key: cord-320455-doup2bqq authors: Werion, Alexis; Belkhir, Leila; Perrot, Marie; Schmit, Gregory; Aydin, Selda; Chen, Zhiyong; Penaloza, Andrea; De Greef, Julien; Yildiz, Halil; Pothen, Lucie; Yombi, Jean Cyr; Dewulf, Joseph; Scohy, Anais; Gérard, Ludovic; Wittebole, Xavier; Laterre, Pierre-François; Miller, Sara E.; Devuyst, Olivier; Jadoul, Michel; Morelle, Johann title: SARS-CoV-2 Causes a Specific Dysfunction of the Kidney Proximal Tubule date: 2020-08-10 journal: Kidney Int DOI: 10.1016/j.kint.2020.07.019 sha: doc_id: 320455 cord_uid: doup2bqq Coronavirus disease 2019 (COVID-19) is commonly associated with kidney damage, and the angiotensin converting enzyme 2 (ACE2) receptor for SARS-CoV-2 is highly expressed in the proximal tubule cells. Whether patients with COVID-19 present specific manifestations of proximal tubule dysfunction remains unknown. To test this, we examined a cohort of 49 patients requiring hospitalization in a large academic hospital in Brussels, Belgium. There was evidence of proximal tubule dysfunction in a subset of patients with COVID-19, as attested by low-molecular-weight proteinuria (70-80%), neutral aminoaciduria (46%), and defective handling of uric acid (46%) or phosphate (19%). None of the patients had normoglycemic glucosuria. Proximal tubule dysfunction was independent of pre-existing comorbidities, glomerular proteinuria, nephrotoxic medications or viral load. At the structural level, kidneys from patients with COVID-19 showed prominent tubular injury, including in the initial part of the proximal tubule, with brush border loss, acute tubular necrosis, intraluminal debris, and a marked decrease in the expression of megalin in the brush border. Transmission electron microscopy identified particles resembling coronaviruses in vacuoles or cisternae of the endoplasmic reticulum in proximal tubule cells. Among features of proximal tubule dysfunction, hypouricemia with inappropriate uricosuria was independently associated with disease severity and with a significant increase in the risk of respiratory failure requiring invasive mechanical ventilation using Cox (adjusted hazard ratio 6.2, 95% CI 1.9-20.1) or competing risks (adjusted sub-distribution hazard ratio 12.1, 95% CI 2.7-55.4) survival models. Thus, our data establish that SARS-CoV-2 causes specific manifestations of proximal tubule dysfunction and provide novel insights into COVID-19 severity and outcome. (i.e. defective tubular handling of uric acid) • Nadir lymphocyte count, peak hsCRP/LDH/D-dimers • Invasive mechanical ventilation (Cox and competing risks models) PT injury, decreased expression of megalin in brush border, particles resembling SARS-CoV-2 in PT cells Independent from comorbidities, glomerular proteinuria, nephrotoxic medications or viral load The recent pandemic of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has major public health and economic impact 1 . The clinical spectrum of COVID-19 is broad, ranging from asymptomatic carrier state to severe, bilateral and diffuse pneumonia, potentially leading to acute respiratory distress syndrome (ARDS), respiratory failure, and/or multiple organ dysfunction 2,3 . Identifying early manifestations of COVID-19 and patients at risk for disease progression and respiratory failure is of crucial importance to alleviate the major stress on healthcare systems. The angiotensin converting enzyme 2 (ACE2), the receptor mediating the entry of SARS-CoV-2 in human cells, is expressed in the lung, heart, intestine and kidney, providing a rationale for the systemic manifestations of the disease [4] [5] [6] [7] . Increasing evidence suggests that COVID-19 may cause kidney damage, as indicated by the occurrence of proteinuria, hematuria and elevated serum creatinine on admission; the high incidence of acute kidney injury (AKI); and a spectrum of pathologic abnormalities including acute tubular necrosis, endothelial damage and capillary occlusions, deposition of complement complex on tubules, and glomerular lesions identified in autopsy reports [8] [9] [10] [11] [12] [13] . Kidney damage may result from hemodynamic factors, dysfunctional immune responses 14 , or direct viral infection of kidney cells, the latter being compatible with the detection of SARS-CoV-2 mRNA and protein in glomerular and tubular cells 15 and of purported viral particles in podocytes and proximal tubule (PT) cells 8, [16] [17] . However, the identification of viral particles by electron microscropy is challenging, as they may easily be mistaken for normal cell organelles such as the endoplasmic reticulum (ER), clathrin-coated vesicles, or multivesicular bodies [18] [19] [20] . Overall, several arguments suggest the PT may be a specific target for SARS-CoV-2 infection. Here, we analyzed the characteristics, extent and structural correlates of PT dysfunction in a cohort of patients admitted with SARS-CoV-2 infection in a large academic J o u r n a l P r e -p r o o f hospital in Brussels, Belgium. We further investigated the association of