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BACKGROUND AND AIMS Vascular dysfunction including vascular calcification (VC), is a consequence of ageing and chronic kidney disease (CKD) that increase the risk of cardiovascular events. Accumulation of senescent cells in arteries can lead to structural and functional alterations and contributes to increased arterial stiffness, reduced compliance and impaired contractility. We tested the hypothesis that senescent cells in the arterial wall are involved in the pathophysiology of vascular calcification in a rat model of CKD and VC induced by a high phosphate diet and vitamin D supplementation. METHOD Eight-week-old Sprague–Dawley rats underwent subtotal nephrectomy (SNx, n = 18), or no surgery (Control, n = 6). Eight weeks after, control animals (n = 6) and part of the SNx group (n = 6) were maintained on the standard diet while the remaining rats underwent a 4-week VC-diet, where a standard diet was supplemented with high phosphate and 1-hydroxy-vitamin D at a dose of 0.1 µg/day (SNx 0.1 group, n = 6) or 0.4 µg/day (SNx 0.4 group, n = 6). At the end of the protocol, renal function, blood pressure and VC (von Kossa) were studied. mRNA and protein expressions of cellular senescence markers (p16, p21, yH2AX), antioxidants (NRF2, Nqo-1), VC inducers/inhibitors and osteoblastic transition drivers were assessed by qPCR, immunohistochemistry (IHC), or immunofluorescence (IF). Correlations between mRNA or protein levels and VC were computed for the VC-diets animals (n = 12). RESULTS A decrease in creatinine clearance (P CONCLUSION In our rat model of induced VC associated with CKD, NRF2 and downstream targets expression was increased in thoracic aorta, confirming the link between NRF2 and VC. This might be a response mechanism as observed with other ‘protective’ factors with increased mRNA expression in this model (data not presented here). Furthermore, we observed that cellular senescence was clearly associated with VC. The co-labelling for p16 and yH2AX in some vascular cells in the media layer, exclusively observed in calcified aortas, is another clue that highlights the link between VC and vascular cell senescence.

BACKGROUND AND AIMS Renal fibrosis is a main feature of chronic kidney disease (CKD) that participates in its progression to the end stage.(1) N-acetylglucosaminyltransferase 5 (MGAT5) adds N-acetylglucosamines to newly synthesized glycoproteins and is overexpressed in tumors participating in epithelial–mesenchymal transition (EMT), invasion and cell migration, some of the features observed in fibrosis.(2) PST3.1a is an inhibitor of MGAT5 that has already been shown to be beneficial in the treatment of brain tumors.(3) We presently evaluated the use of PST3.1a on renal fibrosis in CKD. METHOD Six-week-old Sprague-Dawley rats underwent 5/6th subtotal nephrectomy (SNx) (n = 16) or no surgery (Control) (n = 14). PST3.1a (0.2 mg/mL) or placebo was administered in the drinking water from the day of surgery for the entire protocol. Animals were sacrificed 4 weeks after surgery and the kidney was taken to evaluate fibrosis, MGAT5 expression and activity. Fibrosis was determined both by Sirius red staining and by immunohistochemistry (IHC) staining of three types of collagen (I, III and IV). To measure the activity of MGAT5 immunohistochemistry staining of Phaseolus Vulgaris Leucoagglutinine (PHA-L) and galectin 3 were carried out. RESULTS MGAT5 expression (12.2% ± 1.2 control versus 26.7% ± 2.6 SNx) and renal fibrosis (11.5% ± 0.7 control versus 26.9% ± 1.9 SNx) increased significantly in SNx animals in a preliminary study. In the present study, PST3.1atreatment significantly decreased the expression of collagen 1 (col1) (2.1% ± 0.2 SNx versus 1.3% ± 0.2 SNx + PST3.1a; P CONCLUSION Fibrosis was observed in SNx animals 4 weeks after surgery. PST3.1a significantly decreased MGAT5 activity. Treatment with PST3.1a decreased fibrosis and significantly downregulated col1 and col4. These data show that PST3.1a treatment through MGAT5 inhibition may be a new treatment for fibrosis in the context of CKD.

BACKGROUND AND AIMS Chronic Kidney Disease (CKD) results in modifications in the activity and/or structure of many organs. One of them is the intestine. Intestinal barrier dysfunction may result in turn, in gut leakage and aggravation of the course of CKD. The interaction between the colon and kidneys may be relevant for both, the kidneys as well as the colon. This study aimed to evaluate the impact of CKD and vascular calcification on colon tissue and function. METHOD Sprague–Dawley rats underwent 5/6 nephrectomy (SNx, n = 18) or sham surgery (Control, n = 6). A total of 8 weeks after surgery, SNx rats were divided into three groups: normal chow (SNx, n = 6); high phosphate (1.2%) with high vitamin D; 5 days/week at a dose of 0.1 µg/day (SNx-VC1, n = 6) or 0.4 µg/day (SNx-VC2, n = 6) to induce vascular calcification. Four weeks after dietary intervention, rats were sacrificed and colon tissue was sampled for analysis. The inflammation of colon tissue was scored on haematoxylin-eosin staining (score 1–4) and the presence of macrophages was quantified by immunostaining with CD68 antibody. The mucus production was also quantified by Alcian blue staining. RESULTS Haematoxylin-eosin staining showed increased inflammation in SNx-VC2 rats (2.47 ± 0.26) compared with the Control group (1.52 ± 0.14) (P CONCLUSION CKD was associated with a decrease in mucus production and an increase in inflammation in colon tissue. These changes are more marked in the animals with associated vascular calcification (high vitamin D and high phosphate diet).

