK2 cells treated with indoxyl sulfate or p-cresyl sulfate was significantly increased. Specific inhibition of DNA methyltransferase isoform 1 by 5-aza-2'-deoxycytidine caused demethylation of the Klotho gene and increased Klotho expression in vitro. Thus, inhibition of Klotho gene expression by uremic toxins correlates with gene hypermethylation, suggesting that epigenetic modification of specific genes by uremic toxins may be an important pathological mechanism of disease.     

Accumulation of indoxyl sulfate in OAT1/3-positive tubular cells in kidneys of patients with chronic renal failure.

Indoxyl sulfate shows nephrotoxicity and is a stimulating factor for progression of chronic renal failure (CRF). Indoxyl sulfate is taken up by renal proximal tubular cells through organic anion transporters 1 and 3 (OAT1/3), and is accumulated in the renal proximal tubular cells of uremic rats. To determine whether indoxyl sulfate is accumulated in human OAT1/3 (hOAT1/3)-positive renal proximal tubular cells, localization of indoxyl sulfate and hOAT1/3 in the kidneys of CRF patients was determined by immunohistochemistry. Kidney samples were obtained by autopsy from 9 CRF patients (mean serum creatinine 4.7 mg/dL, ranging from 2.0 to 14.5 mg/dL) and 9 patients with non-kidney disease (mean serum creatinine 0.6 mg/dL, ranging from 0.4 to 0.9 mg/dL). Immunohistochemistry was performed using antibodies against indoxyl sulfate, hOAT1, and hOAT3. Indoxyl sulfate was localized in the hOAT1- and hOAT3-positive renal tubular cells in the kidneys of CRF patients. The indoxyl sulfate-positive area in the kidneys was markedly increased in the kidneys of CRF patients compared with patients with non-kidney disease. The indoxyl sulfate-positive area was positively correlated with serum creatinine. In conclusion, in CRF patients, indoxyl sulfate is accumulated in the tubular cells with hOAT1 and/or hOAT3 localized at the basolateral membrane. The extent of indoxyl sulfate accumulation in the kidneys is more prominent in those patients with more severe CRF.     

Major role of organic anion transporter 3 in the transport of indoxyl sulfate in the kidney

BACKGROUND: Indoxyl sulfate is a uremic toxin that accumulates in the body because of the patient's inability to excrete it and it induces a number of uremic symptoms and leads to chronic renal failure. The functional failure of the excretion system for indoxyl sulfate causes its accumulation in blood. The purpose of the present study was to characterize the transport mechanism responsible for the renal excretion of indoxyl sulfate. METHODS: The [3H]indoxyl sulfate transport mechanism was investigated using an in vivo tissue-sampling single-injection technique, the kidney uptake index (KUI) method. Rat organic anion transporter 3 (rOAT3)-expressing Xenopus laevis oocyte system was used for measuring [3H]indoxyl sulfate uptake activity. RESULTS: Probenecid showed a concentration-dependent inhibitory effect on the uptake of [3H]indoxyl sulfate using the KUI method, and uptake was inhibited by organic anions such as para-aminohippuric acid (PAH) and benzylpenicillin, by weak base such as cimetidine, and by uremic toxins, such as 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) and hippuric acid (HA). However, salicylic acid, indomethacin, 3,5,3'-triiodo-l-thyronine and indole acetic acid (IA) had no effect on the uptake. rOAT3-expressing oocytes exhibited uptake of [3H]indoxyl sulfate by rOAT3 (Km = 158 micromol/L). Moreover, a number of uremic toxins inhibited the uptake of [3H]indoxyl sulfate by rOAT3. CONCLUSIONS: These results suggest that rOAT3 is responsible for the renal uptake of indoxyl sulfate, and uremic toxins share the transport mechanism for indoxyl sulfate. Mutual inhibition of these uremic toxins via OAT3 may accelerate their accumulation in the body and, thereby, the progression of nephrotoxicity in uremia.     

