Eric De Prez

Increased serum levels of endopeptidase 24.11 (‘enkephalinase”) in patients with end-stage renal failure

Endopeptidase 24.11, a widely distributed membrane-bound peptidase is found in low levels in the serum of normal individuals. Although increased levels of the enzyme have been found in sera of patients with sarcoidosis and adult respiratory distress syndrome, the cellular origin of circulating endopeptidase 24.11 remains unknown. As the brush border of the proximal tubular epithelial cells have the highest endopeptidase specific activity, we investigated the possible contribution of the kidney to the release of endopeptidase 24.11 in the systemic circulation. Therefore, we measured serum levels of the enzyme in patients with end-stage renal failure (ESRF) treated by haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). Increased serum levels of endopeptidase 24.11 were observed both in HD patients (mean +/- SEM: 74.6 +/- 20.9 ng/ml) and in CAPD patients (mean +/- SEM: 45.1 +/- 8.1 ng/ml) as compared to normal individuals (mean +/- SEM: 13.6 +/- 1.4 ng/ml). Endopeptidase levels remain stable during haemodialysis sessions on two different dialysis membranes. Finally, serum levels of the enzyme in anephric patients tend to be lower than in ESRF patients, suggesting that the kidney may contribute to the generation of the circulating form of endopeptidase 24.11.

Bioactive Angiotensin (1–8) Is the Main Component of Angiotensin II Immunoreactivity in Human Follicular Fluid

The ovarian renin-angiotensin system is involved in various aspects of human reproduction. As immunoreactive measurement of angiotensin II (ANG II) in follicular fluid (FF) relates to several angiotensin peptides with different biological activities, HPLC was used to characterize the molecular forms of the ANG II immunoreactivity in human FF. Samples of FF, obtained from gonadotropin-stimulated patients for in vitro fertilization, were collected at the time of oocyte retrieval. The C-terminal 2-8 heptapeptide was never detected. HPLC analysis revealed for the first time that the major component of the ANG II-IR in human FF was the biologically active octapeptide ANG II.

Blocking TGF-β Signaling Pathway Preserves Mitochondrial Proteostasis and Reduces Early Activation of PDGFRβ+ Pericytes in Aristolochic Acid Induced Acute Kidney Injury in Wistar Male Rats.

Background The platelet-derived growth factor receptor β (PDGFRβ)+ perivascular cell activation becomes increasingly recognized as a main source of scar-associated kidney myofibroblasts and recently emerged as a new cellular therapeutic target. Aims In this regard, we first confirmed the presence of PDGFRβ+ perivascular cells in a human case of end-stage aristolochic acid nephropathy (AAN) and thereafter we focused on the early fibrosis events of transforming growth factor β (TGFβ) inhibition in a rat model of AAN. Materials and Methods Neutralizing anti-TGFβ antibody (1D11) and its control isotype (13C4) were administered (5 mg/kg, i.p.) at Days -1, 0, 2 and 4; AA (15 mg/kg, sc) was injected daily. Results At Day 5, 1D11 significantly suppressed p-Smad2/3 signaling pathway improving renal function impairment, reduced the score of acute tubular necrosis, peritubular capillaritis, interstitial inflammation and neoangiogenesis. 1D11 markedly decreased interstitial edema, disruption of tubular basement membrane loss of brush border, cytoplasmic edema and organelle ultrastructure alterations (mitochondrial disruption and endoplasmic reticulum edema) in proximal tubular epithelial cells. Moreover, 1D11 significantly inhibited p-PERK activation and attenuated dysregulation of unfolded protein response (UPR) pathways, endoplasmic reticulum and mitochondrial proteostasis in vivo and in vitro. Conclusions The early inhibition of p-Smad2/3 signaling pathway improved acute renal function impairment, partially prevented epithelial-endothelial axis activation by maintaining PTEC proteostasis and reduced early PDGFRβ+ pericytes-derived myofibroblasts accumulation.

Human bone morphogenetic protein-7 does not counteract aristolochic acid-induced renal toxicity

Aristolochic acids (AA) are nephrotoxic and profibrotic agents, leading to chronic kidney disease. As some controversial studies have reported a nephroprotective effect of exogenous recombinant human bone morphogenetic protein (rhBMP)‐7 in several models of renal fibrosis, we investigated the putative effect of rhBMP‐7 to prevent progressive tubulointerstitial damage after AA intoxication in vitro and in vivo. In vitro, the toxicity of AA on renal tubular cells was demonstrated by an increase in vimentin as well as a decrease in β‐catenin expressions, reflecting a dedifferentiation process. Increased fibronectin and interleukin‐6 levels were measured in the supernatants. Enhanced α‐SMA mRNA levels associated to decreased E‐cadherin mRNA levels were also measured. Incubation with rhBMP‐7 only prevented the increase in vimentin and the decrease in β‐catenin expressions. In vivo, in a rat model of AA nephropathy, severe tubulointerstitial lesions induced by AA after 10 and 35 days (collagen IV deposition and tubular atrophy), were not prevented by the rhBMP‐7 treatment. Similarly, rhBMP‐7 did not ameliorate the significant increase in urinary concentrations of transforming growth factor‐β. In summary, our in vitro data demonstrated a poor beneficial effect of rhBMP‐7 to reverse cell toxicity while, in vivo, there was no beneficial effect of rhBMP‐7. Therefore, further investigations are needed to confirm the exact role of BMP‐7 in progressive chronic kidney disease. Copyright

Early detection of acute cisplatin nephrotoxicity: Interest of urinary monitoring of proximal tubular biomarkers

