Uremic Solutes Produced by Colon Microbes.

BACKGROUND: Colon microbes produce a large number of organic compounds that are foreign to mammalian cell metabolism. SUMMARY: Some of the compounds made by microbes are absorbed in the colon and then normally excreted by the kidneys. Accumulation of these compounds in the plasma as uremic solutes may contribute to illness in patients whose kidneys have failed. Mass spectrometry is expanding our knowledge of the chemical identity of the colon-derived uremic solutes, and DNA sequencing technologies are providing new knowledge of the microbes and metabolic pathways by which they are made. Because they are made in an isolated compartment by microbes, their production may prove simpler to suppress than the production of other uremic solutes. KEY MESSAGES: To the extent that they are toxic, suppressing their production could improve the health of renal failure patients without the need for more intensive or prolonged dialysis.

A human serum albumin-thioredoxin fusion protein prevents experimental contrast-induced nephropathy.

Contrast-induced nephropathy (CIN), caused by a combination of the direct tubular toxicity of contrast media, a reduction in medullary blood flow, and the generation of reactive oxygen species, is a serious clinical problem. A need exists for effective strategies for its prevention. Thioredoxin-1 (Trx) is a low-molecular-weight endogenous redox-active protein with a short half-life in the blood due to renal excretion. We produced a long-acting form of Trx as a recombinant human albumin-Trx fusion protein (HSA-Trx) and examined its effectiveness in preventing renal injury in a rat model of ioversol-induced CIN. Compared with saline, a mixture of HSA and Trx, or Trx alone, intravenous HSA-Trx pretreatment significantly attenuated elevations in serum creatinine, blood urea nitrogen, and urinary N-acetyl-ß-D-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx also caused a substantial reduction in the histological features of renal tubular injuries and in the number of apoptosis-positive tubular cells. Changes in the markers 8-hydroxy deoxyguanosine and malondialdehyde indicated that HSA-Trx significantly suppressed renal oxidative stress. In HK-2 cells, HSA-Trx decreased the level of reactive oxygen species induced by hydrogen peroxide, and subsequently improved cell viability. Thus, our results suggest that due to its long-acting properties, HSA-Trx has the potential to effectively prevent CIN.

p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase.

The accumulation of p-cresyl sulfate (PCS), a uremic toxin, is associated with the mortality rate of chronic kidney disease patients; however, the biological functions and the mechanism of its action remain largely unknown. Here we determine whether PCS enhances the production of reactive oxygen species (ROS) in renal tubular cells resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human tubular epithelial cells by enhancing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity. PCS also upregulated mRNA levels of inflammatory cytokines and active TGF-ß1 protein secretion associated with renal fibrosis. Knockdown of p22(phox) or Nox4 expression suppressed the effect of PCS, underlining the importance of NADPH oxidase activation on its mechanism of action. PCS also reduced cell viability by increasing ROS production. The toxicity of PCS was largely suppressed in the presence of probenecid, an organic acid transport inhibitor. Administration of PCS for 4 weeks caused significant renal tubular damage in 5/6-nephrectomized rats by enhancing oxidative stress. Thus, the renal toxicity of PCS is attributed to its intracellular accumulation, leading to both increased NADPH oxidase activity and ROS production, which, in turn, triggers induction of inflammatory cytokines involved in renal fibrosis. This mechanism is similar to that for the renal toxicity of indoxyl sulfate.

Nuclear envelope-localized EGF family protein amphiregulin activates breast cancer cell migration in an EGF-like domain independent manner.

