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BACKGROUND AND AIMS The complex system of human adult renal stem/progenitor cells (ARPCs) and their crucial role in the renewal of epithelial renal cells and regenerative processes in the adult kidneys have been recently discovered. In the human kidney, CD133+/CD24+ ARPCs are present within different segments of the nephron as a population with epithelial expansion, self-renewal and differentiation capabilities. Understanding the factors that regulate the ARPCs behaviour is fundamental to completely take advantage of their potential and to develop treatments for renal injury. Recent studies showed that long no-coding RNA (lncRNAs) are essential to establish developmental patterning and maintain the stem cell pluripotency network, further underscoring their important role in stem cell biology/technology and cellular reprogramming. lncRNAs can be dysregulated in different types of diseases and can also modulate the cellular senescence processes. For the first time, we studied their function in ARPCs showing that they express high levels of a particular lncRNA, HOTAIR, influencing cell senescence. METHOD Whole-genome lncRNA expression was performed by Agilent microarray. lncRNA expression was validated by real-time PCR. CRISPR/Cas9 system has been used to knock-down HOTAIR lncRNA. SA-β-Gal experiments were used to evaluate cellular senescence in normal ARPCs and ARPCs knock-out for HOTAIR. By ELISA, it was evaluated the expression of secreted anti-ageing protein Klotho. Fluorescence-activated cell sorting (FACS) was applied to measure CD133 and protein p15 expression in normal and transfected cells. Chromatin immunoprecipitation assay (ChIP) was used to evaluate H3K27me3 in the promoter of p15. RESULTS We studied the lncRNA profile of renal proximal tubular cells (RPTEC) and tubular ARPCs. We found 611 lncRNAs specifically expressed in ARPCs compared with RPTECs (Fold Change > 2; FDR We discovered that ARPCs produce α-Klotho at much higher levels compared to RPTEC, at a mean concentration of 200 pg/mL versus 33 pg/mL (Fold increase = 6.6; P CONCLUSION These data demonstrated that HOTAIR regulates the self-renewal capacity of ARPCs and prevents them from becoming senescent in the short term. Moreover, HOTAIR influences ARPC ability to secrete high levels of α-Klotho, and those renal progenitors, through α-Klotho secretion, are able to influence its levels in surrounding tissues and to modulate, therefore, kidney ageing.

BACKGROUND AND AIMS Replication of the enveloped SARS-COV2 virus can alter lipidomic composition and metabolism of infected cells [1]. These alterations commonly result in a decline in HDL, total cholesterol and LDL, and an increase in triglyceride levels in COVID-19 patients. Furthermore, the ‘cytokine storm’ subsequent to release of inflammatory cytokines can severely impair lipid homeostasis. Importantly, decreased HDL-cholesterol correlates with severity of COVID-19 infection and represents a significant prognostic factor in predicting poor clinical outcomes [2]. Similarly, it has been observed that COVID-19 patients’ recovery is accompanied by a rise in serum HDL levels. Pharmacological intervention that aims to restore ApoA-1 or functional HDL particles may have beneficial roles for clinical outcome of COVID-19 patients and has recently been approved for compassionate use [3]. SARS-CoV 2 spike proteins S1 and S2 can bind free cholesterol and HDL-bound cholesterol, facilitating virus entry by binding the ACE2 co-receptor Scavenger Receptor-BI (SR-BI) [4]. When activated at the trans-membrane level, SR-BI signalling culminates in Ser1173-eNOS phosphorylation with both anti-inflammatory and anti-apoptotic effect. We hypothesized that SARS-COV2 binding promoted SR-BI internalization, so that it could not exert its essential protective function. Therefore, the aim of this study is to evaluate the effects of CER-001, a mimetic HDL, in antagonizing this