Jeanne Pertijs

Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

The bioartificial kidney (BAK) aims at improving dialysis by developing ‘living membranes’ for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as a fluorescent substrate. Initial ASP+ uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a ‘living membrane’ of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering.

Aging attenuates the vasodilator response to relaxin

Relaxin, an insulin-like growth factor peptide, increases endothelium-dependent vasodilation and vascular compliance and decreases myogenic reactivity. These vascular effects significantly contribute to the physiological circulatory adaptations in pregnancy, particularly in the mesentery and kidney. Aging predisposes to vascular maladaptation and gestational hypertensive disease. We hypothesized that mild aging reduces the vascular responses to relaxin. In 20 young (10-12 wk) and 20 middle-aged (40-46 wk) female Wistar Hannover rats, vascular responses to chronic exposure of relaxin vs. placebo (5 days) were quantified in isolated mesenteric arteries and kidney. Vascular responses were evaluated using pressure-perfusion myograph, wire myograph, and an isolated perfused rat kidney model. Rxfp1 (relaxin family peptide) gene expression was determined by quantitative polymerase chain reaction. In young rats, relaxin stimulated nitric oxide (NO)-dependent flow-mediated vasodilation (2.67-fold, from 48 ± 9 to 18 ± 4 μl/min), reduced myogenic reactivity (from -1 ± 2 to 7 ± 3 μm/10 mmHg), and decreased mesenteric sensitivity to (28%, from 1.39 ± 0.08 to 1.78 ± 0.10 μM) but did not change compliance and renal perfusion flow (RPFF). In aged rats, relaxin did not affect any of the analyzed mesenteric or renal parameters. In aged compared with young placebo-treated rats, all mesenteric characteristics were comparable, while RPFF was lower (17%, from 6.9 ± 0.2 to 5.7 ± 0.1 ml·min⁻¹·100 g⁻¹) even though NO availability was comparable. Rxfp1 expression was not different among young and aged rats. Our findings suggest that moderate aging involves normal endothelial function but blunts the physiological endothelium-dependent and -independent vasodilator response to relaxin.

Impaired vascular responses to relaxin in diet-induced overweight female rats

Relaxin mediates renal and mesenteric vascular adaptations to pregnancy by increasing endothelium-dependent vasodilation and compliance and decreasing myogenic reactivity. Diet-induced overweight and obesity are associated with impaired endothelial dysfunction and vascular remodeling leading to a reduction in arterial diameter. In this study, we tested the hypothesis that local vascular responses to relaxin are impaired in diet-induced overweight female rats on a high-fat cafeteria-style diet for 9 wk. Rats were chronically infused with either relaxin or placebo for 5 days, and vascular responses were measured in isolated mesenteric arteries and the perfused kidney. Diet-induced overweight significantly increased sensitivity to phenylephrine (by 17%) and vessel wall thickness, and reduced renal perfusion flow (RPFF; by 16%), but did not affect flow-mediated vasodilation, myogenic reactivity, and vascular compliance. In the normal weight rats, relaxin treatment significantly enhanced flow-mediated vasodilation (2.67-fold), decreased myogenic reactivity, and reduced sensitivity to phenylephrine (by 28%), but had no effect on compliance or RPFF. NO blockade by l-NAME diminished most relaxin-mediated effects. In diet-induced overweight rats, the vasodilator effects of relaxin were markedly reduced for flow-mediated vasodilation, sensitivity to phenylephrine, and myogenic response compared with the normal diet rats, mostly persistent under l-NAME. Our data demonstrate that some of the vasodilator responses to in vivo relaxin administration are impaired in isolated mesenteric arteries and the perfused kidney in diet-induced overweight female rats. This does not result from a decrease in Rxfp1 (relaxin family peptide receptor) expression but is likely to result from downstream disruption to endothelial-dependent mechanisms in diet-induced overweight animals.

