Appearances Can Be Deceiving - Viral-like Inclusions in COVID-19 Negative Renal Biopsies by Electron Microscopy : Kidney360

Journal Logo

Brief Communications

Appearances Can Be Deceiving - Viral-like Inclusions in COVID-19 Negative Renal Biopsies by Electron Microscopy

Cassol, Clarissa A.1; Gokden, Neriman2; Larsen, Christopher P.1; Bourne, Thomas D.1

Author Information
Kidney360 1(8):p 824-828, August 2020. | DOI: 10.34067/KID.0002692020
  • Free

Since the discovery of the causative agent for the novel severe acute respiratory syndrome (SARS)–like pneumonia syndrome pandemic that started in China in 2019 (1,2), a coronavirus named SARS coronavirus 2 (SARS-CoV-2), electron microscopy images have populated the medical literature (2) and media outlets alike displaying the characteristic 60–140 nm round particles surrounded by a “corona” of 9–12 nm distinctive spikes (2). Although many of these images were obtained after “in vitro” infection of cultured cells with SARS-CoV-2 (2) and are thus likely a true representation of viral particles, we have observed morphologically indistinguishable inclusions within podocytes and tubular epithelial cells both in patients negative for coronavirus disease 2019 (COVID-19) as well as in renal biopsies from the pre–COVID-19 era. Although direct infection of the kidney is theoretically possible, given the presence of angiotensin-converting enzyme 2 (the receptor used by SARS-CoV-2 to gain access to cells) within proximal tubular epithelium (3) and podocytes (4), the virus has not been detected by real-time RT-PCR in urine samples from patients with COVID-19 (5–7). Additionally, for the virus to have access to kidney parenchyma, viremia should occur, and this has only been detected in a minority of patients (6–8).

We would, therefore, like to issue a note of caution for inferring viral tissue infection by morphology alone using electron microscopy images from tissues obtained from biopsies or autopsy material in patients with COVID-19. Moreover, caution should be used when interpreting immunohistochemical results, especially within proximal tubules, which are prone to nonspecific staining by a variety of antibodies due to their intense reabsorptive capacity. Additionally, more specific techniques such as immunoelectron microscopy using specific viral antigens (9), or in situ hybridization for viral RNA, are likely necessary to undoubtedly confirm tissue infection in these cases.

Indeed, in two recent reports of collapsing glomerulopathy in patients with COVID-19, viral RNA was not detected in the kidney by in situ hybridization (10,11). Additionally, immunohistochemical analysis using a SARS-CoV-2 nucleoprotein antibody previously shown to have positive staining in the kidney of patients with COVID-19 showed nonspecific positive staining in the renal parenchyma of all kidneys in our laboratory (10).

We postulated that endogenous mimickers could be present that are morphologically indistinguishable from SARS-CoV-2 virions ultrastructurally. To confirm this, we reviewed cases under the electron microscope, looking for round cytoplasmic inclusions in podocytes, tubular epithelial cells, or endothelial cells, each with an individual diameter between 60 and 140 nm, present either in isolation or in groups. Approval for this study was obtained by the Solutions Institutional Review Board, and the ethical principles highlighted by the Declaration of Helsinki were followed. To optimize ultrastructural morphology, we excluded cases that were rapidly processed and only reviewed cases for which the renal biopsy tissue underwent routine processing (this included overnight polymerization and standard grid staining). Five cases were from the pre–COVID-19 era and ten cases were recent (biopsy dates from February to April 2020). Eight cases were allografts and six were native kidney biopsies. Case details can be found in Table 1. Viral-like inclusions, consisting both of single vesicles with diameters between 50 and 139 nm, as well as packed groups within larger vesicles, were found in all 15 cases, either in podocytes, tubular epithelium, or vascular endothelial cells (Figure 1).

