key: cord-0837392-3utki5cz
authors: Kapp, Meghan E.; Fogo, Agnes B.; Roufouse, Candice; Najafian, Behzad; Radhakrishnan, Jai; Mohan, Sumit; Miller, Sara E.; D’Agati, Vivette D.; Silberzweig, Jeffrey; Barbar, Tarek; Gopalan, Tulasi; Srivatana, Vesh; Mokrzycki, Michele H.; Benstein, Judith A.; Ng, Yue-Harn; Lentine, Krista L.; Aggarwal, Vikram; Perl, Jeffrey; Salenger, Page; Koyner, Jay L.; Josephson, Michelle A.; Heung, Michael; Velez, Juan Carlos; Ikizler, Alp; Vijayan, Anitha; William, Preethi; Thajudeen, Bijin; Slepian, Marvin J.
title: Renal Considerations in COVID-19: Biology, Pathology, and Pathophysiology
date: 2021-10-04
journal: ASAIO J
DOI: 10.1097/mat.0000000000001530
sha: 8b045a7a3aec004d2af1c11a2a97e8975f8d0e69
doc_id: 837392
cord_uid: 3utki5cz

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emerged into a worldwide pandemic of epic proportion. Beyond pulmonary involvement in coronavirus disease 2019 (COVID-19), a significant subset of patients experiences acute kidney injury. Patients who die from severe disease most notably show diffuse acute tubular injury on postmortem examination with a possible contribution of focal macro- and microvascular thrombi. Renal biopsies in patients with proteinuria and hematuria have demonstrated a glomerular dominant pattern of injury, most notably a collapsing glomerulopathy reminiscent of findings seen in human immunodeficiency virus (HIV) in individuals with apolipoprotein L-1 (APOL1) risk allele variants. Although various mechanisms have been proposed for the pathogenesis of acute kidney injury in SARS-CoV-2 infection, direct renal cell infection has not been definitively demonstrated and our understanding of the spectrum of renal involvement remains incomplete. Herein we discuss the biology, pathology, and pathogenesis of SARS-CoV-2 infection and associated renal involvement. We discuss the molecular biology, risk factors, and pathophysiology of renal injury associated with SARS-CoV-2 infection. We highlight the characteristics of specific renal pathologies based on native kidney biopsy and autopsy. Additionally, a brief discussion on ancillary studies and challenges in the diagnosis of SARS-CoV-2 is presented.

The American Society for Artificial Organs (ASAIO) has long been focused on systems, advances, and strategies for management of organ dysfunction through use of support or replacement therapeutics. With the emergence of COVID-19, ASAIO initially assembled a whitepaper addressing advanced pulmonary and cardiac support. 7 With recognition of the significance and emerging spectrum of renal involvement in COVID-19, ASAIO has reached out broadly to the wider renal community, and has established a broad writing group to assemble knowledge and provide a resource document focused on the evolving understanding of renal involvement in COVID- 19 . We here address the biology, pathology, and pathophysiology of SARS-CoV-2 infection and associated renal involvement. We particularly report herein on new pathologic observations based on biopsy and autopsy studies.

Coronavirus was first described and named by Tyrrell et al. 8 in 1967, as a generally round, enveloped, nonsegmented virus of 80-125 nm in size with a large genome of 30 kb in size. Coronaviruses early on were recognized to cause respiratory illness, though also resulting in gastroenteritis and necrotizing enterocolitis in children and hepatitis in mice. [9] [10] [11] Over time four genera of enveloped positive-sense single-stranded ribonucleic acid (RNA) coronaviruses have been described: alpha, beta, gamma, and delta. In recent years, new emergent beta coronaviruses have been described including SARS and Middle Eastern respiratory virus (MERS). Some patients with MERS and SARS also experienced AKI but were not reported to show nephrotic syndrome and the spectrum of glomerular lesions described below for COVID-19, and risks potentially related to apolipoprotein L-1 (APOL1) risk allele variants for those infections are not described.