Humoral signalling compounds in remote ischaemic preconditioning of the kidney, a role for the opioid receptor

BACKGROUND Renal ischaemia-reperfusion injury (IRI) is a common clinical problem associated with significant mortality and morbidity. One strategy to reduce this damage is remote ischaemic preconditioning (RIPC), in which brief ischaemia of a limb protects the kidney against a prolonged ischaemic insult. The mechanism of renal RIPC has not yet been elucidated. Here, we address the gap in our understanding of renal RIPC signalling, using a rat model of renal IRI and RIPC by brief hind limb ischaemia. METHODS Rats were treated with either no RIPC, RIPC+vehicle or RIPC+ an inhibitor or antagonist of one of the following candidate signalling molecules: noradrenalin, cannabinoids, glucocorticoids, inducible nitric oxide synthase, calcitonin gene-related peptide, ganglion-mediated signalling, haem oxygenase and free radicals. Subsequently, the animals underwent 25 min of renal ischaemia and 2 days of reperfusion, after which renal function and damage were assessed. RESULTS RIPC by three 4 min cycles of hind limb ischaemia effectively reduced renal IRI. Pre-treatment with the opioid receptor antagonist naloxone completely blocked this protective effect, when compared with animals treated with RIPC+vehicle; serum creatinine and urea increased (307.8±43.7 versus 169.5±16.7 µmol/L and 42.2±4.9 versus 27.6±2.2 mmol/L, respectively), as did the renal histological damage (score 4.2±0.7 versus 2.8±0.5) and expression of kidney injury molecule-1 (KIM-1; relative-fold increase in mRNA expression 164±18 versus 304±33). All other antagonists were without effect. CONCLUSIONS Renal RIPC by brief hind limb ischaemia may be the result of endorphin release from the hind limb. The importance of opioid signalling in renal RIPC provides vital clues for its successful translation to the clinical setting.

Impaired effect of relaxin on vasoconstrictor reactivity in spontaneous hypertensive rats

Relaxin is thought to be involved in vasodilation to pregnancy by increasing endothelium-dependent vasodilation and compliance, and decreasing myogenic reactivity. Primary (essential) hypertension predisposes to circulatory maladaptation and subsequent gestational hypertensive disease. This study aimed to determine that vascular responses to chronic exposure to relaxin are impaired in young female rats with primary hypertension. In 10-12 weeks old Wistar-Hannover rats (WHR) and spontaneous hypertensive rats (SHR), we determined vascular responses in isolated kidney and mesenteric arteries after 5-days of chronic exposure to relaxin (4 μg/h) or placebo. SHR show decreased sensitivity to phenylephrine (by 67%, p<0.01) and renal perfusion flow (RPFF, by 19%, p<0.01), but no changes in flow-mediated vasodilation, myogenic reactivity or vascular compliance. In WHR, relaxin stimulated flow-mediated vasodilation (2.67 fold, from 48 ± 9 to 18 ± 4 μl/min, p = 0.001), inhibited myogenic reactivity (from -1 ± 2 to 7 ± 3 μm/10 mmHg, p = 0.01), and decreased sensitivity to phenylephrine (28%, from 1.39 ± 0.08 to 1.78 ± 0.10 μM, p<0.01), but left compliance and RPFF unchanged. NO-blockade by L-NAME diminished most relaxin-mediated responses. In SHR, the vasodilator effects of relaxin were blunted for myogenic reactivity and sensitivity to phenylephrine, with similar effects on flow-mediated vasodilation, compliance, RPFF and equal Rxfp1 (relaxin family peptide receptor) gene expression, as compared to WHR. Primary hypertension blunts both the relaxin-induced inhibition of myogenic reactivity and α-adrenergic vasoconstrictor response, independent from Rxfp1 gene expression, while the relaxin-dependent enhanced flow-mediated vasodilation remains intact. This implies selective resistance to relaxin in young subjects suffering from primary hypertension.