process. METHOD Endothelial and tubular (RPTEC) cells were exposed to S1, S2 and S1 + S2 (50–250 nM) with or without CER-001 (CER-001 50–500 ug/mL) and cholesterol (10–50 uM). Apoptosis tests (MTT and AnnV/PI) were performed. Internalization of SR-BI, ACE2 with S1 and activation of eNOS was evaluated by FACS analysis. SR-BI and ACE2 expression were evaluated on kidney biopsies from COVID-19 patients. RESULTS At concentrations used, the exposition of S1, S2 and S1 + S2 in the presence of CER-001 and cholesterol did not induce apoptosis of endothelial cells and RPTEC. Endothelial and tubular cells stimulated by S1, in presence of cholesterol, showed an increased intracellular level of SR-BI and ACE-2, with significantly reduced eNOS phosphorylation compared to baseline (P < 0.05). The treatment with CER-001 reversed trans-membrane SR-BI levels and eNOS phosphorylation to baseline values. The detection of S1 spike protein by endothelial cells immunohistochemistry revealed an increased level in S1-exposed cells with cholesterol and reduced S1 intracellular positive staining in CER-001-exposed cells (P < 0.05). Interestingly, S1-exposed cells without cholesterol appeared not to be capable of mediating S1 spike protein internalization. Consistent with in vitro results, analysis of renal biopsies from COVID-19 patients with proteinuria showed increased SR-BI and ACE-2 cytoplasmic signals and reduced expression at the apical domain of injured tubules. CONCLUSION Our data confirmed the key role of lipid profile in SARS-COV2 infection, evaluating the molecular signalling involved in HDL metabolism and inflammatory processes, and could offer new therapeutic strategies for COVID-19 patients.

Background and Aims EVs (Extracellular vesicles) are circulating microparticles able to mediate cell-to cell communication by carrying proteins, DNA, RNA or antibodies on their surface. EV are emerging as pivotal in renal Antibody-mediated rejection (AMR), one of the major causes of transplant failure associated to a massive complement activation. AMR is characterized by endothelial to mesenchymal transition (EndMT) and the occurrence of tubular accelerated senescence process, known as graft “Inflammaging”. However, the potential role of EVs in accelerating the renal aging and graft fibrosis after AMR is not well understood. Method Plasma EVs were isolated from 10 Acute AMR (AAMR), 10 Chronic AMR (CAMR) patients, 5 stable graft transplanted patients and 5 healthy volunteers. EVs were isolated by ultracentrifugation at 100.000 g for 1h at 4°C, quantified by Nanoparticle Tracking Analysis (Nanosight, NTA EV/ml) and characterized by FACS (Attune Nxt). RPTEC and endothelial cell culture were incubated with EVs (5e+4 EVs/cells target for 24h. MTT test was performed to assess cell viability. Cellular senescence was investigated by qPCR for p21, p53, Klotho and CYP1B1 and SA-β-gal staining were performed. To assess EndMT analysis for CD31, VE-cadherin, Vimentin, Collagen I was performed by FACS. mRNA level of C3 and CFH were also measured by qPCR and C4d deposits were evaluated in endothelial cell colture by IF. Renal biopsies were then analyzed for inflammaging (p16INK4a) and EndMT markers (CD31/αSMA) by IHC and IF. Results The Nanosight analysis showed significant differences in the amount of-EVs count per ml of plasma in AMR patients compared to healthy subjects. EVs appeared to be significantly augmented in acute AMR, in a higher manner than chronic AMR (NTA, p=0.0154). By cytofluorimetric analysis, the endothelial markers CD31 and VE- cadherin appeared to be significantly increased compared of healthy control, indicating a predominant endothelial EV origin (p Conclusion These results suggest a putative role for circulating AMR derived-EVs in inducing the tubular inflammaging by local complement activation and early fibrosis by EndMT. The EVs cargo characterization that is ongoing might highlight novel targets for therapeutic intervention.