BACKGROUND AND AIMS Chronic kidney disease (CKD) increases the risk of developing cognitive dysfunction and it has been identified as a modifiable risk factor for Alzheimer's disease (AD) [1]. Regenerating family member 1 alpha (Reg-1α) protein, originally identified as a 16 kDa polypeptide in the pancreas, is increased in cerebrospinal fluid (CSF) and in neurons of AD patients [2, 3]. Reg-1α is also elevated in blood in inflammatory conditions and chronic illnesses such as diabetes and interestingly, in patients with renal function impairment [4, 5]. It is not known whether Reg-1α is synthesized in the kidney or the modifications observed in CKD are merely due to a decline in the glomerular filtration rate. The aim of our study was to localize and quantify the Reg-1α protein in the kidneys of normal rats or CKD rats (5/6 nephrectomized rats) and determine, in a second time, if serum Reg-1α level is a suitable marker for assessment of renal function. As Reg-1a is able to cross the altered blood-brain barrier, it might be a new interesting biomarker at the interface between renal impairment and neurodegenerative diseases. METHOD A total of 16 3-month-old rats were randomly allocated to 5/6 nephrectomy (Nx) or Sham operation. Three months after surgery, serum creatinine and urea, systolic blood pressure (SBP) were measured and renal fibrosis (Sirius Red) was quantified. The expression of Reg-1α was evaluated by double-immunofluorescence using anti-Reg-1α and anti-Aquaporin-1 (Aqp-1) (as a proximal tubule marker) antibodies to determine the localization of Reg-1α in the kidney. RESULTS 5/6Nx rats showed increased serum creatinine levels (97.3 ± 17.6 versus Sham 26.3 ± 0.8 µmol/L; P < 0.05) and serum urea levels (23.9 ± 8.9 versus Sham 4.7 ± 0.1 mmol/L). They also had a higher SBP (158 ± 12 versus Sham 133 ± 5 mmHg) and renal fibrosis increased in the 5/6Nx group (8.1 ± 1.5 versus Sham 2.7 ± 0.2% staining). The double-immunofluorescence revealed an increased expression of Reg-1α of 5/6Nx rats compared with the Sham group. This expression seemed to be specific to the cytoplasm of the proximal tubules, with a ring-shaped distribution (preliminary results, Figure 1). CONCLUSION Our data show, for the first time, a proximal tubule-specific accumulation in Reg-1α in CKD suggesting an influence of impaired renal function on Reg-1α accumulation. Further assessment of Reg-1a in the blood, the blood-brain barrier and neuroinflammation status in this model is needed to better understand the potential effect of kidney accumulation of Reg-1α on brain dysfunction. Should this effect be confirmed, Reg-1α would be a new actor involved in the kidney-brain dialogue.

