Patrick C. D’Haese

Inhibitors of HMG-CoA Reductase Reduce Receptor-mediated Endocytosis in Human Kidney Proximal Tubular Cells

The proximal tubular cells of the kidney are responsible for reabsorption of proteins from the tubular lumen. In a study using Opossum kidney (OK) cells, receptor-mediated protein endocytosis was reduced by statins, inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, which are widely used for therapeutic reduction of plasma cholesterol levels. To explore the possible clinical relevance of the observations in OK cells, protein endocytosis in human kidney tubular cells was investigated in the presence and absence of statins. The uptake of FITC-labeled albumin in these cultures of human kidney tubular cells was investigated by microscopy, flow cytometry and spectrofluorometry. Protein uptake occurred selectively into proximal tubular cells while it was absent in distal tubular/collecting duct cells. Three statins (simvastatin, pravastatin, and rosuvastatin) significantly inhibited the uptake of protein in a concentration-dependent way. This inhibitory effect of statins could be prevented by the co-addition of mevalonate, the product of HMG-CoA reductase. This effect was not the result of a statin-induced cytotoxicity since cell-viability was unaffected. Finally, it was demonstrated that statins strongly inhibited cholesterol synthesis in the human kidney tubular cells. These data suggest that statins have the potential to inhibit albumin uptake by the human proximal nephron as a result of inhibition of HMG-CoA reductase in the proximal tubule cells. Taken into account the data of the accompanying manuscript this inhibitory effect most probably results from a reduced prenylation of some proteins critically involved in endocytosis. It is suggested that these data help to explain the occurrence of proteinuria in some patients treated with high statin doses.

Lanthanum: A Safe Phosphate Binder

Accumulation of inorganic phosphate due to renal functional impairment contributes to the increased cardiovascular mortality observed in dialysis patients. Phosphate plays a causative role in the development of vascular calcification in renal failure; treatment with calcium‐based phosphate binders and vitamin D can further increase the Ca × PO4 product and add to the risk of ectopic mineralization. The new generation of calcium‐free phosphate binders, sevelamer and lanthanum, can control hyperphosphatemia without adding to the patients calcium load. In this article, the metabolism of lanthanum carbonate and its effects in bone, liver and brain are discussed. Although lanthanum is a metal cation its effects are not comparable to those of aluminum. Indeed, in clinical studies no toxic effects of lanthanum have been reported after up to four years of follow‐up. The bioavailability of lanthanum is extremely low. The effects observed in bone are due to phosphate depletion, with no signs of direct bone toxicity yet observed in rats or humans. The liver is the main route of excretion for lanthanum carbonate, which can be localized in the lysosomes of hepatocytes. No lanthanum could be detected in brain tissue.

Adequate phosphate binding with lanthanum carbonate attenuates arterial calcification in chronic renal failure rats

BACKGROUND Hyperphosphataemia is a risk factor for arterial calcification contributing to the high cardiovascular mortality in patients with chronic kidney disease. Calcium-based phosphate binders can induce hypercalcaemia and are associated with progression of vascular calcification. Therefore, the effect of lanthanum carbonate, a non-calcium phosphate binder, on the development of vascular calcification was investigated in uraemic rats. METHODS Chronic renal failure (CRF) was induced by feeding rats an adenine-enriched diet for 4 weeks. After 2 weeks, 1% or 2% lanthanum carbonate was added to the diet for 6 weeks. Calcification in the aorta, carotid and femoral arteries was evaluated histomorphometrically, biochemically and by ex vivo micro-CT. Chondro-/osteogenic conversion of vascular smooth muscle cells was also analysed in the rat aorta. RESULTS Treatment with 1% lanthanum carbonate (1% La) did not reduce vascular calcification, but in the 2% lanthanum carbonate (2% La) group vascular calcium content and area% Von Kossa positivity were decreased compared with control CRF rats. The aortic calcified volume measured with ex vivo micro-CT was significantly reduced in rats treated with 2% La. Although calcification was inhibited by treatment with 2% La, the chondrocyte transcription factor sox-9 was abundantly expressed in the aorta. CONCLUSION Treatment of CRF rats with 2% La reduces the development of vascular calcification by adequate phosphate binding resulting in a decreased supply of phosphate as a substrate for vascular calcification.

Al and Si: their speciation, distribution, and toxicity.