Amphiregulin (AREG), an EGF family protein, is synthesized as a type I transmembrane precursor (proAREG) and expressed on the cell surface with an extracellular EGF-like domain and an intracellular short cytoplasmic tail. The ectodomain shedding yields a soluble EGF receptor ligand (soluble AREG) which binds to EGF receptor (EGFR) and concomitantly induces migration of unshed proAREG from the plasma membrane to the nuclear envelope (NE). AREG is known to play a potential role in breast cancer and has been intensively investigated as an EGF receptor ligand, while the function of the NE-localized proAREG remains unknown. In this study we used a truncated mutant that mimics NE-localized proAREG without shedding stimuli to discriminate between the functions of NE-localized and plasma membrane-localized proAREG and demonstrate that NE-localized proAREG activates breast cancer cell migration, but suppresses cell growth. Moreover, the present study shows that induction of cell migration by NE-localized proAREG does not require the extracellular growth factor domain or EGF receptor function. Collectively these data demonstrate a novel function mediated by the intracellular domain of proAREG and suggest a significant role for NE-localized proAREG in driving human breast cancer progression.

Characterization and quantification of proliferating cell patterns in endocapillary proliferation.

BACKGROUND: Endocapillary proliferation (EP) is a common pathological finding in proliferative glomerulonephritis (GN). Its appearance indicates the presence of active lesions of GN. In this study, we reinvestigated the pathological features of EP. METHODS: Cell markers that included CD15, CD68, CD45RO, CD31 and alpha-smooth muscle actin (alpha-SMA) were used to identify the intraglomerular cells in renal biopsy tissues collected from patients with post-streptococcal acute GN (PSAGN), methicillin-resistant Staphylococcus aureus-associated GN (MRSAGN) with or without EP, membranoproliferative GN (MPGN) with or without EP, Henoch-Schönlein nephritis, immunoglobulin A nephropathy, membranous nephropathy and minimal change nephrotic syndrome. Proliferating cells and apoptotic cells were investigated simultaneously. RESULTS: The glomerular infiltrating polymorphonuclear leukocytes, macrophages, T cells, mesangial cells and endothelial cells were enumerated. In PSAGN, the glomerulus was enlarged and all cell types were greatly increased. In MRSAGN EP, the glomerulus was slightly enlarged with abundant infiltrating leukocytes and monocyte/macrophages and had moderate mesangial cell proliferation with negligible endothelial cell proliferation. In MPGN, the glomerulus markedly enlarged with multiple monocyte/macrophages and remarkable mesangial proliferation. The mesangial cells in EP glomeruli were highly activated as evidenced by alpha-SMA expression, particularly in PSAGN. CONCLUSIONS: This is the first report to use monoclonal antibodies specific for cell markers to quantitatively analyze and compare the proliferating cell types in EP glomeruli in different forms of GN. The results suggest that identification of the presence of polymorphonuclear leukocytes in the capillary lumen might help in differentiating between glomerular global and segmental EP.

Effects of cinacalcet treatment on serum soluble Klotho levels in haemodialysis patients with secondary hyperparathyroidism.

BACKGROUND: Klotho is a transmembrane protein that acts as a cofactor for fibroblast growth factor 23 (FGF23). Klotho also exists as a soluble circulating protein, but its role in secondary hyperparathyroidism (SHPT) is largely unknown. METHODS: We measured serum soluble Klotho levels in 51 haemodialysis patients, who participated and completed a 52-week, multicentre, open-label single-arm trial that examined the effectiveness of cinacalcet for treating SHPT. RESULTS: After 12 weeks of cinacalcet treatment, serum soluble Klotho decreased significantly (P = 0.03) but only marginally from 398 pg/mL [interquartile range (IQR), 268-588 pg/mL] to 378 pg/mL (IQR, 266-568 pg/mL) and returned to baseline levels. There were no significant associations between the changes in soluble Klotho levels and changes in any other parameters of mineral metabolism, including serum calcium, phosphorus, intact parathyroid hormone and FGF23. CONCLUSION: Despite significant alterations in mineral and bone metabolism during treatment with cinacalcet, this resulted in only small and transient reductions in serum levels of soluble Klotho.

Role of uremic toxins and oxidative stress in the development of chronic kidney disease-mineral and bone disorder.