Table 1. - Biopsy characteristics including date, indication, brief clinical history, and final diagnosis
Case Biopsy Type Biopsy Date Biopsy Indication Clinical History Biopsy Diagnosis
1 N April 2020 Proteinuria (10 g), increased Cr (1.7) 33 yr F with DM2, CHF Diffuse and nodular glomerulosclerosis, consistent with diabetic nephropathy, class 3
2 N April 2020 AKI (Cr 2.7), low C4 75 yr M with HTN, hyperlipidemia, CVA, lung cavitary lesions Mesangiopathic immune complex disease, most consistent with resolving-phase infectious glomerulonephritis
3 N April 2020 AKI (Cr 8.4) 46 yr M with hematuria, hemoptysis, and weakness Diffuse crescentic and necrotizing GN, pauci-immune (ANCA-associated) type
4 N April 2020 AKI (Cr 1.5) 33 yr M with h/o methamphetamine abuse Thrombotic microangiopathy
5 N March 2020 Rapid decline of renal function 72 yr M with DM2, HTN on hydralazine. Positive ANA (1:640) and ANCA Mesangiopathic immune complex disease, suspicious for autoimmune diseases such as lupus or lupus-like conditions
6 N March 2020 History of IgAN, now nephrotic-range proteinuria 40 yr, Cr 1.3;UA: 3+ blood, 3+ protein IgAN with focal cellular crescents and fibrinoid necrosis
7 T March 2020 AKI (Cr 7.9) 47 yr M with ESKD from DM/HTN Acute cellular rejection, Banff IB
Acute vascular rejection, Banff IIB
C4d negative
SV-40 negative
8 T March 2020 AKI (Cr 2.7) 55 yr M with ESKD from RCC requiring bilateral nephrectomies Borderline changes by Banff criteria (suspicious for acute cellular rejection)
C4d negative
SV-40 negative
9 T March 2020 AKI (Cr 4.3), anemia, leukopenia 28 yr M with ESKD of unknown etiology Acute cellular rejection, Banff IB
Acute vascular rejection, Banff IIB
C4d negative
SV-40 negative
10 T February 2020 AKI (Cr 5.1) 36 yr F with h/o ESKD due to SLE. Admitted with pulmonary HTN, acute decompensated HFpEF, and AKI. DSAs negative Negative for rejection
Findings favor thrombotic microangiopathy
C4d negative
SV-40 negative
11 T October 2019 AKI (Cr 6.5) 49 yr M with ESKD from DM/HTN s/p DDKT with DGF Negative for rejection
C4d negative
SV-40 negative
12 T October 2019 AKI (Cr 1.7) 53 yr with ESKD from DM2 Borderline changes by Banff criteria
C4d negative
SV-40 negative
13 T October 2019 AKI (Cr 4.6) 38 yr M with HTN, AFib, HLPD, s/p kidney transplant with diarrhea Acute cellular rejection, Banff IA
C4d positive
SV-40 negative
14 T October 2019 Pain over allograft, AKI (Cr 1.8) 40 yr M with ESKD from HTN/DM BK polyomavirus nephropathy
Negative for rejection
C4d negative
SV-40 positive
15 T October 2019 AKI (Cr 2.7) 67 yr F with ESKD from DM/HTN Borderline changes by Banff criteria
C4d negative
SV-40 negative
N, native kidney; Cr, creatinine (all values are in mg/dl; reference range, 0.6–1.3 mg/dl); F, female; DM2, type 2 diabetes mellitus; CHF, congestive heart failure; M, male; HTN, hypertension; CVA, cerebrovascular accident; h/o, history of; ANA, anti-nuclear antibodies; IgAN, IgA nephropathy; UA, urinalysis; T, transplant kidney; SV-40, simian virus 40; RCC, renal cell carcinoma; HFpEF, heart failure with preserved ejection fraction; DSA, donor-specific antibody; s/p, status post; DDKT, deceased donor kidney transplant; DGF, delayed graft function; AFib, atrial fibrillation; HLPD, hyperlipidemia.

Figure 1.:
Viral-like particles in non-COVID19 patients biopsies. Electron microscopy images of viral-like particles within podocytes in a case of thrombotic microangiopathy in a (A) native kidney biopsy specimen and (B) acute cellular rejection in an allograft. Note the presence in both cases of single vesicles with an electron-dense rim likely representing endocytic coated vesicles, as well as larger multivesicular bodies (arrows), which could be confounded with vesicle packets containing virions. Inset in (A): the individual small coated pits in the exterior of the vesicle bear resemblance to a viral corona. (C) Similar intracytoplasmic vesicles within tubules in an allograft with changes suspicious for acute cellular rejection.