OBJECTIVES In dialysis patients both aluminum (AI) and silicon (Si) may accumulate. Whereas the toxic effects of AI within this population are clearly established, little is known on the role of Si in the development/protection of particular dialysis-related diseases. A clear insight in the protein binding and speciation of trace elements is important to better understand the mechanisms underlying their toxicity/essentiality. Research in this field however is complex and often prone to analytical difficulties and inaccuracies. DESIGN AND METHODS In the first part of this review techniques used for speciation studies of AI and Si in biological fluids are discussed. Notwithstanding recent technical advances (a) extraneous metal contamination, (b) unrecognized aspecific binding of metals to proteins, and (c) unwanted interactions with separation equipment such as chromatography columns and ultrafiltration membranes remain important pitfalls and often lead to erroneous conclusions. The factors that determine the speciation of AI and Si and their ultimate tissue distribution and toxicity are dealt with in the second part. Here, experimental data obtained with various speciation techniques are linked to in vivo data on the tissue distribution, localization/toxicity of both elements. CONCLUSIONS A model in which the AI tissue distribution/toxicity is mediated by either its citrate or transferrin bound form is proposed.

Partial Prevention of Long-Term Femoral Bone Loss in Aged Ovariectomized Rats Supplemented with Choline-Stabilized Orthosilicic Acid

Silicon (Si) deficiency in animals results in bone defects. Choline-stabilized orthosilicic acid (ch-OSA) was found to have a high bioavailability compared to other Si supplements. The effect of ch-OSA supplementation was investigated on bone loss in aged ovariectomized (OVX) rats. Female Wistar rats (n = 58, age 9 months) were randomized in three groups. One group was sham-operated (sham, n = 21), and bilateral OVX was performed in the other two groups. OVX rats were supplemented orally with ch-OSA over 30 weeks (OVX1, n = 20; 1 mg Si/kg body weight daily) or used as controls (OVX0, n = 17). The serum Si concentration and the 24-hour urinary Si excretion of supplemented OVX rats was significantly higher compared to sham and OVX controls. Supplementation with ch-OSA significantly but partially reversed the decrease in Ca excretion, which was observed after OVX. The increase in bone turnover in OVX rats tended to be reduced by ch-OSA supplementation. ch-OSA supplementation increased significantly the femoral bone mineral content (BMC) in the distal region and total femoral BMC in OVX rats, whereas lumbar BMC was marginally increased. Femoral BMD was significantly increased at two sites in the distal region in OVX rats supplemented with ch-OSA compared to OVX controls. Total lumbar bone mineral density was marginally increased by ch-OSA supplementation. In conclusion, ch-OSA supplementation partially prevents femoral bone loss in the aged OVX rat model.

Co-exposure to lead increases the renal response to low levels of cadmium in metallurgy workers.

PURPOSE Research on the effect of co-exposure to Cd and Pb on the kidney is scarce. The objective of the present study was to assess the effect of co-exposure to these metals on biomarkers of early renal effect. METHODS Cd in blood (Cd-B), Cd in urine (Cd-U), Pb in blood (Pb-B) and urinary renal biomarkers, i.e., microalbumin (μ-Alb), beta-2-microglobulin (β₂-MG), retinol binding protein (RBP), N-acetyl-β-d-glucosaminidase (NAG), intestinal alkaline phosphatase (IAP) were measured in 122 metallurgic refinery workers examined in a cross-sectional survey. RESULTS AND CONCLUSIONS The median Cd-B, Cd-U, Pb-B were: 0.8 μg/l (IQR = 0.5, 1.2), 0.5 μg/g creatinine (IQR = 0.3, 0.8) and 158.5 μg/l (IQR = 111.0, 219.3), respectively. The impact of Cd-B on the urinary excretion of NAG and IAP was only evident among workers with Pb-B concentrations ≥ 75th percentile. The association between Cd-U and the renal markers NAG and RBP was also evidenced when Pb-B ≥ 75th percentile. No statistically significant interaction terms were observed for the associations between Cd-B or Cd-U and the other renal markers under study (i.e., μ-Alb and β2-MG). Our findings indicate that Pb increases the impact of Cd exposure on early renal biomarkers.