Molecular insights into preventive effects of AST-120 on the progression of renal failure

Circulating uremic toxins are considered to be involved in the progression of chronic renal failure (CRF). An oral adsorbent AST-120 (Kremezin) is effective in removing circulating uremic toxins from the digestive tract, and retards the progression of CRF. The administration of AST-120 combined with an angiotensin-converting enzyme inhibitor or a low-proein diet has an additive effect on the progression of CRF. In a variety of experimental models of CRF, AST-120 attenuates the progression of interstitial fibrosis and inflammation, as well as attenuating that of glomerular sclerosis. However, the precise mechanism by which AST-120 delays the progression of CRF had not been clear. Indoxyl sulfate, a dietary protein metabolite, is a circulating uremic toxin that stimulates the progression of CRF. AST-120 reduces the serum and urine levels of indoxyl sulfate and the accumulation of indoxyl sulfate in remnant tubular cells, by adsorbing its precursor, indole, in the intestine. The administration of indoxyl sulfate to uremic rats stimulated the expression of transforming growth factor (TGF)-β1, tissue inhibitor of metalloproteinase (TIMP)-1, and pro-α1(I)collagen in the kidneys. The administration of AST-120 to uremic rats reduced the extent of glomerular sclerosis and interstitial fibrosis, as well as reducing the renal expression of TGF-β1 and TIMP-1, by alleviating the overload of indoxyl sulfate in remnant tubular cells. We propose the protein metabolite theory, i.e., that endogenous protein metabolites such as indoxyl sulfate play an important role in the progression of CRF, and that AST-120 is effective in retarding the progression of CRF by adsorbing these protein metabolites in the intestine. Received: August 31, 2001 / Accepted: September 11, 2001     

Does uremia cause vascular dysfunction?

Vascular dysfunction induced by uremia has 4 main aspects. (1) Atherosclerosis is increased. Intima-media thickness is increased, and animal studies have established that uremia accelerates atherosclerosis. Uremic toxins are involved in several steps of atherosclerosis. Leukocyte activation is stimulated by guanidines, advanced glycation end products (AGE), p-cresyl sulfate, platelet diadenosine polyphosphates, and indoxyl sulfate. Endothelial adhesion molecules are stimulated by indoxyl sulfate. Migration and proliferation of vascular smooth muscle cells (VSMC) are stimulated by local inflammation which could be triggered by indoxyl sulfate and AGE. Uremia is associated with an increase in von Willebrand factor, thrombomodulin, plasminogen activator inhibitor 1, and matrix metalloproteinases. These factors contribute to thrombosis and plaque destabilization. There is also a decrease in nitric oxide (NO) availability, due to asymmetric dimethylarginine (ADMA), AGE, and oxidative stress. Moreover, circulating endothelial microparticles (EMP) are increased in uremia, and inhibit the NO pathway. EMP are induced in vitro by indoxyl sulfate and p-cresyl sulfate. (2) Arterial stiffness occurs due to the loss of compliance of the vascular wall which induces an increase in pulse pressure leading to left ventricular hypertrophy and a decrease in coronary perfusion. Implicated uremic toxins are ADMA, AGE, and oxidative stress. (3) Vascular calcifications are increased in uremia. Their formation involves a transdifferentiation process of VSMC into osteoblast-like cells. Implicated uremic toxins are mainly inorganic phosphate, as well as reactive oxygen species, tumor necrosis factor and leptin. (4) Abnormalities of vascular repair and neointimal hyperplasia are due to VSMC proliferation and lead to severe reduction of vascular lumen. Restenosis after coronary angioplasty is higher in dialysis than in nondialysis patients. Arteriovenous fistula stenosis is the most common cause of thrombosis. Uremic toxins such as indoxyl sulfate and some guanidine compounds inhibit endothelial proliferation and wound repair. Endothelial progenitor cells which contribute to vessel repair are decreased and impaired in uremia, related to high serum levels of β(2)-microglobulin and indole-3 acetic acid. Overall, there is a link between kidney function and cardiovascular risk, as emphasized by recent meta-analyses. Moreover, an association has been reported between cardiovascular mortality and uremic toxins such as indoxyl sulfate, p-cresol and p-cresyl sulfate.     