Additionally, we have performed in situ hybridization for SARS-CoV-2 RNA in eight biopsies from patients with active COVID-19 who had evidence of kidney disease and were unable to detect virus RNA in renal tissue, despite adequate positive controls. This appears to be in contrast with a recent study that showed SARS-CoV-2 RNA could be detected by RT-PCR within renal tissue in 13 of 22 autopsied kidneys; however, it should be noted that the viral RNA levels detected were quite low (close to the lowest limit of detection of one copy per cell), and could potentially represent viral RNA within renal blood vessels (12). Microdissection by renal compartment was done in only six cases, and of those only three showed positive viral RNA within the glomerular compartment, which again could still potentially be secondary to viral particles in the blood. Spatially resolved in situ hybridization in one example provided shows positive SARS-CoV-2 RNA within tubular epithelium and glomeruli, but it is not clear in how many cases this was present, and whether this corresponded to intact viral particles.

A number of potential natural mimickers that can generate intracellular groups of round vesicles mimicking SARS-CoV-2 virions could be listed, the most likely being endocytic vesicles and endosomal compartment components such as microvesicular bodies containing exosomes, among others. Endocytosis leads to the formation of 60–120 nm vesicles, which is within the size range described for SARS-CoV-2 (60–140 nm) (2). These endocytic vesicles may be coated by different proteins, one of the most common being clathrin (13). The presence of coating proteins may be responsible for the presence of an electron-dense area surrounding these vesicles, giving the appearance of a viral corona. The presence of clathrin-mediated endocytosis is well described in proximal tubule cells. Podocytes also rely on both clathrin-mediated as well as clathrin-independent endocytic processes to maintain the filtration barrier by regulating the uptake of integrins and lipoproteins (14). During podocyte development or after injury, the clathrin-independent pathway of raft-mediated endocytosis of nephrin and podocin has been shown to be important for proper slit diaphragm spatiotemporal orientation (14). Given its role in nephrin and podocin trafficking and distribution, it is possible that the formation of endocytic vesicles is increased in proteinuric states that are associated with loss of filtration barrier function and podocyte cytoskeletal and basement membrane remodeling. Indeed, Farquahr et al. (15) in their seminal studies of the glomerular ultrastructure, in the 1950s, described an increased number of cytoplasmic vesicles in children with nephrotic syndrome. Moreover, albumin endocytosis by podocytes has been demonstrated, in vitro and in vivo, in a mouse model of puromycin-induced nephrotic syndrome (16) and could contribute to an increased number of cytoplasmic vesicles in albuminuric diseases.

Proteinuria is a common finding in COVID-19, and has been described in up to 63% of patients at some point during the disease course (17). Moreover, the development of kidney injury in patients with COVID-19 has been associated with increased in-hospital mortality (18). Therefore, it is possible that autopsy series are enriched with those patients, which thus increases the probability of finding endocytic vesicles due to podocyte injury; however, specific data regarding proteinuria was mostly not available in the largest series reported to date which focused on renal pathologic findings (19). Proximal tubular cells also strongly rely on endocytic processes to fulfill their function of reabsorbing filtered macromolecules, which can be accomplished both through receptor-mediated endocytosis as well as fluid-phase endocytosis (20).