Severe acute respiratory syndrome coronavirus 2 also belongs to the beta coronavirus group. 6 The surface of SARS-CoV-2 has club-like projections which afford its crown-like structure. The spike protein in coronavirus determines tissue tropism of the virus, enabling viral entry promoting cell to cell spread. 12 Severe acute respiratory syndrome coronavirus 2 enters the body mainly through the nasal and laryngeal mucosa, subsequently reaching the lungs where it replicates rapidly. Although the principal mode of transmission is droplets spread by coughing or sneezing, other routes of transmission, that is, fecal-oral route, are possible. Major target organs for the virus that have been described, beyond the lung, include but are not limited to heart, gastrointestinal tract, and kidney. 7 The average incubation period for COVID-19 ranges between 2 and 14 days, with a mean of 5.2 days, and a mean case fatality rate of 3.31% among confirmed cases. 13 The host receptor responsible for providing access to SARS-CoV-2 into cells is the angiotensin-converting enzyme 2 (ACE2) receptor. 14 The kidneys have abundant ACE2 receptors, predominantly located in podocytes, mesangial cells, parietal epithelium of Bowman's capsule, proximal cell brush border, and collecting duct. 6 Angiotensin-converting enzyme 2 expression in kidney tissue is higher than that of lung tissue and the binding affinity of SARS-CoV-2 to ACE2 receptors is 10-20 times higher than SARS-CoV. 15 Angiotensin-converting enzyme 2 mediates the cleavage of angiotensin-I and angiotensin-II into angiotensin-(1-9) and angiotensin-(1-7), respectively. Angiotensin-(1-7) exerts vasodilatory, antiproliferative, and anti-inflammatory activity counterbalancing the adverse effects of angiotensin-IIlike vasoconstriction, proliferation, and inflammation. Binding of SARS-CoV-2 to ACE2 leads to depletion of ACE2 activity disrupting the physiologic balance ACE/ACE2 with consequential loss of the protective effects of angiotensin-(1-7) and accumulation of angiotensin-II. This promotes glomerular dysfunction, vasoconstriction, and inflammation resulting in AKI. [16] [17] [18] Other potential mechanisms for organ involvement include a receptor related to the transmembrane serine protease (TMPRSS) gene and invasion through CD147-spike protein. Transmembrane serine protease primes viral surface spike protein promoting the fusion of virus and host cell membrane, whereas CD147spike protein is a transmembrane glycoprotein expressed on proximal tubular epithelial cells and inflammatory cells. 19 

Risk factors for development of AKI include ventilator support, use of vasopressor drugs, increased age, male sex, multiple comorbidities (especially diabetes mellitus, hypertension, and cardiovascular disease), severe disease, higher body mass index, non-O blood group type, and African American race. [20] [21] [22] Other features of increased risk include leukocytosis, lymphopenia, thrombocytopenia, prolonged activated partial thromboplastin time, and higher levels of inflammatory markers: D-dimer, procalcitonin, aspartate aminotransferase, and lactic dehydrogenase. 23 Most AKI occurred in 7 days from admission to hospital with faster onset in patients who had elevated serum creatinine at baseline. 24 Acute kidney injury was reported in nearly 3-fold more patients with pre-existing kidney disease (11.4% vs. 4%). 24 Acute kidney injury is also more common in those with higher systolic blood pressure and potassium levels and lower serum albumin. 25 Higher rates of AKI have been documented in critically ill patients including those with higher sequential organ failure assessment (SOFA) score, renal SOFA score, cardiovascular SOFA score, and lower partial pressure of oxygen/fraction of inspired oxygen. 26

The pathophysiology of renal involvement in COVID-19 is not fully understood. The most common causes of AKI in COVID-19 infection appears to be a combination of volume depletion, synergistic effects of virus-induced direct cytotropic effect, and cytokine-induced systemic inflammatory response. These result in intrinsic injury to the kidney in the form of tubular injury, acute interstitial nephritis, and de novo glomerular disease. Indirect processes associated with AKI include downstream consequences of infection such as endothelial injury, rhabdomyolysis, ischemic thrombi, inflammation, and complement dysregulation. 27 Hemodynamic instability (caused by hypovolemia or hypotension) or injury caused by nephrotoxic drugs likely contribute to AKI as well. Potential mechanisms of tubular injury include hemodynamic disturbances such as central venous pressure elevation, increased intrathoracic pressure, and high positive end-expiratory pressure (PEEP) which result in reduction of effective renal perfusion pressure. 28 Hemodynamic decompensation and shock compound respiratory decompensation leading to ischemic acute tubular injury (ATI). 20, 29 Cardiorenal syndrome from right ventricular failure secondary to COVID-19 pneumonia or left ventricular dysfunction lead to tubular injury over time. Other causes of tubular injury include macrophage activation syndrome, microemboli and microthrombi as a result of dysregulation of coagulation homeostasis, and endothelialitis, leading to renal microcirculatory dysfunction. Impairment of gas exchange and severe hypoxemia seen in acute respiratory distress syndrome patients have been associated with AKI. 6, 20, 29, 30 African American individuals with COVID-19 may present with new nephrotic range proteinuria and AKI along with respiratory illness. 31 Collapsing glomerulopathy appears to be a manifestation of an inflammatory response by a susceptible host, that is, with risk allele variants of APOL1, rather than direct parenchymal injury caused by the virus.