转化生长因子β1对肾小管上皮细胞中结缔组织生长因子基因启动子活性的影响

目的探讨转化生长因子[β1（TCF-β1）对人近端肾小管上皮细胞系HK-2中结缔组织生长因子（CTGF）基因启动子活性的调控作用，以及丝裂原激活蛋白激酶（MAPK）途径对该生长因子作用的影响。方法构建含有人类CTGF基因启动子的报告基因pCTGF-luc，将其瞬时转染HK-2细胞。通过检测荧光素酶的活性观察TGF-β1和MAPK途径抑制剂对CTGF基因启动子活性的影响。结果TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子的活性。最佳刺激浓度是5ng／ml，最佳刺激时间为12h，荧光素酶相对活性分别为对照组的1．82倍和2．10倍（P〈0．05）。应用PD98059、SB203580和SP600125分别特异性抑制MAPK途径的胞外信号调节蛋白激酶（ERK）、蛋白激酶p38（p38MAPK）和c-Jun-氨基末端激酶（JNK）通路，对TGF-β1上调CTGF启动子活性的作用有不同影响。PD98059显著增加HK-2中pCTGF-luc的基础活性．并在一定浓度范围内（0．5～10μmol／L）促进TGF-β1的上调作用。SB203580对pCTGF-luc基础活性无影响，但以剂量依赖方式显著抑制TGF-β1的激活效应。而SP600125对基础状态和TGF-β1刺激下CTGF基因启动子活性无影响。结论TGF-β1以剂量和时间依赖方式上调HK-2中CTGF基因启动子活性，在转录水平调节CTGF表达。MAPK途径的ERK和p38MAPK通路可影响TGF-β1的这一调控作用。     

Inhibitory effect of oral sorbent on accumulation of albumin-bound indoxyl sulfate in serum of experimental uremic rats

Serum indoxyl sulfate, which is markedly accumulated in uremic patients, cannot be removed efficiently by hemodialysis due to its albumin binding. To determine if oral adsorbent (AST-120) can decrease its serum concentration in uremic state, oral adsorbent was administered to experimental nephrectomized uremic rats. Uremic rats fed with oral adsorbent showed a significantly lower serum concentration of indoxyl sulfate compared to control uremic rats, even when serum concentrations of urea nitrogen and creatinine were not significantly decreased in the uremic rats fed with oral adsorbent. Indoxyl sulfate was detected only at a lower concentration in bile as compared with the serum of uremic rats. These results suggest that oral adsorbent adsorbs indole, a precursor of indoxyl sulfate, in the intestine and prevents the accumulation of indoxyl sulfate in uremic rats.     

Roles of organic anion/cation transporters at the blood–brain and blood–cerebrospinal fluid barriers involving uremic toxins

The blood–brain barrier (BBB) and blood–cerebrospinal fluid barrier (BCSFB) play key roles in the influx and efflux transport of endogenous substrates in the brain and cerebrospinal fluid. The organic anion transporter (OAT) 3 and organic cation transporter (OCT) 3, which belong to the solute carrier (SLC) 22A family, are expressed at the BBB and BCSFB, and regulate the excretion of endogenous and exogenous organic anions and cations. Our recent research provides novel molecular and functional evidence that indoxyl sulfate, an anionic uremic toxin, undergoes efflux transport at the BBB via OAT3 and creatinine, a uremic guanidino compound, undergoes efflux transport at the BCSFB via OCT3. Renal impairment is associated with the accumulation of uremic toxins in blood and uremic encephalopathy. It is conceivable that uremic encephalopathy is related to inhibition or dysfunction of efflux transport systems for uremic toxins in the brain. Here, we review the function of OAT3 and OCT3 at the BBB and BCSFB in the context of their roles in the progression of renal failure.     

