No evidence of occult hepatitis C or E virus infections in liver‐transplant patients with sustained virological response after therapy with direct acting agents

International audienceBackground and aims: It has been recently suggested that occult hepatitis C virus (HCV) infection and hepatitis E virus (HEV) reactivation might occur after direct acting antiviral agent-induced (DAA-induced) sustained virological response (SVR). The aim of our study was to identify occult HCV and HEV infection in a cohort of organ transplant patients who had achieved SVR and had persistent elevation in liver-enzyme levels.Patients and method: Sixty-six liver and/or kidney transplant patients were treated with DAAs. All but one achieved SVR12. Twenty-nine (8-39) months post-SVR12, 8 of the 65 patients (12.3%) who achieved SVR12 had persistently elevated liver enzyme levels. In 1 patient, this was related to hepatitis B virus reactivation. In the 7 remaining patients, blood samples (n = 7), liver biopsies (n = 4), and peripheral blood mononuclear cells (PBMCs) (n = 7) were collected simultaneously in order to identify occult HCV or HEV infection.Results: Hepatitis C virus RNA and HEV RNA were not detected in serum, liver tissues, or PBMCs. No HEV reactivation was observed after HCV clearance in patients who had anti-HEV IgG.Conclusion: Our study suggests that there is no occult HCV or HEV infection in transplant patients after successful treatment of HCV infection with DAAs, even in patients with a persistent elevation of liver enzyme levels. However, due to the small number of patients included in our study, this finding should be confirmed in a larger cohort

Immunological and translational key challenges in systemic lupus erythematosus: A symposium update.

The first LBMR-Tim (Toulouse Referral Medical Laboratory of Immunology) symposium convened on December 16, 2022 in Toulouse, France to address challenging questions in systemic lupus erythematosus (SLE). Special focus was put on (i) the role played by genes, sex, TLR7, and platelets on SLE pathophysiology; (ii) autoantibodies, urinary proteins, and thrombocytopenia contribution at the time of diagnosis and during follow-up; (iii) neuropsychiatric involvement, vaccine response in the COVID-19 era, and lupus nephritis management at the clinical frontline; and (iv) therapeutic perspectives in patients with lupus nephritis and the unexpected adventure of the Lupuzor/P140 peptide. The multidisciplinary panel of experts further supports the concept that a global approach including basic sciences, translational research, clinical expertise, and therapeutic development have to be prioritized in order to better understand and then improve the management of this complex syndrome

Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells.

Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2) were subjected to FSS (0.5 Pa) for 48 h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1), Par polarity complex (Pard6), adherens junctions (E-Cadherin, β-Catenin) and the primary cilium (α-acetylated Tubulin) were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and β-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months) mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and β-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium

Kidney inflammaging is promoted by CCR2+ macrophages and tissue-derived micro-environmental factors

International audienceAbstract The incidence of disorders associated with low inflammatory state, such as chronic kidney disease, increases in the elderly. The accumulation of senescent cells during aging and the senescence-associated secretory phenotype, which leads to inflammaging, is known to be deleterious and account for progressive organ dysfunction. To date, the cellular actors implicated in chronic inflammation in the kidney during aging are still not well characterized. Using the DECyt method, based on hierarchical clustering of flow cytometry data, we showed that aging was associated with significant changes in stromal cell diversity in the kidney. In particular, we identified two cell populations up-regulated with aging, the mesenchymal stromal cell subset (kMSC) expressing CD73 and the monocyte-derived Ly6C + CCR2 + macrophage subset expressing pro-inflammatory cytokines. Aged CD73 + kMSCs depicted senescence associated features with low proliferation rate, increased DNA damage foci and Ccl2 expression. Using co-cultures experiments, we showed that aged CD73 + kMSC promoted monocyte activation and secretion of inflammatory cytokines albeit less efficiently than young CD73 + kMSCs. In the context of ageing, increased frequency of CD73 + kMSC subpopulations could provide additional niche factors to newly recruited monocytes favoring a positive regulatory loop in response to local inflammation. Interfering with such partnership during aging could be a valuable approach to regulate kidney inflammaging and to limit the risk of developing chronic kidney disease in the elderly

Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells

<div><p>Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent <i>in vitro</i> studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated <i>in vitro</i> and <i>in vivo</i> the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2) were subjected to FSS (0.5 Pa) for 48h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1), Par polarity complex (Pard6), adherens junctions (E-Cadherin, β-Catenin) and the primary cilium (α-acetylated Tubulin) were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and β-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed <i>in vivo</i> in a uninephrectomy (8 months) mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and β-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both <i>in vitro</i> and <i>in vivo</i>. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium.</p></div

FSS-exposed cells exhibit loss of the primary cilium without marked change in actin cytoskeleton organization.

<p>Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. <b>A/</b> α-acetylated Tubulin was analyzed by immunofluorescence to visualize the primary cilium. White arrows show primary cilia. <b>B/</b> Phalloidin was used to stain the actin cytoskeleton (basal [left] and subapical [right]). Cells were counterstained with DAPI. Pictures display representative areas of staining from 5 independent experiments. Green, α-acetylated Tubulin; red, Phalloidin; blue, DAPI-nuclei. Bars indicate 20 μm.</p

Uninephrectomy as an animal model of increased urinary FSS.

<p>Sham- and UNx-mice were analyzed 8 months after surgery. <b>A</b>/ Renal function was evaluated by measuring glomerular filtration rate (GFR), single kidney GFR (skGFR) and urinary albumin/creatinine ratio (UACR). <b>B</b>/ Renal corpuscule surface was measured on PAS-stained kidney slices. Pictures display representative areas of staining and bars indicate 200 μm. Data represent mean ± SEM from 6 animals per group. *p<0.05, ***p<0.01 <i>versus</i> sham.</p

Alteration of adherens junctions in response to FSS.

<p>Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. <b>A/</b> Immunofluorescence detection of β-Catenin. Cells were counterstained with DAPI. Pictures display representative areas of staining from three independent experiments. Green, β-Catenin; blue, DAPI-nuclei. Bar indicates 20 μm. <b>B/</b> Real-time PCR and Western blot analysis of E-Cadherin mRNA and protein, respectively. <b>C/</b> Real-time PCR was used for evaluation of mRNA levels encoding Snail1 or Snail2. In B and C, results are expressed as the fold induction compared to static condition and data represent mean ± SEM of 4–7 experiments. *p<0.05, **p<0.01 <i>versus</i> FSS 0.</p

FSS does not induce a mesenchymal phenotype in tubular cells.

<p>Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. <b>A</b>/ Transcript level of mesenchymal markers such as Vimentin and αSMA (cytoskeleton), Fibronectin and Collagen I (ECM), N-Cadherin (intercellular junction) was quantified by real-time PCR. Results are expressed as the fold induction compared to static condition. <b>B</b>/ Protein level of Vimentin, αSMA and Fibronectin was measured by Western blot. Results are expressed as the fold induction compared to static condition and data represent mean ± SEM of 4 experiments. *p<0.05, **p<0.01 <i>versus</i> FSS 0.</p

Effect of uninephrectomy-mediated FSS on epithelial gene expression and the density of primary cilia.

<p>Sham- and UNx-mice were analyzed 8 months after surgery. <b>A</b>/ The expression of ZO-1, E-cadherin and β-Catenin mRNA was quantified by real-time PCR from total RNA extracted from kidney cortex. Results are expressed as the fold induction compared to sham. <b>B</b>/ Immunofluorescence detection of α-acetylated Tubulin for quantification of the primary cilium. Kidney sections were counterstained with WGA and DAPI. Pictures in the left panel display representative areas of staining. Red, α-acetylated Tubulin; green, WGA-cell membranes; blue, DAPI-nuclei. Bar indicates 20 μm and white arrows show primary cilia. Graph in the right panel displays quantification of primary cilia by cortex tubular section. Data represent mean ± SEM from 6 animals per group. *p<0.05 <i>versus</i> sham.</p