Background and Aims Sepsis-induced acute kidney injury (AKI) is a growing health care problem, refractory to conventional treatments. This disease is characterized by an overwhelmed immune response against a primary insult that become responsible for renal dysfunction and poor outcome. Therapeutic strategies based on blood purification have been developed for the treatment of this disease. The use of polymethyl methacrylate (PMMA) membrane hemofilter in continuous hemodiafiltration (CHDF) modality showed better hemodynamic stability and efficient renal support in chronic dialysis maintenance. Here we investigated the efficacy of Hemofeel PMMA membrane (TORAY, Japan) in interfering with Complement activation and renal damage in a swine model of sepsis-induced AKI. Method After 3 hours from LPS infusion, 7 hours of PMMA-CVVH treatment or 7 hours of polysulfone (PSF)-CVVH were performed. Animals were sacrificed after 24h from LPS infusion. Histologic and renal function parameters were analyzed in all pigs. Pentraxin-3 (PTX3) and C5b-9 deposits were assessed on renal biopsies. Systemic Complement activation was evaluated by Wieslab kit. Gene expression profile was obtained from isolated PBMCs by Agilent SurePrint G3 Porcine Gene Expression Microarrays. Genespring and R software were used for the analysis. Results were validated by Real-time PCR. Results Analysis of renal biopsies from septic pigs presented increased interstitial leucocyte infiltrate, extensive collagen deposition and diffuse glomerular thrombi compared to healthy pigs (p2 ; false discovery rate Conclusion Our data suggest that LPS induced AKI is characterized by activation of Classical and alternative Complement pathways resulting in significant renal tissue damage. By interfering with complement activation and inflammatory response, PMMA membrane might prevent dysfunctional activation of resident renal cells with prevention of sepsis-induced AKI.

Background and Aims Cisplatin, is a nonspecific cytotoxic agent that primarily interferes with cellular DNA replication and the cell cycle, nevertheless it lacks tumor selectivity and acts also in normal cells. The most serious adverse reaction of cisplatin is Acute Kidney Injury (AKI), limiting its use and efficacy in chemotherapy. Cisplatin nephrotoxicity is observed in more than 30% of older patients, however the mechanism of nephrotoxicity remains unclear and specific preventive measures are not available. Today, there is an urgent need for specific nephroprotective strategies to be used during cisplatin chemotherapy. Recently, we found that tubular stem/progenitor cells (tARPC) are able to protect the tubular epithelial (RPTEC) from cisplatin induced injury, preserving their proliferation and inhibiting apoptosis. The aim of this study was to identify the molecular mechanisms involved in tARPC-mediated resistance to cisplatin. Method Co-cultures of RPTEC cells and tARPCs were exposed to cisplatin (2.5 µM) for 6 h and then kept in culture for 96 h. Gene expression profile was obtained from tARPCs and RPTECs by Agilent SurePrint G3 Human Gene Expression Microarrays. Genespring and R software were used for the analysis. Gene expression data were validated by Real-time PCR. Extracellular vesicles were isolated from cell culture supernatant by miRCURY Exosome Cell/Urine/CSF Kit (Qiagen) and RNA contained in extracellular vesicles was purified, analyzed in quality by Bioanalyzer (RNA nano) and evaluated by qPCR. The BrdU assay and caspase 3 were used to measure proliferation and apoptosis levels. Immunohistochemical expression of activated caspase-3 was used as a marker of apoptosis in RPTECs. Results By a whole-genome gene expression analysis, we found 107 genes specifically modulated by RPTECs in response to cisplatin and, among these, 30 genes induced by ARPCs following the cisplatin damage. In particular, we found a strong upregulation of the CYP1B1 gene (false discovery rate corrected p value These data support the hypothesis that ARPCs are sensor of cisplatin damaged-RPTEC and confers cisplatin resistance by transferring CYP1B1 gene in extracellular vesicles. Conclusion This is the first evidence of a cisplatin-induced overexpression of CYP1b1 in renal epithelial cells as a defense mechanism against cisplatin toxicity. This is consistent with our previous data showing that renal progenitors are resistant to cisplatin. The findings may have biological and clinical significance in terms of their implications in cellular communications and potential use of CYP1B1 as biomarkers for AKI induced by cisplatin or as protective agent.