Abstract Background Renal toxicity induced by cisplatin (CisPt) is a clinical issue in patients with or without chronic kidney disease (CKD). Proximal tubular injury can result in acute kidney injury (AKI), which may compromise the course of chemotherapy and the prognosis. The purpose of this study was to investigate the time course of urinary markers of acute tubulotoxicity and to assess the usefulness of such monitoring in a routine clinical setting. Methods This work is an open prospective pilot study carried out among 23 patients receiving a platinum-based chemotherapy. Individual comorbidities, plasma parameters of kidney function (urea, creatinine) and estimated glomerular filtration rate were registered. Urinary excretion of leucine aminopeptidase, neutrophil gelatinase-associated lipocalin, cystatin C, liver fatty acid-binding protein and interleukin-18 were monitored during successive chemotherapy cycles. Episodes of AKI were identified according to KDIGO (Kidney Disease Improving Global Outcomes) 2012 guidelines. Results A total of 28 patients were recruited; among them 23 agreed to be part of the study, of whom 18 received CisPt and 5 carbo- or oxaliplatin. Of the 18 CisPt patients, 12 had a preexisting CKD. Sixteen AKI episodes were observed in 13 patients receiving CisPt with a pejorative evolution in seven cases (partial recovery of the renal function); a transient but dramatic increase in urinary biomarkers was observed 3 h after chemotherapy initiation, whereas plasma creatinine rise appeared 72 h after the end of CisPt treatment. Identified precipitating factors included: dehydration due to lack of fluid intake or diuretic use, exposure to high CisPt doses, regular use of nonsteroidal anti-inflammatory drugs and/or iodinated contrast agents and sepsis. Conclusion Even if numerous precipitating factors could be avoided, the monitoring of urinary markers seemed helpful for the early detection of subclinical AKI induced during CisPt chemotherapy.

CD4 + and CD8 + T Cells Exert Regulatory Properties During Experimental Acute Aristolochic Acid Nephropathy

Experimental aristolochic acid nephropathy is characterized by transient acute proximal tubule necrosis and inflammatory cell infiltrates followed by interstitial fibrosis and tubular atrophy. The respective role of T-cell subpopulations has never been studied in the acute phase of the mouse model, and was heretofore exclusively investigated by the use of several depletion protocols. As compared to mice injected with aristolochic acids alone, more severe acute kidney injury was observed after CD4+ or CD8+ T-cells depletion. TNF-alpha and MCP-1 mRNA renal expressions were also increased. In contrast, regulatory T-cells depletion did not modify the severity of the aristolochic acids induced acute kidney injury, suggesting an independent mechanism. Aristolochic acids nephropathy was also associated with an increased proportion of myeloid CD11bhighF4/80mid and a decreased proportion of their counterpart CD11blowF4/80high population. After CD4+ T-cell depletion the increase in the CD11bhighF4/80mid population was even higher whereas the decrease in the CD11blowF4/80high population was more marked after CD8+ T cells depletion. Our results suggest that CD4+ and CD8+ T-cells provide protection against AA-induced acute tubular necrosis. Interestingly, T-cell depletion was associated with an imbalance of the CD11bhighF4/80mid and CD11blowF4/80high populations.

CD44-positive cells and hyaluronan are a hallmark of a rat model of aristolochic acid nephropathy

Background: Aristolochic acid (AA) Nephropathy (AAN) is characterized by tubulointerstitial injury leading to fibrosis and tubular atrophy. Defective regeneration of tubular cells was hypothesized as a mechanism leading to Proximal Tubular Epithelial Cell (PTEC) atrophy. Here, we examined the distribution of CD44, an adhesion molecule involved in differentiation, and its main ligand, Hyaluronan (HA), in an experimental AAN model. Methods: Wistar rats were injected daily with AA or vehicle during 35 days. Tubular enzymuria and tissue expression of CD44 and HA were evaluated at days 3, 7, 10, 21 and 35. Co-expressions of CD44 and PCNA (proliferation), vimentin (mesenchymal phenotype), a-SMA (myofibroblasts), and CD133 (progenitor cells) were investigated. Results: AA induced acute tubular damage followed by PTEC atrophy. In controls, CD44 was limited to the basolateral membrane of collecting ducts while HA expression was confined to the medullary interstitium. CD44 was overexpressed as soon as 3 days after AA exposure, spreading to the apical and basolateral membranes of dedifferentiated PTECs and appearing on numerous interstitial cells along with marked HA accumulation around necrotic tubules. Both CD44 and HA expressions increased steadily throughout the 1-month follow-up. A large amount of tubular and interstitial CD44+ cells were proliferative (PCNA+) and mesenchymal-like (vimentin+). Few CD44+ cells expressed a-SMA or CD133. Conclusions: Our results suggest CD44 is a strong marker of dedifferentiation, and thus of regeneration, in a rat model of AAN. Concomitant HA accumulation around necrotic tubules may create a sustained crosstalk between CD44+ cells and their interstitial ligand. PTEC atrophy is accompanied by overexpression, not deficiency, of the CD44-HA axis.

Cleavage of atrial natriuretic peptide by a kidney membrane-bound carboxypeptidase A.

An enzymatic activity that cleaved the C-terminal Tyr of ANP (1-28) was characterized in human kidney microvillar membranes by using 125I-labeled rat ANP as substrate. This activity was inhibited by potato carboxypeptidase inhibitor (PCI) and 1.10 phenanthroline, suggesting that it corresponded to a metallo-carboxypeptidase. Solubilization experiments indicated that the carboxypeptidase activity could be recovered in the supernatant after 1% Triton X-100 extraction. As separation by ion exchange chromatography revealed several peaks of enzyme activity, PCI coupled to Sepharose was used for purification. This step resulted in a single protein band at 30 kDa, as analyzed by SDS-PAGE.