Severe acute respiratory syndrome coronavirus 2 activates macrophages which induce cytokine storm leading to the activation of coagulation factors and aggregation of erythrocytes leading to microvascular damage which compounds the hypercoagulable state triggered by the inflammatory milieu related to the infection. 27 Levels of proinflammatory cytokines that are elevated include interleukin-1β, interleukin-1RA, interleukin-7, interleukin-8, interleukin-9, interleukin-10, fibroblast growth factor, granulocyte-macrophage colony-stimulating factor, interferon-γ, granulocyte colony-stimulating factor, interferon-γ-inducible protein, monocyte chemoattractant protein, macrophage inflammatory protein one alpha, plateletderived growth factor, tumor necrosis factor α, vascular endothelial growth factor. 32 These cytokines induce endothelial and tubular dysfunction in the kidneys. Histopathological evidence based on postmortem examination of kidneys has demonstrated the presence of cluster of differentiation 68 (CD68)positive macrophage infiltration in the tubules and interstitium as well as complement 5b-9 deposition, CD8+ T lymphocyte cells, and CD56+ (natural killer) cells. 33, 34 

The clinical presentation of COVID-19-related kidney disease reflects the underlying pathology. Patients with AKI may present with proteinuria or hematuria in addition to elevations of serum creatinine and blood urea nitrogen. 5 The reported rates of AKI are variable, with estimates of >20% of hospitalized patients and >50% of patients in the intensive care unit. 35 Such patients are not typically biopsied since their clinical presentation and disease course resemble other patients with AKI in the setting of critical illness. In a study of 442 hospitalized patients with COVID-19 in China, proteinuria was present in 43.9% (with 30% having ≥2+ on dipstick) with significant hematuria demonstrated in 11.3% patients. 23 Nephrotic syndrome is uncommon in COVID-19 and such patients are likely biopsied more commonly, and thus glomerular diseases appear to be over-represented in biopsy series. For example, in the series of 17 patients reported by Kudose et al., 36 88% presented with AKI or AKI superimposed on chronic kidney disease, and 53% had nephrotic syndrome. A variety of pathologic entities were reported including 10 glomerular diseases. Collapsing glomerulopathy is the entity that appears to be directly related to a COVID-19-associated hyperinflammatory state in predisposed individuals with APOL1 risk alleles. The striking similarity in kidney pathology between human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) and the collapsing glomerulopathy of COVID-19 has led to the suggestion that the eponym "COVAN," that is, COVID-associated nephropathy, should be applied. 31 Patients with COVAN present with AKI and nephrotic syndrome. In some series, manifestations of COVID-19 were mild or resolving at the time of kidney biopsy. 37 The clinical course is variable; however, a significant proportion of patients were either dialysis-dependent or with chronic kidney disease at the end of follow-up. 36, 38 None of the patients were treated with immunosuppressive therapy specifically target to COVAN.

In patients with non-COVAN biopsy findings, for example, with membranous nephropathy, it is likely that they had preexisting kidney disease and were biopsied incidentally during COVID-19. However, since COVID-19 represents a state of altered immunity, immune-mediated renal disease could potentially flare or present for the first time in this situation, for example, the multisystem inflammatory syndrome in children.

