Investigation of non-linear Mate1-mediated efflux of trimethoprim in the mouse kidney as the mechanism underlying drug-drug interactions between trimethoprim and organic cations in the kidney

The purpose of this study was to elucidate the involvement of Mate1 in the tubular secretion of trimethoprim and saturation of Mate1-mediated efflux to address the mechanisms underlying the pharmacokinetic drug interactions with trimethoprim. Trimethoprim is a more potent inhibitor of MATE2-K than MATE1 with K_i values (μM) of 0.030–0.28 and 2.4–5.9, respectively. Trimethoprim is a substrate of human MATE1 and MATE2-K with K_m values of 2.3 ± 0.9 and 0.018 ± 0.004 μM, and mouse Mate1, but not human OCT2, mouse Oct1 and Oct2. Pyrimethamine significantly reduced the renal clearance (CL_R) of trimethoprim (mL/min/kg) from 40.0 ± 5.1 to 20.1 ± 3.7 (p < 0.05). Trimethoprim was given to mice at three infusion rates (150, 500, and 1500 nmol/min/kg). Together with an increase in the plasma concentrations of trimethoprim, the CL_R (mL/min/kg) of trimethoprim decreased to 25.9 ± 3.2, 13.5 ± 5.7, and 8.92 ± 1.50 at the respective rates. Trimethoprim decreased the CL_R of rhodamine 123 in an infusion rate-dependent manner: 11.5 ± 1.3 (control), 5.17 ± 1.55, 1.31 ± 0.50, and 0.532 ± 0.180. These results suggest that Mate1 mediates the tubular secretion of trimethoprim, and at therapeutic doses, MATEs-mediated efflux can be saturated, and thereby, cause drug interactions with other MATE substrates.     

Familial Juvenile Nephronophthisis in Two Siblings — Histological Findings at an Early Stage —

We present two female siblings with familial juvenile nephronophthisis (FJN) which was diagnosed at the early stage of renal failure. Diagnosis was made during the investigation of anemia in case 1 and by a subsequent family survey in case 2. Most patients with FJN are not identified until the terminal stage of renal failure and such cases have rarely been reported in Japan. Case 2 had a reduction in the maximum urinary concentration ability but no azotemia, and among the FJN patients reported in Japan so far she has the least advanced renal disease. Histological examination of the renal biopsy in case 1 showed typical findings of FJN, such as thickening and lamination of the tubular basement membrane (TBM), interstitial fibrosis, and round cell infiltration of the interstitium. In case 2, renal biopsy revealed an irregular marked thickening of the TBM with trivial interstitial changes and a normal glomerular appearance. The histology of these two cases suggests that the TBM may be the primary site affected in FJN.     

Proteinuria and other renal functions in Wilson’s disease

Renal lesions have repeatedly been described in Wilson’s disease (WD). We investigated the excretion of total protein, albumin, low (LMW) and high molecular weight (HMW) proteins, N-acetyl-β-D-glucosaminidase (NAG), and calcium, as well as creatinine clearance, in 24-h urine samples of 41 patients with WD aged 6 – 37 (mean 17) years who had been treated for a period of 0 – 15 (mean 4.5) years with D-penicillamine (900 mg/day). The amount of all protein excreted was significantly increased compared with controls, 39% of patients presenting with total proteinuria more than two standard deviations from the mean of controls. The changes in protein excretion depended on the duration of treatment. LMW proteinuria was elevated almost exclusively in the first 2 years after the start of treatment, indicating early tubular damage. This is supported by an initially high excretion of β₂-microglobulin, NAG, and calcium. Increased excretion of HMW proteins, including albumin, persisted over longer periods, which suggests glomerular injury in some patients, possibly related to the use of D-penicillamine. Creatinine clearance remained roughly within normal limits. We propose that renal function should regularly be checked in patients with WD. Received October 26, 1995; received in revised form August 27, 1996; accepted September 20, 1996     

当归或三七对兔甘油致急性肾衰的保护作用及其机制研究

目的：用甘油复制兔急性肾小管坏死(ATN)模型，观察当归、三七对ATN的保护作用，并初步探讨其机制。方法：40只健康新西兰白色家兔，等分为四组：当归组、三七组、对照组、正常组。当归、三七、对照三组均用50％甘油等渗盐水15ml/kg注入兔双后肤皮下，复制ATN模型，当归组于注射甘油后即刻，第3小时，第6小时，肌注当归注射液50mg／kg，三七组于相同时点肌注三七皂甙50mg／kg，正常组则于兔双后肢皮下注射生理盐水15ml/kg，并于相同时点注射5％葡萄糖注射液。于实验第24小时取血液、尿液和肾脏，检测血清尿素氮、肌酐、尿液总渗透压、尿总氨基酸、肾组织MDA、SOD、GSH—PX、ATP酶、Ca2^ 、肾组织病理形态学检查。结果：各组与正常组比，肌酐、尿素氮明显升高，说明模型复制成功。当归、三七纪元l例死亡，对照组死士率为30％，血清肌酐、尿素氮、丙二醛、尿总氨基酸、滤过钠排泄分数和肾组织钙含量均明显低于对照组(P＜0．01)，而肾组织ATP酶、SOD、GSH-PX活性均高于对照组(P＜0.01)，病理形态学变化轻于对照组。以上指标，当归与三七组无显著差异(P＞0．05)。结论：当归、三七对甘油所致的ATN有明显保护作用，二者效果无差异，其机制可能与二者均有改善微循环、血液流变学、抑制脂质过氧化反应、保护肾脏抗氧化酶和ATP酶活性及减轻肾组织钙超载有关。     