Role of organic anion transporters in the tubular transport of indoxyl sulfate and the induction of its nephrotoxicity

In uremic patients, various uremic toxins are accumulated and exert various biologic effects on uremia. Indoxyl sulfate (IS) is one of uremic toxins that is derived from dietary protein, and serum levels of IS are markedly increased in both uremic rats and patients. It has been previously reported that the accumulation of IS promotes the progression of chronic renal failure (CRF). This study demonstrates the role of rat organic anion transporters (rOATs) in the transport of IS and the induction of its nephrotoxicity. The administration of IS to 5/6-nephrectomized rats caused a faster progression of CRF, and immunohistochemistry revealed that IS was detected in the proximal and distal tubules where rOAT1 (proximal tubules) and/or rOAT3 (proximal and distal tubules) were also shown to be localized. In in vitro study, the proximal tubular cells derived from mouse that stably express rOAT1 (S2 rOAT1) and rOAT3 (S2 rOAT3) were established. IS inhibited organic anion uptake by S2 rOAT1 and S2 rOAT3, and the Ki values were 34.2 and 74.4 microM, respectively. Compared with mock, S2 rOAT1 and S2 rOAT3 exhibited higher levels of IS uptake, which was inhibited by probenecid and cilastatin, organic anion transport inhibitors. The addition of IS induced a decrease in the viability of S2 rOAT1 and S2 rOAT3 as compared with the mock, which was rescued by probenecid. These results suggest that rOAT1 and rOAT3 play an important role in the transcellular transport of IS and the induction of its nephrotoxicity.     

Protein-Bound Uremic Toxins Induce Tissue Remodeling by Targeting the EGF Receptor

Indoxyl sulfate and p-cresol sulfate have been suggested to induce kidney tissue remodeling. This study aimed to clarify the molecular mechanisms underlying this tissue remodeling using cultured human proximal renal tubular cells and half-nephrectomized mice treated with indoxyl sulfate or p-cresol sulfate as study models. Molecular docking results suggested that indoxyl sulfate and p-cresol sulfate dock on a putative interdomain pocket of the extracellular EGF receptor. In vitro spectrophotometric analysis revealed that the presence of a synthetic EGF receptor peptide significantly decreased the spectrophotometric absorption of indoxyl sulfate and p-cresol sulfate. In cultured cells, indoxyl sulfate and p-cresol sulfate activated the EGF receptor and downstream signaling by enhancing receptor dimerization, and increased expression of matrix metalloproteinases 2 and 9 in an EGF receptor–dependent manner. Treatment of mice with indoxyl sulfate or p-cresol sulfate significantly activated the renal EGF receptor and increased the tubulointerstitial expression of matrix metalloproteinases 2 and 9. In conclusion, indoxyl sulfate and p-cresol sulfate may induce kidney tissue remodeling through direct binding and activation of the renal EGF receptor.     

Indoxyl sulfate induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells

Skeletal resistance to parathyroid hormone (PTH) is well known to the phenomenon in chronic renal failure patient, but the detailed mechanism has not been elucidated. In the process of analyzing an animal model of renal failure with low bone turnover, we demonstrated decreased expression of PTH receptor (PTHR) accompanying renal dysfunction in this model. In the present study, we focused on the accumulation of uremic toxins (UTx) in blood, and examined whether indoxyl sulfate (IS), a UTx, is associated with PTH resistance. We established primary osteoblast cultures from mouse calvariae and cultured the cells in the presence of IS. The intracellular cyclic adenosine 3',5' monophosphate (cAMP) production, PTHR expression, and free radical production in the primary osteoblast culture were studied. We found that the addition of IS suppressed PTH-stimulated intracellular cAMP production and decreased PTHR expression in this culture system. Free radical production in osteoblasts increased depending on the concentration of IS added. Furthermore, expression of organic anion transporter-3 (OAT-3) that is known to mediate cellular uptake of IS was identified in the primary osteoblast culture. These results suggest that IS taken up by osteoblasts via OAT-3 present in these cells augments oxidative stress to impair osteoblast function and downregulate PTHR expression. These finding strongly suggest that IS accumulated in blood due to renal dysfunction is at least one of the factors that induce skeletal resistance to PTH.     