Finally, evidence of isolated proximal tubular dysfunction has also been reported in COVID-19. In a cohort of 49 hospitalized patients, proximal tubule dysfunction was present in a significant proportion of patients and included low-molecularweight proteinuria (70-80%), neutral aminoaciduria (46%), and defective handling of uric acid (46%), or phosphate (19%). These manifestations were independent of pre-existing comorbidities, glomerular proteinuria, nephrotoxic medications, or viral load. Biopsy findings showed prominent tubular injury, including in the initial part of the proximal tubule. 39

Kidney biopsies performed in patients with COVID-19 have revealed diverse glomerular and tubular disorders. The most common findings in biopsy series are ATI and de novo collapsing glomerulopathy, and more rarely, thrombotic microangiopathy (TMA). Other diverse immune-mediated glomerular disorders have also been described, and in some cases, development of SARS-CoV-2 infection causes exacerbation of preexisting autoimmune and alloimmune conditions.

In two biopsy series reported, four of 14 native kidney biopsies and two of three allograft biopsies reported from Columbia University Medical Center and five of 10 biopsies performed in hospitalized patients with COVID-19 from Northwell Hospital Systems showed ATI as the sole pathologic process. In both series combined, totaling biopsies from 27 patients, rhabdomyolysis and myoglobin casts were an identifiable etiology in only two cases of ATI (one from each center). 36, 40 Other potential etiologies included exposure to nephrotoxins, hypoxemia, and severe hemodynamic instability. However, in several cases, no obvious etiology could be found, and COVID-19 symptomatology was not considered severe, implicating yet unidentified triggers. Importantly, no case of ATI had evidence of direct viral infection of renal tubular cells by electron microscopy (EM) or in situ hybridization for SARS-CoV-2 RNA or by definitive virus by EM (discussed later). 36, 40, 41 Interestingly, both series described ATI as a background finding in association with other glomerular and vascular processes, discussed in detail later. The two patients with TMA had other potential underlying causes, including exposure to gemcitabine and complement disorder, suggesting that COVID-19 may potentiate endothelial injury leading to overt TMA in patients with predisposing conditions. 40

Collapsing glomerulopathy was the first glomerular disease to be identified in SARS-CoV-2-infected patients. Collapsing glomerulopathy occurring in association with COVID-19 has been reported in patients of African descent from the United States, France, and Switzerland. 31, 36, 40, [42] [43] [44] [45] Collapsing glomerulopathy is a highly aggressive form of focal segmental glomerulosclerosis (FSGS) defined by implosive wrinkling and collapse of glomerular capillaries associated with hypertrophy and hyperplasia of glomerular epithelial cells, sometimes forming pseudocrescents that obliterate the urinary space (Figure 1) . The tubulointerstitium displays ATI and focal tubular microcysts. Collapsing glomerulopathy was first well characterized in HIV-infected patients, where it is termed HIVAN. Patients typically present with nephrotic range proteinuria and acute renal failure, often accompanied by nephrotic syndrome. There may be rapid progression to irreversible renal failure while some patients present in a relatively chronic phase of collapsing glomerulopathy. 38 Similar to HIVAN, patients with so-called COVAN often have endothelial tubuloreticular inclusions, which are known as "interferon footprints" because they are induced in endothelial cells by exposure to ambient interferon. Interferon mRNA is not detected in kidney biopsies with collapsing glomerulopathy, consistent with renal pathology resulting from a systemic innate immune response to COVID-19. 31 Collapsing glomerulopathy secondary to COVID-19 is thought to be induced by cytokine storm, including massive release of interferon and interleukins, in patients with a susceptible genetic background. Many collapsing glomerulopathy patients have relatively mild symptoms of COVID-19 when they present with severe AKI and nephrotic proteinuria. Collapsing glomerulopathy occurring in the native kidney in the setting of COVID-19 has thus far only been identified in patients of African descent who carry two APOL1 risk allele variants, G1 or G2. , 31, 36, 42, 43 Such homozygosity or compound heterozygosity (G1/G1, G1/G2, or G2/G2) occurs in approximately 13-14% of African Americans. 46 The presence of APOL1 risk alleles confers 29-fold higher odds of developing HIVAN in the African American population and 89-fold higher odds ratio in South African blacks. 46, 47 An exception is a single case report of collapsing glomerulopathy associated with COVID-19 occurring in the renal transplant recipient of a donor kidney carrying one APOL1 risk allele. 48 Recent data suggest that even a single APOL1 risk allele can cause dosedependent-dominant cytotoxicity. 49 Activation of a viral program in podocytes bearing APOL1 risk alleles upregulates APOL1 expression and may cause podocyte injury and cell death via alteration in autophagy, mitochondrial function, energy metabolism, and potassium efflux. 49 Apolipoprotein L-1 risk alleles also confer risk for collapsing glomerulopathy among patients with other interferon-mediated forms of this lesion, including systemic lupus erythematosus with podocytopathy, hemophagocytic syndrome, and collapsing glomerulopathy in the setting of other viral infections such as parvovirus B19, cytomegalovirus, and Epstein-Barr virus.