The kidney plays an important role in the regulation of mineral metabolism. As kidney function declines, there is a progressive deterioration in mineral homeostasis, along with various abnormalities, including bone disease and vascular calcification, which has recently been named as "Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD)." Although the precise mechanisms of this systemic disorder remain to be elucidated, accumulating evidence suggest that uremic toxins contribute substantially to the development of CKD-MBD, partly through evoking oxidative stress in the bone and cardiovascular systems. This brief review summarizes recent work on the role of uremic toxins and oxidative stress in the development of CKD-MBD.

[Hormonal regulation of phosphate balance].

Phosphate homeostasis is maintained by intestine, bone, and kidney, which is regulated by many hormones. In addition to classical hormones such as parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25 (OH) (2)D] , fibroblast growth factor 23 (FGF23) and Klotho have been discovered as novel factors for phosphate metabolism. FGF23 is a circulating hormone that is synthesized by osteocytes and osteoblasts in bone. The physiological actions of FGF23 are to promote phosphaturia and decrease production of 1,25 (OH) (2)D. Klotho was initially identified as an anti-aging protein, but subsequent research revealed multiple functions of Klotho in phosphate metabolism. Klotho directly binds to FGF receptor 1 (FGFR1) and contributes to FGFR1-Klotho complex-mediated signal transduction with higher affinity than FGFR1 or Klotho alone. In addition, the extracellular domain of Klotho is clipped by enzymes and secreted into blood, which exerts phosphaturic effects independently of FGF23. In the regulation of phosphate metabolism, this FGF23-Klotho axis constitutes feedback loops with classical factors such as PTH and 1,25 (OH) (2)D. However, the precise feedback system in the phosphate metabolism remains to be determined and further research is required to provide a greater understanding of the phosphate metabolism system.

Interrelationships Between Sclerostin, Secondary Hyperparathyroidism, and Bone Metabolism in Patients on Hemodialysis.

CONTEXT: Sclerostin is an osteocyte-derived inhibitor of bone formation and is increased in kidney failure, but its role in the pathogenesis of renal bone disease remains unknown. OBJECTIVE: We aimed to explore the association of serum sclerostin with bone metabolism in patients undergoing hemodialysis, with a particular focus on parathyroid hormone (PTH)-dependent and PTH-independent pathways. METHODS: This cross-sectional and prospective cohort study included 654 patients undergoing hemodialysis at 10 facilities in Japan. We employed multivariable linear regression to explore whether sclerostin levels were associated with metacarpal bone mineral density (BMD), intact PTH, bone alkaline phosphatase (BAP), and tartrate-resistant acid phosphatase-5b (TRACP-5b). We employed mediation analyses to explore whether and to what extent the association of PTH with bone turnover markers is mediated by sclerostin. We also compared sclerostin levels between patients with and without previous or incident fractures. RESULTS: The median sclerostin level in hemodialysis patients was 3- to 4-fold higher than that in healthy individuals. Higher sclerostin levels were associated with higher metacarpal BMD and lower levels of intact PTH, BAP, and TRACP-5b. However, the relationships of sclerostin with bone turnover markers were substantially attenuated after adjustment for PTH. Mediation analysis suggested that the effects of PTH on bone turnover markers were mainly direct rather than mediated by sclerostin. Sclerostin levels were not associated with previous or incident fractures. CONCLUSION: These findings suggest that in patients undergoing dialysis, sclerostin has only a limited role in bone metabolism and may not mediate the effect of PTH on bone turnover.

[Frontiers in vitamin D; basic research and clinical application. Vitamin D and secondary hyperparathyroidism].

In chronic kidney disease (CKD) , phosphate retention and low 1, 25-dihydroxyvitamin D [1, 25 (OH) 2D] due to a decrease in the number of functioning nephrons had long been considered to contribute to the pathogenesis of secondary hyperparathyroidism (SHPT) . The discovery of fibroblast growth factor 23 (FGF23) drastically changed our understanding and provided new implication of the development of SHPT. In addition, CKD impairs the maintenance of normal 25-hydoxyvitamin D [25 (OH) D] levels which can act directly on the parathyroid glands to repress PTH synthesis. Active vitamin D therapy is widely used for the treatment of SHPT. However, the active form of vitamin D, 1, 25 (OH) 2D and its analogs increase the calcium and phosphate levels by increasing the intestinal calcium and phosphate absorption. To suppress PTH secretion without these potential complications, several vitamin D analogs including paricalcitol, maxacalcitol and doxercalciferol are already in clinical use. A novel therapeutic concept in new vitamin D therapies are expected to improve patient outcome.