Urinary proteomic profiling reveals diclofenac-induced renal injury and hepatic regeneration in mice

Diclofenac (DF) is a widely used non-steroidal anti-inflammatory drug for the treatment of rheumatic disorders, but is often associated with liver injury. We applied urinary proteomic profiling using MALDI-TOF MS to identify biomarkers for DF-induced hepatotoxicity in mice. Female CH3/HeOUJIco mice were treated with 75mg/kg bw DF by oral gavage and 24h urine was collected. Proteins identified in urine of DF-treated mice included epidermal growth factor, transthyretin, kallikrein, clusterin, fatty acid binding protein 1 and urokinase, which are related to liver regeneration but also to kidney injury. Both organs showed enhanced levels of oxidative stress (TBARS, p<0.01). Kidney injury was confirmed by histology and increased Kim1 and Il-6 mRNA expression levels (p<0.001 and p<0.01). Liver histology and plasma ALT levels in DF-treated mice were not different from control, but mRNA expression of Stat3 (p<0.001) and protein expression of PCNA (p<0.05) were increased, indicating liver regeneration. In conclusion, urinary proteome analysis revealed that DF treatment in mice induced kidney and liver injury. Within 24h, however, the liver was able to recover by activating tissue regeneration processes. Hence, the proteins found in urine of DF-treated mice represent kidney damage rather than hepatic injury.

Contribution of different local vascular responses to mid-gestational vasodilation

OBJECTIVE At-term pregnancy-induced vasodilation is the resultant of endothelium-dependent vasodilation, decreased myogenic reactivity, increased compliance, and reduced sensitivity to vasoconstrictor agents. We hypothesized that these vascular changes are already present at mid-gestation. STUDY DESIGN In 20 mid-pregnant and 20 nonpregnant Wistar Hannover rats, we measured vascular responses of isolated mesenteric arteries and kidney. RESULTS In the pregnant rats compared with the nonpregnant rats, mesenteric flow-mediated vasodilation and renal perfusion flow increased 1.52-fold (from 47±5 to 31±4 μL/min) and 1.13-fold (from 12.8±0.1 to 14.4±0.1 mL/min), respectively. Nitric oxide inhibition reduced mesenteric flow-mediated vasodilation to a similar extent in the pregnant and nonpregnant rats; it completely blocked the pregnancy-induced increase in renal perfusion flow. Pregnancy did not change mesenteric artery sensitivity to phenylephrine, myogenic reactivity, nor vascular compliance. CONCLUSION At mid-gestation, alterations in rat mesenteric vascular tone depend primarily on flow-mediated endothelium-dependent changes and not on changes in α-adrenergic vasoconstrictor sensitivity, myogenic reactivity, or vascular compliance.

Switch in FGFR3 and -4 expression profile during human renal development may account for transient hypercalcemia in patients with Sotos syndrome due to 5q35 microdeletions

CONTEXT Sotos syndrome is a rare genetic disorder with a distinct phenotypic spectrum including overgrowth and learning difficulties. Here we describe a new case of Sotos syndrome with a 5q35 microdeletion, affecting the fibroblast growth factor receptor 4 (FGFR4) gene, presenting with infantile hypercalcemia. OBJECTIVE We strove to elucidate the evanescent nature of the observed hypercalcemia by studying the ontogenesis of FGFR3 and FGFR4, which are both associated with fibroblast growth factor (FGF) 23-mediated mineral homeostasis, in the developing human kidney. DESIGN Quantitative RT-PCR and immunohistochemical analyses were used on archival human kidney samples to investigate the expression of the FGFR signaling pathway during renal development. RESULTS We demonstrated that renal gene and protein expression of both FGFRs increased during fetal development between the gestational ages (GAs) of 14-40 weeks. Yet FGFR4 expression increased more rapidly as compared with FGFR3 (slope 0.047 vs 0.0075, P = .0018). Moreover, gene and protein expression of the essential FGFR coreceptor, Klotho, also increased with a significant positive correlation between FGFR and Klotho mRNA expression during renal development. Interestingly, we found that perinatal FGFR4 expression (GA 38-40 wk) was 7-fold higher as compared with FGFR3 (P = .0035), whereas in adult kidney tissues, FGFR4 gene expression level was more than 2-fold lower compared with FGFR3 (P = .0029), thus identifying a molecular developmental switch of FGFR isoforms. CONCLUSION We propose that the heterozygous FGFR4 deletion, as observed in the Sotos syndrome patient, leads to a compromised FGF23 signaling during infancy accounting for transient hypercalcemia. These findings represent a novel and intriguing view on FGF23 mediated calcium homeostasis.