Relative weight of glucose, insulin and parathyroid hormone in the urinary loss of phosphate by chronically diabetic rats

This report deals with the relationships between glucose (G) and insulin on the tubular transport of phosphate (P) in chronically diabetic rats with high plasma levels of parathyroid hormone (PTH). Alloxan-induced diabetes leads to phosphorus depletion of the soft tissues. This phenomenon appears associated with weight loss and negative P balances caused by the increased urinary P excretion. Administration of 2 IU of insulin/100 g body weight (bw) to diabetic rats normalized their P balance and body weight. The effect of parathyroid function on the P metabolism of diabetic rats was investigated with balance experiments. Diabetic rats, intact or thyroparathyroidectomized (TPTX), have a greater urinary excretion of P than their controls. However, in control rats, the ratio intact:TPTX for urinary P is 1.0:0.76, showing the antiphosphaturic effect of parathyroid ablation. For diabetic animals, on the other hand, the ratio is 1.0:1.44. The simultaneous deficit of insulin and PTH thus quadruples the urinary P loss, instead of compensating for each other. The contribution of insulin deficit and hyperglycemia to the defect in tubular reabsorption (TRP) was investigated with clearance experiments (done on anesthetized, perfused rats). Five experimental groups were used: Controls (C), diabetics (D), controls+glucose (C+G), diabetics+insulin (D+I) and diabetics+insulin+glucose (D+I+G). All experimental groups showed a linear relationship between the TRP of P and G. The regression equation for C is significantly different (F=40.1, P<0.001) from that of D animals. The slope value measure the number of μmoles of P per μmol of G reabsorbed. For C and D rats, the ratio P:G approximates 1:4 and 1:20, respectively. The increase in P:G ratios represents the competition between both substrates for tubular resorption. Glycemias up to 11 mM (C and D+I) exist concurrent with the P:G ratio 1:4. Glycemias above 25 mM (D, C+G and D+I+G) produce a P:G ratio of 1:20. Fractional excretion of P (FEP) increased significantly in untreated, chronically diabetic rats (0.47± 0.12 vs controls=0.05±0.01, P<0.001). After a single intramuscular injection of insulin, the FEP decreased as a function of insulin levels. To normalize the FEP of diabetic rats in short-term experiments, insulin had to be administered in doses that produce plasma insulin levels 25 times greater than normal. The general information afforded by the present experiments shows that in untreated, chronically diabetic rats, insulin deficit plays an indirect role. The absence of PTH enhances the effect of hyperglycemia. The latter and the concurrent tubular overload of glucose are the cause of hyperphosphaturia in these animals. Received: 10 September 1996 / Accepted in revised form: 18 April 1997     

Angiogenesis of glioma: evaluation of ultrastructural characteristics of microvessels and tubular bodies (Weibel–Palade) in endothelial cells and immunohistochemical findings with VEGF and p53 protein

Capillary endothelial proliferation is often a prominent feature of malignant gliomas. The understanding of structural and functional characteristics of the vascular microenvironment in gliomas is essential for the design of future therapeutic strategies against this tumor. Electron microscopic analysis of the capillary endothelial proliferation in malignant gliomas indicated that the complex vascular structures within the tumor were composed essentially of immature capillaries. Immature capillaries had a narrow slitlike lumen composed of endothelial cells with their high nuclear:cytoplasmic ratio and the relative paucity of organelles. They resembled capillary buds seen in normal repair tissue. Immature microvessels caused by angiogenesis were found more frequently in marginal zone of the tumors with increased microvessels. The tubular body was an organelle observed in vascular endothelial cells and was used frequently as a marker of the endothelial cell. Tubular bodies were evaluated by quantitative measurement of the mean percent (%) ratio of the number of endothelial cells with tubular bodies to all endothelial cells in microvessels of tumors. In glioblastomas it yielded a value of 32.4% in the margin, about two times as high as that in the center of the tumors. However, it was lower in all locations of astrocytomas. Tubular bodies in endothelial cells could be increased in proportion to neovascularization, and they might serve as a marker for increasing microvessels in astrocytic tumors. Tumor angiogenesis may be regulated by growth factors with angiogenic activities that are secreted by tumor cells. Vascular endothelial growth factor (VEGF) may play a critical role in the regulation of vascular endothelial proliferation. We found that 86% of 29 glioblastomas and 79% of 14 anaplastic astrocytomas demonstrated immunoreactivity for VEGF in their tumor cells. There tended to be a correlation between VEGF and vascularity. A correlation existed between the grade of immunoreactivity for VEGF and the grade of p53 protein expression in the malignant gliomas. However, the MIB-1 indices did not increase in correlation with increase in the extent of immunoreactivity for VEGF.     