Demonstration of lanthanum in liver cells by energy-dispersive X-ray spectroscopy, electron energy loss spectroscopy and high-resolution transmission electron microscopy

The appearance of lanthanum in liver cells as a result of the injection of lanthanum chloride into rats is investigated by advanced transmission electron microscopy techniques, including electron energy loss spectroscopy and high‐resolution transmission electron microscopy. It is demonstrated that the lysosomes contain large amounts of lanthanum appearing in a granular form with particle dimensions between 5 and 25 nm, whereas no lanthanum could be detected in other surrounding cellular components.

High-Resolution X-Ray Microtomography Is a Sensitive Method to Detect Vascular Calcification in Living Rats With Chronic Renal Failure

Objective—Chronic renal failure (CRF) is associated with a 10- to 20-fold increase in cardiovascular risk. Vascular calcification is a prominent feature of cardiovascular disease in patients with end-stage renal failure and contributes to the excess mortality in this population. In this study, we explored in vivo X-ray microtomography (micro-CT) as a tool to detect and follow-up vascular calcifications in the aorta of living rats with adenine-induced CRF. Methods and Results—With in vivo micro-CT, calcification of the aorta in uremic rats was clearly discernible on transversal virtual cross-sections. Micro-CT findings correlated well with tissue calcium content and histology. Repetitive scans in animals with light, moderate, and severe vascular calcification showed good reproducibility with minimal interference of motion artifacts. Moreover, both calcified volume and area could be quantified with this method. Conclusions—In vivo micro-CT scanning is a sensitive method to detect vascular calcifications in CRF rats, allowing follow-up and quantification of the development, and potential reversal during treatment, of vascular calcifications in living animals.

Selenium, Lead, and Cadmium Levels in Renal Failure Patients in China

Whole blood and serum samples of Chinese stable chronic renal failure (CRF) patients (n = 81), hemodialysis patients (n = 135), posttransplant patients (n = 60), and subjects with normal renal function (NRF; N = 42) were collected, as well as water and dialysate samples from five dialysis centers. The concentration of selenium (Se), lead (Pb), and cadmium (Cd) was measured by atomic absorption spectrometry. The mean serum Se levels in patients with different degrees of renal failure were significantly lower than those of subjects with NRF (p < 0.01). Pb levels were not increased in renal failure patients, while the Cd levels in patients with various degrees of renal failure were higher than in subjects with NRF (p < 0.05). After correcting the results of Pb and Cd for hematocrit (Hct) however, Pb levels of dialysis patients were also increased. In the dialysis population under study, blood Pb and Cd levels were closely related to the time on dialysis, while contamination of the final dialysate may also contribute to the increased blood Cd and to a less extent Pb levels. Correction for Hct may be recommended to accurately compare blood Pb and Cd levels in dialysis patients and CRF patients with varying degrees of anemia to those of subjects with NRF.

From skeletal to cardiovascular disease in 12 steps—the evolution of sclerostin as a major player in CKD-MBD

Canonical Wnt signaling activity contributes to physiological and adaptive bone mineralization and is an essential player in bone remodeling. Sclerostin is a prototypic soluble canonical Wnt signaling pathway inhibitor that is produced in osteocytes and blocks osteoblast differentiation and function. Therefore, sclerostin is a potent inhibitor of bone formation and mineralization. Accordingly, rodent sclerostin-deficiency models exhibit a strong bone phenotype. Moreover, blocking sclerostin represents a promising treatment perspective against osteoporosis. Beyond the bone field novel data definitely associate Wnt signaling in general and sclerostin in particular with ectopic extraosseous mineralization processes, as is evident in cardiovascular calcification or calciphylaxis. Uremia is characterized by parallel occurrence of disordered bone mineralization and accelerated cardiovascular calcification (chronic kidney disease – mineral and bone disorder, CKD-MBD), linking skeletal and cardiovascular disease—the so-called bone-vascular calcification paradox. In consequence, sclerostin may qualify as an emerging player in CKD-MBD. We present a stepwise review approach regarding the rapidly evolving field sclerostin participation in CKD-MBD. Starting from data originating in the classical bone field we look separately at three major areas of CKD-MBD: disturbed mineral metabolism, renal osteodystrophy, and uremic cardiovascular disease. Our review is intended to help the nephrologist revise the potential importance of sclerostin in CKD by focusing on how sclerostin research is gradually evolving from the classical osteoporosis niche into the area of CKD-MBD. In particular, we integrate the limited amount of available data in the context of pediatric nephrology.