various markers of PT dysfunction with COVID-19 severity and outcome. J o u r n a l P r e -p r o o f The detection of proteinuria of unknown mechanism by routine analysis among ~80 % of patients in the early phase of the pandemic prompted us to perform specific urinalyses in 49 patients with documented SARS-Cov-2 infection hospitalized at the Cliniques universitaires Saint-Luc in Brussels, Belgium, between March 31, 2020 and April 18, 2020 (Suppl. Fig. 1 ). Patients on kidney replacement therapy at the time of admission were excluded. Baseline characteristics of the 49 patients with specific urinalysis are presented in Table 1 . Median age (interquartile range, IQR) at admission was 64 years (54-74) and 69% were males; 86% were of Caucasian and 12% of Sub-Saharan African origin. Eighteen percent, 47%, 20%, and 14% had a history of cardiovascular disease, hypertension, diabetes and chronic kidney disease, respectively. Chronic kidney disease was ascribed to hypertension (n=2, 29%), chronic interstitial nephritis (n = 2, 29%), nephron mass reduction (n=1, 14 %), or unknown origin (n=2, 29%). Forty percent were treated with a renin angiotensin system inhibitor. Eight percent were on chronic immunosuppressive treatment and another 8% on anti-cancer therapy. A detailed list of these medications is provided in Table 1 . Patients were admitted a median (IQR) of 7 days (3-9) after the onset of symptoms, mainly with fever (80%), dyspnea (71%) and cough (59%) ( 1A; Fig. 1C ; Table 2 ). Hypophosphatemia with inappropriate phosphaturia (FE P >20%) was observed in 19% (6/32). Aminoaciduria was detected in 6 out of 13 (46%) tested COVID-19 patients ( Fig. 1A ; Table 2 ), and was restricted to neutral amino acids ( Fig. 1E ; Suppl. Fig. 4A ,C,D). Transcriptomic analysis of specific segments of the mouse nephron confirmed the enrichment of genes encoding for the SARS-Cov-2 receptor ACE2, its homologue TMEM27 (collectrin), uric acid transporter URAT1 (SLC22A12), sodium-dependent phosphate cotransporter NaPi-IIa (SLC34A1), and B0AT1 in PT segments (Suppl. Fig. 4B ). Three out of the 43 patients tested had positive dipstick glucosuria upon admission. Two of them had a history of type 2 diabetes, concomitant hyperglycemia and significant glucosuria (blood glucose 215 mg/dl and 331 mg/dl; and dipstick glucosuria 2+ and 4+, respectively), while the remaining individual had prediabetes (HbA1c 6.0%), slightly elevated blood glucose (127 mg/dl) and mild glucosuria (1+). In contrast with other signs of PT dysfunction, none of the patients in the cohort showed normoglycemic glucosuria (Table 2) . Altogether, these observations showed that a specific dysfunction of the PT develops Biological signs of PT dysfunction were not associated with demographics, disease presentation or severity, or viral load, estimated from real time polymerase chain reaction and cycle threshold (Ct) values at admission. They were also independent from comorbidities, medications interfering with uric acid production or with potential PT toxicity, including antiviral or immunomodulatory drugs ( Table 8 ). To characterize structural alterations associated with PT dysfunction, we analyzed 6 kidney autopsy samples from patients who died of COVID-19, for whom tissue samples were wellpreserved with absence of autolysis at the optical level. The patients' age ranged between 57 and 82 years; 4 were males; all had abnormal proteinuria and 2 developed AKI requiring kidney replacement therapy (Suppl. Table 9 ). None of the patients received anti-viral medications. Post-mortem examination of the COVID-19 kidneys showed prominent and diffuse PT damage in all patients, with dilation of the tubular lumen containing cellular debris, denuded basement membranes and major alterations of the brush border ( Fig PT dysfunction, related e.g. to sepsis, aminoglycosides, or hepatorenal syndrome 31 . The fact that PT cells highly express ACE2 suggests that they could be targeted by SARS-CoV-2 at an early stage of disease. The binding affinity of SARS-CoV-2 spike glycoprotein to ACE2 is a major determinant of disease severity 32 . Besides its role in the renin angiotensin system, ACE2 facilitates the trafficking of B 0 AT1 to the apical membrane 23 . Recent studies reported the cryo-electron microscopy structure of the full-length human ACE2, forming heterodimers with B 0 AT1 6 and similar morphology to coronaviruses. They are located inside vacuoles or ER lumens and show faint hints of projections in contact with the vacuolar contents -not the cell cytoplasm 19 . Also, the particles contain dense dots (10-15 nm), forming either crisp circles or larger, smudgy dots, that may correspond to sections across the tube-or helix-forming of the virus nucleocapsid 38, 39 . Of note, most dots inside these particles appear larger than nucleocapsids observed in properly fixed tissue cultures 38, 39 . These dots are approximately the size of ribosomes (~20 nm); however, only arenaviruses appear to contain ribosomes and, to our knowledge, no