甲状旁腺素对人肾小管上皮细胞细胞外基质合成与降解的影响

目的 研究甲状旁腺素（PTH）对人肾小管上皮细胞（HK-2）合成分泌Ⅲ型胶原、纤连蛋白以及对纤溶酶原激活物抑制物1（PAI-1）、基质金属蛋白酶1（MMP-1）、金属蛋白酶1组织抑制剂（TIMP-1）基因表达的影响,以探讨PTH在肾间质纤维化发病机制中的作用。 方法 HK-2细胞培养于含5％ FBS的DMEM-F12培养液中,加入终浓度为0、10-12、10-11、10-10、10-9、10-8 mol/L PTH的培养液刺激HK-2细胞48 h,或10-8 mol/L PTH刺激HK-2细胞不同时间（0、12、24、48、72 h）。应用半定量RT-PCR法检测细胞中Ⅲ型胶原、纤连蛋白、PAI-1、MMP-1、TIMP-1基因的表达;Western印迹法检测HK-2细胞中Ⅲ型胶原的蛋白表达;ELISA法检测细胞培养上清液中纤连蛋白的含量。 结果 PTH 呈剂量和时间依赖性促进HK-2细胞中Ⅲ型胶原、纤连蛋白、PAI-1、TIMP-1 mRNA的表达,抑制MMP-1 mRNA的表达,MMP-1/TIMP-1 比值降低。PTH呈剂量和时间依赖性增加HK-2细胞中Ⅲ型胶原的蛋白表达及细胞培养上清液中纤连蛋白的含量。 结论 PTH通过促进HK-2细胞中PAI-1、TIMP-1的基因表达,抑制MMP-1的基因表达,导致细胞外基质合成增加而降解减少。     

Characterization of uremic toxin transport by organic anion transporters in the kidney

BACKGROUND: Harmful uremic toxins, such as indoxyl sulfate (IS), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoleacetate (IA), and hippurate (HA), accumulate to a high degree in uremic plasma. IS has been shown to be a substrate of rat organic anion transporter 1 (rOat1) and rOat3. However, the contribution of rOat1 and rOat3 to the renal uptake transport process of IS and other uremic toxins in the kidney remains unknown. METHODS: The cellular uptake of uremic toxins was determined using stable transfectants of rOat1/hOAT1 and rOat3/hOAT3 cells. Also, the uptake of uremic toxins by rat kidney slices was characterized to evaluate the contribution of rOat1 and rOat3 to the total uptake by kidney slices using inhibitors of rOat1 (p-aminohippurate) and rOat3 (pravastatin and benzylpenicillin). RESULTS: Saturable uptake of IS, CMPF, IA, and HA by rOat1 was observed with Km values of 18, 154, 47, and 28 micromol/L, respectively, whereas significant uptake of IS and CMPF, but not of IA or HA, was observed in rOat3-expressing cells with Km values of 174 and 11 micromol/L, respectively. Similar parameters were obtained for human OAT1 and OAT3. Kinetic analysis of the IS uptake by kidney slices revealed involvement of two saturable components with Km1 (24 micromol/L) and Km2 (196 micromol/L) values that were comparable with those of rOat1 and rOat3. The Km value of CMPF uptake by kidney slices (22 micromol/L) was comparable with that of rOat3, while the corresponding values of IA and HA (42 and 33 micromol/L, respectively) were similar to those of rOat1. PAH preferentially inhibited the uptake of IA and HA by kidney slices, while pravastatin and benzylpenicillin preferentially inhibited the uptake of CMPF. The effect of these inhibitors on the uptake of IS by kidney slices was partial. CONCLUSIONS: rOat1/hOAT1 and rOat3/hOAT3 play major roles in the renal uptake of uremic toxins on the basolateral membrane of the proximal tubules. Both OAT1 and OAT3 contribute almost equally to the renal uptake of IS. OAT3 mainly accounts for CMPF uptake by the kidney, while OAT1 mainly accounts for IA and HA uptake.     