The early phase of experimental acute renal failure

Summary Experiments were designed to determine whether leakage of substances across the tubular epithelium, which are impermeant in the normal kidney, falsifies the measurement of glomerular filtration rate in acute renal failure. Permeability to those substances most commonly used for filtration rate determination, polyfructosan, inulin and ferrocyanide, was estimated by measuring their recoveries following perfusion through various nephron segments in haeme pigment, ischaemic and nephrotoxic models of actue renal failure. Late proximal recovery of¹⁴C ferrocyanide was only marginally decreased compared to controls, by a maximum of 6%. Distal recovery of polyfructosan,¹⁴C and³H inulin were depressed somewhat more, by a maximum of 11%. Urinary recovery of¹⁴C inulin was reduced by only 15% in kidneys showing severely restricted renal function. It is concluded that tubular leakage is not a feature of significance in the early phase of moderate acute renal failure, that ferrocyanide and inulin are reliable markers for the determination of nephron filtration rate and water reabsorption, and that the reduction in whole kidney inulin or polyfructosan clearance reflects primarily a reduction in glomerular filtration rate.     

Effect of hypothermia on renal sodium reabsorption

Summary The relationship between sodium reabsorption and oxygen consumption was studied in an isolated rabbit kidney preparation perfused with blood at 37, 28 and 19° C. When the temperature was lowered from 37° C to 28° C and to 19°C the rate of oxygen consumption and of the maximal P.A.H. excretion (Tm P.A.H.) decreased more than that of sodium reabsorption.TheQ ₁₀ for sodium reabsorption is about 1.8, while that for maximal P.A.H. excretion is 2.5. Some hypothesis on the possible mechanisms of the lowQ ₁₀ of the Na⁺ reabsorption are forwarded.Preliminary reports have been published [Boll. Soc. Ital. Biol. Sper.43, 1019–1023 (1966) and44, 1784–1787 (1967);45, 860–862 (1969) and45, 863–865 (1969)].     

Selective planting of cationized,haptenized ovalbumin on the rat tubular basement membrane

We developed an experimental protocol for planting exogenous antigens with different molecular weights and charges on the constituents of the renal tubulointerstitium. The cationized antigens were injected selectively into the left renal arteries of Wistar rats. Antigen localization was documented by immunohistochemistry on frozen sections. Cationized bovine serum albumin (BSA; 68 kDa, isoelectric point =9.5) localized almost exclusively along the glomerular capillary wall. After application of highly cationic polyethyleneimine, cationized BSA given subsequently was found in a linear distribution along the glomerular capillary wall and along the peritubular capillaries. The fate of highly cationized ovalbumin conjugated with trinitrophenol (TNP-OA), subjected to gel filtration to obtain monomers (42 kDa, isoelectric point >10) differed; it was deposited in a linear pattern on the tubular basement membrane (TBM) and Bowman's capsule, and remained up to 36 h after injection. Noncationized, monomeric TNP-OA (42 kDa, isolectnic point =4.6) showed fine granular deposition in the tubular epithelium exclusively. These findings indicate that the barrier of the glomerular BM acts selectively on antigens with different molecular weights. They either settle on the peritubular capillaries, after passing the glomerular, or reach the urinary space, after which they are reabsorbed by the tubular epithelial cells to reach the TBM.     

Phosphate transport in brush border membrane vesicles isolated from renal cortex of young growing and adult rats

Recent clearance studies have demonstrated that the maximal tubular reabsorption of inorganic phosphate (Pi) per ml of glomerular filtrate (max. TRPi/ml GF) of the whole kidney is markedly lower in adult than in young growing rats fed either normal (0.8 g%) or low (0.2 g%) phosphorus diet. In addition, in adult rats clearance studies indicate that enhancement of max. TRPi/ml GF is observed 21 days but not 8 days after starting the low (0.2%) phosphorus diet. In the present work we have studied in the same experimental condition the Na⁺-dependent Pi uptake in brush border membrane vesicles (BBMV) isolated from renal cortex of either young growing or adult rats. The results of this study indicate that under the low (0.2%) but not under the normal (0.8%) phosphorus diet the Na⁺-dependent Pi uptake by BBMV was significantly depressed in adult as compared to young growing rats. In adult rats the Pi transport response to Pi restriction monitored at the brush border membrane level was different from that observed by clearance studies in the whole kidney. Indeed, the Pi uptake by BBMV was already enhanced after 8 days of Pi restriction and it did not increase further when studied 21 days after starting the low (0.2%) phosphorus diet. These results suggest that the regulation of the overall transfer of Pi across the renal epithelium may involve other additional modulating factors than the Na⁺-dependent Pi transport system present in the luminal membrane of the proximal tubule.