normal cell structures appear like this. Alternatively, the larger-than-expected (for a nucleocapsid) and irregularly shaped dense dots could be related to poor fixation and delay in processing 38, 39 . In cells containing these virus-like structures, the particles are more uniform in size The following data were extracted from electronic medical records: demographics, symptoms at admission, unit where the patient was hospitalized first (intensive care unit versus conventional unit), vital signs, biological and imaging data, and outcome. We recorded routine biological data obtained at admission, as well as the nadir lymphocyte count, and the peak values for highly sensitive C-reactive protein, lactate dehydrogenase and D-dimers The following antibodies were used: rabbit anti-human Gc-globulin (also known as VDBP, A0021, Dako); mouse anti-human ACE2 (AMAB91262, Sigma); rabbit anti-human ACE2 (AF933, R&D); rabbit anti-human AQP1 (ab2219, Millipore); sheep anti-LRP2 (gift from P. Verroust and R. Kozyraki, INSERM, Paris, France); rabbit anti-human UP1 (A0257, Dako); rabbit anti-human LMW proteins (A0126, Dako). Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblot were performed as previously described 46 . Urine samples were thawed on ice, diluted in Laemmli buffer and proteins separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (7.5%, 12% or 16% acrylamide slabs) in non-reducing conditions. The volume of urine loaded in each lane was normalized for creatinine concentration. After blotting onto nitrocellulose and blocking, membranes were incubated overnight at 4°C with primary antibody, washed, incubated with peroxidase-labeled secondary antibody, and visualized with enhanced chemiluminescence (ECL, Pierce, Aalst, Belgium). Pathologic evaluation was performed on kidney samples obtained from autopsies of 6 COVID-19 cases. All specimens were well preserved without autolysis, and post-mortem interval was less than 6 hours. None of the patients received antiviral or nephrotoxic drug. Sections of formaldehyde-fixed paraffin-embedded blocks were stained with hematoxylin and Immunostaining was performed on formaldehyde-fixed paraffin-embedded sections of normal human kidney and kidney from patients with active COVID-19 using a sequential staining protocol as described previously 46 The segmental expression of specific markers in mouse kidney was performed as described 48 . Kidneys of C57BL6J mice were digested with type-2 collagenase and tubules were isolated manually according to the morphological differences, before lysis in RNA extraction buffer Table 11 ). The relative changes in targeted genes over Gapdh mRNAs were calculated using the 2 -∆∆Ct formula. The connectivity network of the interactions between human SLC6A19 (B 0 AT1) and other proteins including the SARS-Cov-2 receptor ACE2 and the related protein TMEM27 (collectrin), was established using the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database 49 . Results are presented as means ± SD or median (IQR) for continuous variables and as numbers and proportions for categorical variables. Continuous variables were expressed in their natural units without standardization. Comparisons between groups were performed using unpaired t-test, Kruskal-Wallis, or χ2 test, as appropriate. Spearman's rank test was performed to assess the correlation between serum uric acid level and urinary fractional excretion of uric acid. Time to event analyses were performed using Cox proportional hazard regressions (where events were defined as the need for invasive mechanical ventilation or death) and a competing risk approach. In the latter, the hazard ratio for invasive mechanical ventilation and its 95% confidence interval was estimated by including each specific PT defect and baseline characteristics as covariates and by considering death and discharge as competing events. Multivariable time to event analyses have been adapted to adjust for pre-specified relevant covariates including age, gender, baseline lymphocyte count, LDH and hsCRP levels. Collinearity between variables was quantified using variance inflation factors, and variance inflation factors >10 suggested excessive correlation between variables. All statistical analyses were performed using GraphPad Prism (version 8.0) or Stata (version 16.0) software. All tests were two-tailed and a P-value <0.05 was considered significant. The authors declare no potential conflict of interest relevant to this article. Supplementary Figures S1-S5 Supplementary Tables S1-S11 Supplementary information is available at Kidney International's website. 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(%) corticosteroids (2), methotrexate (1), rituximab (1). b Anti-cancer drugs included cyclophosphamide (1), doxorubicine (1), vincristine (1), venetoclax (1), cisplatin (1), and cytarabine (1). c Immunomodulatory drugs for COVID-19 (one or more per patient) blood pressure; eGFR, CKD-EPI estimated glomerular filtration rate; hsCRP, highly-sensitive C-reactive protein