BACKGROUND AND AIMS Vascular calcification is a clinical sequelae of chronic kidney disease (CKD) that is associated with high cardiovascular-related mortality in end-stage kidney failure (ESKF) patients. Increased senescent cell burden and dysregulation of the NRF2 pathway, the master regulator of antioxidant genes, have been suggested to play an important role in the onset of multiple non-communicable burden-of-lifestyle diseases, including but not limited to diabetes, obesity and CKD. Thus, we investigated if senescence and NRF2 pathways may serve as drivers of uraemia-induced vascular calcification in human arteries. METHOD ESKF patients without epigastric artery calcification and coronary artery calcification (CAC) (n = 8) were matched with patients with severely calcified epigastric arteries and the presence of CAC (n = 8). In all subjects, samples of epigastric arteries were obtained during living donor kidney transplantation surgery. Gene (qPCR) and protein expression (immunohistochemistry) of cellular senescence markers (p16, p21), NRF2 pathway candidates (NRF2, NQO1, CAT, SOD1) and calcification markers (RUNX2, ALPL) were assessed. In addition, four groups of aortic vascular smooth muscle cells (VSMCs) were treated as follows: i) control media; ii) osteogenic media (Pi = 2.5mM); iii) osteogenic media + pooled serum from non-ESKD subjects; or iv) osteogenic media + pooled uraemic serum from ESKF patients that had severely calcified epigastric arteries (n = 8). Calcification in cell culture experiments was assessed using BoneTag Optical Probe and normalized for protein content, while gene expression analysis for a selection of calcification, senescence and NRF2-related markers, and SA-beta-GAL staining, were performed (n = 4 each). RESULTS At the gene expression level, severely calcified epigastric arteries as assessed by von Kossa staining (Figure 1a) had preserved ALPL expression but significantly increased RUNX2 expression (P < 0.01; Figure 1b) when compared with non-calcified vessels. Gene expression of p16, but not p21, was increased in the calcified compared with non-calcified group (P < 0.001, Figure 1c), while both p16 and p21 protein expressions were elevated in the media layer of calcified vessels (Figure 1c). NRF2 and downstream target gene expressions remained similar between the two groups, however, NRF2 protein expression was increased in the non-calcified group. In cell culture studies, uraemic serum-induced VSMC calcification was increased when compared to non-uraemic serum and osteogenic controls (P < 0.0001, Figure 2a). This was further accompanied by increased ALPL gene expression (P < 0.0001, Figure 2b) and reduced NRF2, SOD1 and NQO1 gene expression (all P < 0.01, Figure 2c). SA-beta-gal staining and p16/p21 gene expression analysis revealed that uraemic serum-induced calcification was not associated with increased senescent cell burden. CONCLUSION Established calcification in ESKF patients is associated with increased senescent cell burden, but preserved NRF2 pathway markers, in epigastric arteries. In contrast, induced calcification was accompanied by the diminished activity of the NRF2 pathway, but not senescence pattern, using an in vitro approach in VSMCs. Our translational data indicate that established and induced calcification may reflect different pathophysiological processes, supporting data from a parallel study using 5/6 rats (Laget, Hobson et al., unpublished data). Future studies should consider the timing of initiation of NRF2 agonist/senolytic treatment when targeting calcification in ESKF patients.

BACKGROUND AND AIMS Obesity is an important risk factor for chronic kidney disease (CKD). It carries hemodynamic and morphologic changes in the kidney resulting in renal function reduction. CKD is associated with glomerulosclerosis and tubulointerstitial fibrosis that involve collagen deposition. The aim of the study was to evaluate the impact of obesity on kidney fibrosis in a model of 5/6th nephrectomized rats fed a cafeteria diet. METHOD Twenty four rats were fed acafeteria diet and 10 rats received a standard diet for 6 months. For each diet group, half of the rats underwent 5/6 nephrectomy (SNx) at 4 months. Expression of collagen type 1, 3, 4 was quantified by immunohistochemistry in kidney sections at 6 months. Adipocyte size was measured in epididymal adipose tissue (eWAT), and the presence of macrophages was determined by CD68 immunostaining. RESULTS Rats fed the cafeteria diet were hyperglycemic and hypertensive. Their body weight was higher than those fed the standard diet. SNx rats had increased serum creatinine levels, which were even higher in the cafeteria group (71 ± 10 µM) compared with standard diet group (54 ± 5 µM) (P CONCLUSION Cafeteria diet-induced obesity worsened the 5/6 nephrectomy-induced CKD. The expression of collagens 3 and 4 as well as the number of macrophages were increased in kidneys from the cafeteria SNx group suggesting an exacerbation by pre-existing obesity of CKD-induced renal inflammation and fibrosis.

Background and Aims Vascular calcification (VC) is a consequence of ageing that confers development of future cardiovascular events. Accumulation of senescent cells can lead to structural and functional abnormalities in vessel wall towards increased stiffness, reduced compliance and impaired contractile and dilatory capacity. Thus, accumulation of senescent cells in the arterial wall could also contribute to the pathophysiology of VC. To test this hypothesis, the presence of cellular markers of senescence, p16, p21 and NRF2, was assessed in aorta from rat model of VC associated with chronic kidney disease (CKD). Method Six-week-old Sprague-Dawley rats underwent 5/6th subtotal nephrectomy (SNx, n=6) or no surgery (Control, n=1). After 8 weeks of renal failure, the regular chow was supplemented with high phosphate (1.2%) and vitamin D (1 µg/day, 5 days per week) for 1 or 4 weeks to initiate vascular calcification (SNx-VC group). At the end of the protocol (Figure 1), blood pressure was measured, and thoracic aorta was taken for determination of calcification by Von Kossa staining, and protein and gene expressions of p16, p21, and NRF2 by immunostaining and qPCR, respectively. Results After 4 weeks of dietary intervention, SNx rats showed an increase in pulse pressure (88±15mmHg vs 35 mmHg for the control). Marked VC was also observed in these animals, 17% of calcified area vs Conclusion This pilot study suggests that uraemia-induced cellular senescence accompanies VCs, as suggested by the modified expression of p16 or NRF2 genes. Our observations deserve exploration in larger studies using additional senescence markers for validation. If so, cellular senescence kinetics will be evaluated in order to test whether senolytics compounds could be a therapeutic option to arrest VC in CKD.