An Enlarged Profile of Uremic Solutes.

Better knowledge of the uremic solutes that accumulate when the kidneys fail could lead to improved renal replacement therapy. This study employed the largest widely available metabolomic platform to identify such solutes. Plasma and plasma ultrafiltrate from 6 maintenance hemodialysis (HD) patients and 6 normal controls were first compared using a platform combining gas and liquid chromatography with mass spectrometry. Further studies compared plasma from 6 HD patients who had undergone total colectomy and 9 with intact colons. We identified 120 solutes as uremic including 48 that had not been previously reported to accumulate in renal failure. Combination of the 48 newly identified solutes with those identified in previous reports yielded an extended list of more than 270 uremic solutes. Among the solutes identified as uremic in the current study, 9 were shown to be colon-derived, including 6 not previously identified as such. Literature search revealed that many uremic phenyl and indole solutes, including most of those shown to be colon-derived, come from plant foods. Some of these compounds can be absorbed directly from plant foods and others are produced by colon microbial metabolism of plant polyphenols that escape digestion in the small intestine. A limitation of the metabolomic method was that it underestimated the elevation in concentration of uremic solutes which were measured using more quantitative assays.

A Retrospectively Diagnosed Case of IgG4-Related Tubulointerstitial Nephritis Showing Good Renal Outcome and Pathological Progress.

A 74-year-old man was hospitalized for diabetic nephropathy evaluation and assessment of the effect of treatment on his tubulointerstitial nephritis (TIN). When he was 62 years old, he developed polyarthralgia and had superficial lymph node swelling, mildly increased serum creatinine concentration, hypergammaglobulinemia, hypocomplementemia, high serum IL-2R level, and positive titer of antinuclear antibody. Several tissues were biopsied. Mild chronic sialadenitis and reactive lymphadenitis were identified. Renal specimen showed mild glomerular ischemia, extensive storiform fibrosis, and abundant infiltrating monocytes and plasma cells. He was treated with oral prednisolone and cyclophosphamide. After the treatment, most of his clinical parameters quickly returned to within the reference range. However, he developed diabetes mellitus soon after steroid therapy. At the time of rebiopsy, a high level of serum IgG4 was detected. The second renal biopsy showed diabetic nephropathy without any tubulointerstitial damage. The first biopsied tissues were retrospectively investigated. Large numbers of IgG4-positive plasma cells were detected in the kidneys and lymph nodes. A retrospective diagnosis of IgG4-related TIN with lymph node involvement was made. In conclusion, this paper describes a retrospectively diagnosed case of IgG4-related TIN with lymph node involvement, showing good clinical and pathological prognosis.

p-Cresyl sulfate induces osteoblast dysfunction through activating JNK and p38 MAPK pathways.

Recent data suggest that several uremic toxins may contribute to the development of bone abnormalities in chronic kidney disease. p-Cresyl sulfate (PCS), the sulfate conjugate of p-cresol, is a protein-bound uremic toxin associated with the progression of chronic kidney disease, cardiovascular risk, and mortality. However, the effects of PCS on bone metabolism remain unclear. In the present study, we evaluated the toxic effects of PCS on primary mouse osteoblasts, compared with an extensively studied uremic toxin indoxyl sulfate (IS). Pre-treatment of osteoblasts with PCS at 0.125 mM and above significantly decreased parathyroid hormone (PTH)-induced cAMP production in a dose-dependent manner. PCS also induced a significant increase in intracellular production of reactive oxygen species (ROS) at 0.25 mM and above, but not at lower concentrations. PCS at 0.125 mM (a concentration that did not induce significant ROS increase) decreased cell viability by augmenting DNA fragmentation and reducing cell proliferation. Inhibition of JNK and p38 mitogen-activated protein kinase (MAPK) abolished the PCS-induced increase in DNA fragmentation and decrease in cAMP production in osteoblastic cells. Compared with PCS, IS induced ROS production at 0.05 mM but did not reduce cAMP production from 0.05 to 0.5 mM. IS induced decrease in cell viability and increase in DNA fragmentation at 0.5mM only. These results suggest that PCS damages osteoblastic cells through not only increasing ROS production but also activating JNK/p38 MAPKs, which is different from the mechanism of injury by IS. These damages of osteoblasts induced by PCS may play a critical role in impairing bone metabolism in patients with chronic kidney disease in whom PCS accumulates.