Alternatively, the viral-like inclusions could represent microvesicular bodies containing exosomes before their release onto the cell surface. Exosomes form within the endosomal compartment as intraluminal vesicles within microvesicular bodies, which eventually fuse with the plasma membrane and are released (21). Virtually all segments of the kidney in contact with the urinary space can give rise to exosomes, including podocytes (22). Recently, a model of albumin transcytosis has been proposed, through which albumin is initially endocytosed at the capillary aspect of the podocyte and subsequently exocytosed through the apical podocyte membrane within exosomes that can be detected in the urine (16). The podocytic origin of these exosomes is confirmed by the presence of proteins typical of the podocyte cell body, such as podocalyxin (16). This again could contribute to increased numbers of cytoplasmic vesicles within podocytes in patients with COVID-19 who are proteinuric, and could lead to the mistaken assumption that these represent virions. Individual exosome sizes vary, but they are generally between 30 and 150 nm (21), which falls within the size range reported for SARS coronaviruses (9). The potential for confusion of coronavirus particles with normal cellular components was in fact highlighted in a detailed ultrastructural study by the Centers for Disease Control and Prevention (CDC) of the SARS-CoV responsible for the 2003 SARS outbreak (9). The authors recommended that, in clinical specimens, the viral nature of inclusions should be confirmed by immunoelectron microscopy for viral antigens or ultrastructural viral RNA in situ hybridization (9).

Recognition of this pitfall of “viral-like particles” actually dates back to the 1970s, when the potential for mistakenly assuming that normal cellular components, such as phagocytic vacuoles, microvesicular bodies, or extracellular breakdown products, could represent viral particles was emphasized after a proliferation of studies claiming to have found ultrastructural viral particles within different types of cancer cells and fluids (23). Thus, we would like to echo the CDC (9) and earlier authors’ observations (15,23) and issue a note of caution regarding the use of ultrastructural images as evidence of SARS-CoV-2 tissue infection without confirmatory evidence of viral proteins or RNA in the tissue through immunoelectron microscopy or in situ hybridization.


C. Larsen reports grants from National Institutes of Health during the conduct of the study, outside the submitted work. All remaining authors have nothing to disclose.



Author Contributions

T. Bourne was responsible for formal analysis and validation; T. Bourne and C. Cassol provided supervision; T. Bourne, C. Cassol, and C. Larsen conceptualized the study; T. Bourne and N. Gokden were responsible for data curation; C. Cassol was responsible for project administration and wrote the original draft; C. Larsen was responsible for resources; and all authors reviewed and edited the manuscript.


1. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL, Chen HD, Chen J, Luo Y, Guo H, Jiang RD, Liu MQ, Chen Y, Shen XR, Wang X, Zheng XS, Zhao K, Chen QJ, Deng F, Liu LL, Yan B, Zhan FX, Wang YY, Xiao GF, Shi ZL: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579: 270–273, 2020
2. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang D, Xu W, Wu G, Gao GF, Tan W; China Novel Coronavirus Investigating and Research Team: A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 382: 727–733, 2020
3. Zou X, Chen K, Zou J, Han P, Hao J, Han Z: Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection. Front Med 14: 185–192, 2020
4. Ye M, Wysocki J, William J, Soler MJ, Cokic I, Batlle D: Glomerular localization and expression of Angiotensin-converting enzyme 2 and Angiotensin-converting enzyme: Implications for albuminuria in diabetes. J Am Soc Nephrol 17: 3067–3075, 2006
5. Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, Tan W: Detection of SARS-CoV-2 in different types of clinical specimens. JAMA 323: 1843–1844, 2020
6. Lescure F-X, Bouadma L, Nguyen D, Parisey M, Wicky PH, Behillil S, Gaymard A, Bouscambert-Duchamp M, Donati F, Le Hingrat Q, Enouf V, Houhou-Fidouh N, Valette M, Mailles A, Lucet JC, Mentre F, Duval X, Descamps D, Malvy D, Timsit JF, Lina B, van-der-Werf S, Yazdanpanah Y: Clinical and virological data of the first cases of COVID-19 in Europe: A case series [published correction appears in Lancet Infect Dis 20: e148, 2020]. Lancet Infect Dis 20: 697–706, 2020
7. Wölfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Müller MA, Niemeyer D, Jones TC, Vollmar P, Rothe C, Hoelscher M, Bleicker T, Brünink S, Schneider J, Ehmann R, Zwirglmaier K, Drosten C, Wendtner C: Virological assessment of hospitalized patients with COVID-2019. Nature 581: 465–469, 2020
8. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X, Cheng Z, Yu T, Xia J, Wei Y, Wu W, Xie X, Yin W, Li H, Liu M, Xiao Y, Gao H, Guo L, Xie J, Wang G, Jiang R, Gao Z, Jin Q, Wang J, Cao B: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China [published correction appears in Lancet 395: 496, 2020]. Lancet 395: 497–506, 2020
9. Goldsmith CS, Tatti KM, Ksiazek TG, Rollin PE, Comer JA, Lee WW, Rota PA, Bankamp B, Bellini WJ, Zaki SR: Ultrastructural characterization of SARS coronavirus. Emerg Infect Dis 10: 320–326, 2004
10. Larsen CP, Bourne TD, Wilson JD, Saqqa O, Sharshir MA: Collapsing glomerulopathy in a patient with coronavirus disease 2019 (COVID-19). Kidney Int Rep 5: 935–939, 2020
11. Peleg Y, Kudose S, D’Agati V, Siddall E, Ahmad S, Kisselev S, Gharavi A, Canetta P: Acute kidney injury due to collapsing glomerulopathy following COVID-19 infection. Kidney Int Rep 5: 940–945, 2020
12. Puelles VG, Lütgehetmann M, Lindenmeyer MT, Sperhake JP, Wong MN, Allweiss L, Chilla S, Heinemann A, Wanner N, Liu S, Braun F, Lu S, Pfefferle S, Schröder AS, Edler C, Gross O, Glatzel M, Wichmann D, Wiech T, Kluge S, Pueschel K, Aepfelbacher M, Huber TB: Multiorgan and renal tropism of SARS-CoV-2 [published online ahead of print May 13, 2020]. N Engl J Med doi:10.1056/NEJMc2011400
13. Kaksonen M, Roux A: Mechanisms of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 19: 313–326, 2018
14. Inoue K, Ishibe S: Podocyte endocytosis in the regulation of the glomerular filtration barrier. Am J Physiol Renal Physiol 309: F398–F405, 2015
15. Farquhar MG, Vernier RL, Good RA: An electron microscope study of the glomerulus in nephrosis, glomerulonephritis, and lupus erythematosus. J Exp Med 106: 649–660, 1957
16. Castrop H, Schießl IM: Novel routes of albumin passage across the glomerular filtration barrier. Acta Physiol (Oxf) 219: 544–553, 2017
17. Naicker S, Yang CW, Hwang SJ, Liu BC, Chen JH, Jha V: The Novel Coronavirus 2019 epidemic and kidneys. Kidney Int 97: 824–828, 2020
18. Cheng Y, Luo R, Wang K, Zhang M, Wang Z, Dong L, Li J, Yao Y, Ge S, Xu G: Kidney disease is associated with in-hospital death of patients with COVID-19. Kidney Int 97: 829–838, 2020
19. Su H, Yang M, Wan C, Yi LX, Tang F, Zhu HY, Yi F, Yang HC, Fogo AB, Nie X, Zhang C: Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China. Kidney Int 98: 219–227, 2020
20. Schuh CD, Polesel M, Platonova E, Haenni D, Gassama A, Tokonami N, Ghazi S, Bugarski M, Devuyst O, Ziegler U, Hall AM: Combined structural and functional imaging of the kidney reveals major axial differences in proximal tubule endocytosis. J Am Soc Nephrol 29: 2696–2712, 2018
21. Karpman D, Ståhl AL, Arvidsson I: Extracellular vesicles in renal disease. Nat Rev Nephrol 13: 545–562, 2017
22. Pisitkun T, Shen RF, Knepper MA: Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A 101: 13368–13373, 2004
23. Dalton AJ: Microvesicles and vesicles of multivesicular bodies versus “virus-like” particles. J Natl Cancer Inst 54: 1137–1148, 1975

clinical nephrology; COVID-19; endosomes; exosomes; inclusions; microscopy; electron; microscopy; immunoelectron; pandemics; reverse transcriptase polymerase chain reaction; RNA; viral; SARS-CoV-2; ultrastructure; virions

Copyright © 2020 by the American Society of Nephrology