金雀异黄素在甲状旁腺激素诱导的人近曲小管上皮细胞结缔组织生长因子表达中的调控作用

目的 探讨金雀异黄素（Gen）对甲状旁腺激素（PTH）引起的人近曲小管上皮细胞分泌结缔组织生长因子（CTGF）的调控作用。 方法 应用实时定量-聚合酶链反应（real time-PCR）、Western蛋白印迹、报告基因等技术,观察Gen对PTH诱导人近端肾小管上皮细胞系HK-2细胞CTGF表达的影响。使用MAPK通路抑制剂U0126阻断信号通路以明确Gen发挥作用的机制。 结果 HK-2细胞有基础量的CTGF mRNA和蛋白表达,PTH刺激后其表达量显著增加（P ＜ 0.05）。10-10 mol/L PTH作用12 h后,荧光素酶活性较对照组明显升高（1.8884±0.0780比0.9891±0.0300,P ＜ 0.01）。Gen剂量依赖性下调PTH诱导的HK-2细胞CTGF表达。正常HK-2细胞有少量磷酸化（p）ERK1/2表达,PTH刺激后p-ERK1/2表达明显升高,以10-10 mol/L PTH作用30 min时效应最强。U0126作用后,CTGF mRNA、蛋白表达均明显下降（P ＜ 0.05）。Gen抑制PTH所致的HK-2细胞ERK1/2活化。 结论 Gen可通过阻断MAPK信号通路抑制PTH诱导的HK-2细胞CTGF表达。     

甲状旁腺激素通过丝裂原活化蛋白激酶信号通路诱导人近曲小管上皮细胞结缔组织生长因子表达

目的 观察甲状旁腺激素（PTH）对人肾小管上皮细胞分泌结缔组织生长因子（CTGF）的影响，并探讨丝裂原活化蛋白激酶（MAPK）信号途径在此过程中的作用。 方法 采用实时定量PCR、Western印迹、报告基因等技术，观察PTH诱导人近端肾小管上皮细胞系HK-2细胞CTGF表达的情况。使用信号通路抑制剂PD98059、U0126阻断信号通路以明确PTH发挥作用的信号途径。 结果 正常HK-2细胞有基础水平的CTGF mRNA和蛋白表达，PTH刺激后其表达水平显著增加。10-10 mol/L PTH作用12 h后，荧光素酶活性较对照组明显升高[（1.8884±0.0780）比（0.9891±0.0300） A，P ＜ 0.01]。正常HK-2细胞有少量p-ERK1/2表达，PTH刺激后p-ERK1/2表达明显升高，以10-10 mol/L PTH作用30 min时效应最强；MAPK通路抑制剂PD98059、U0126作用后，CTGF mRNA、蛋白、基因启动子表达均明显下降。 结论 PTH可诱导HK-2细胞CTGF表达，其作用可能是通过MAPK信号通路来实现的。     

Uremic Toxins in Acute Kidney Injury

Acute kidney injury (AKI) is a serious and frequent condition which may fully resolve but is associated with markedly increased mortality. Mortality in AKI is caused by nonrenal, distant organ failure. Renal recovery from AKI is often not achieved on account of new injuries in the repair phase. Uremic toxins may be the missing link between AKI and nonrenal organ failure, tubular and endothelial injury. Compared with chronic kidney disease (CKD), research of uremic toxins in AKI is in its infancy. This review presents the current knowledge on uremic toxins in AKI which is predominately derived from experimental studies. Most uremic toxins investigated have previously been identified in CKD. The review focuses on those uremic toxins with biologic effect on the respective nonrenal organs failing in AKI and on the renal tubule and the endothelium. These uremic toxins may insofar be specific mediators of pathophysiological processes in AKI.     

Suppressed serum and urine levels of indoxyl sulfate by oral sorbent in experimental uremic rats.

In uremic patients, the serum concentration of indoxyl sulfate is markedly increased. To determine if oral sorbent (AST-120) suppresses the endogenous synthesis of indoxyl sulfate, it was administered to experimental uremic rats, and the serum concentration and urinary excretion of indoxyl sulfate were quantified by high-performance liquid chromatography. Oral sorbent decreased both the serum concentration and urinary excretion of indoxyl sulfate, suggesting that there was suppression of the endogenous synthesis of indoxyl sulfate by the oral sorbent. Oral sorbent did not decrease the serum concentration and urinary excretion of hippuric acid, but it did alleviate the deterioration of renal function in the experimental uremic rats.