Novel electrochemiluminescence immunoassay exclusively for full-length parathyroid hormone during treatment with cinacalcet for secondary hyperparathyroidism.

A novel, electrochemiluminescence immunoassay that exclusively measures full-length parathyroid hormone (PTH), called Elecsys PTH (1-84) assay, is currently under development for clinical use. We measured serum PTH levels using this novel assay, as well as the Elecsys Intact PTH assay and the Whole PTH immunoradiometric assay, in 53 hemodialysis patients who participated in a 52-week clinical trial of cinacalcet. At baseline, serum PTH (1-84) levels measured with the Elecsys PTH (1-84) assay and those with the Whole PTH assay were comparable, and both values were significantly lower than Elecsys Intact PTH levels. After 52 weeks of cinacalcet treatment, Elecsys PTH (1-84) levels and Whole PTH levels decreased significantly by 56% and 60% from baseline, respectively. These results indicate that the Elecsys PTH (1-84) assay provides comparable data to the Whole PTH assay for monitoring parathyroid function in patients receiving hemodialysis. Introduction of this novel automated immunoassay would provide more widespread measurements of full-length PTH (1-84) in clinical practice.

Atypical thymic carcinoid associated with Cushing's syndrome.

A 56-year-old Japanese woman with adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome (CS) was admitted to hospital, where she was diagnosed as having a mediastinal tumor with ectopic ACTH production. The tumor and associated lymph node metastases were resected endoscopically, and the pathological diagnosis was atypical thymic carcinoid. Radiation therapy and administration of metyrapone, an inhibitor of 11b-hydroxylase to decrease the cortisol level, were attempted, but the levels of ACTH and cortisol were unresponsive. Bilateral adrenalectomy and hydrocortisone replacement were performed to ameliorate the patient's hypercortisolism. She subsequently developed multiple vertebral metastases, but was unwilling to undergo chemotherapy. Her condition deteriorated progressively, and she died of heart and respiratory failure 3 years and 6 months after the first admission. Immunostaining for ACTH, chromogranin A, synaptophysin, and neuron-specific enolase was positive in the carcinoid cells. Since somatostatin (SS) and SS analogues inhibit the growth of carcinoid via the SS receptor (SSTR) 2, we evaluated the expression of SSTR2 in the carcinoid cells using reverse transcription-polymerase chain reaction, and this confirmed the expression of SSTR2 in the carcinoid cells. Our experience of this patient with CS due to an ectopic ACTH-producing atypical thymic carcinoid suggests that SS analogues may be useful for treatment of carcinoid showing expression of SSTR2.

Patient with eight metachronous gastrointestinal cancers thought to be hereditary nonpolyposis colorectal cancer (HNPCC).

An 81-year-old woman presented with a chief complaint of swelling of both lower legs. She had a history of surgery for cancers of the stomach, rectum and colon. Among her immediate family members, her son had colon and rectal cancers, and her sister had ovarian cancer. After close examination the patient was diagnosed with small intestine cancer and ascending colon cancer. Gene mutation analyses did not reveal any mutations in DNA mismatch repair genes, but MSH-2 protein expression was lost only in the cancer lesions. Here, we report this rare case of eight metachronous gastrointestinal cancers thought to be HNPCC.