To-date, no viral particles have been definitively identified in glomerular or tubular cells in patients with collapsing glomerulopathy due to COVID-19, using various techniques including in situ hybridization for viral RNA, RNAScope, immunohistochemistry for viral spike and nucleocapsid proteins, or demonstration of virions by EM. 31, 36, 40, 42, 43, 50 This represents a major difference between COVAN and HIVAN, in which HIV-1 viral RNA and DNA have been detected in glomerular podocytes, parietal epithelial cells, and tubular epithelial cells, leading to dysregulation of host genes governing cell cycle and differentiation. 51

Two biopsy series were reported from the New York epicenter of the pandemic, one from Columbia University Medical Center and the other from Northwell Hospital Systems. 36, 40 A major difference between the two series is that all biopsies were performed on hospitalized patients in the Northwell series, where ATI predominated, whereas most biopsies were performed on an outpatient basis in the Columbia series, where glomerular disease predominated. In addition to five cases of collapsing glomerulopathy, a single case of minimal change disease associated with COVID-19 was reported in the Columbia series. 36 This African American patient presented with nephrotic syndrome and AKI. Genotyping revealed highrisk APOL1 genotype; nonetheless, the patient responded to glucocorticoid therapy. Other glomerulopathies included two cases of membranous glomerulopathy of uncertain duration, one crescentic transformation of longstanding lupus nephritis, and one de novo antiglomerular basement membrane antibody nephritis. 36 While these cases may represent detection of renal disease during the time of COVID, some findings warrant postulation of an association. Antiglomerular basement membrane antibody nephritis is an extremely rare glomerulonephritis, yet a 5-fold increased rate of new cases was described in London, United Kingdom, during the COVID-19 pandemic, of which half had detectable antibody to SARS-CoV-2. 52 Pulmonary injury due to COVID-19, as proposed for influenza and other infectious insults, has been postulated to precede onset of antiglomerular basement membrane antibody nephritis by exposing the cryptic target Goodpasture antigen, consisting of particular epitopes in COL4A3, in damaged alveolar capillary basement membranes. Thus, COVID-19 could initiate an aberrant adaptive immune response targeting the basement membrane. Hypercytokinemia due to COVID-19 may also promote heightened adaptive autoimmune and alloimmune responses that trigger crescentic transformation of longstanding stable lupus nephritis and acute T cell-mediated rejection in allografts of patients with preformed donor-specific antibodies. 36 

Postmortem examinations of patients who died from SARS-CoV-2 infection likely represent more severe manifestations of COVID-19, yet have shed light on the causes of AKI and are summarized in Table 1 . Acute tubular injury is the leading cause of renal dysfunction, and in most cases, there are no distinguishing features of the ATI in this setting on light microscopy. There is tubular epithelial cell flattening, detachment of epithelial cells from the tubular basement membrane, and formation of granular casts (Figure 2) . On occasion, myoglobin casts are noted (Figure 3 ) and a few reports have noted isometric tubular epithelial cell cytoplasmic vacuolation. 2, 41, 55 In Su et al, 55 this could be attributed to mannitol and intravenous immunoglobulin administration, whereas Farkash et al. 41 hypothesize that this could be related to viral infection of tubular epithelial cells; however, identification of virus in the tubular epithelial cell using a variety of methods has proven controversial. 41, 55 Interstitial inflammation is minor and confined to areas of interstitial fibrosis, without tubulitis; therefore, renal failure cannot be attributed to active tubulointerstitial nephritis. A substantial proportion of cases show areas of tubulointerstitial fibrosis, often in association with global glomerulosclerosis and arterial intimal thickening and was therefore attributed to hypertensive nephropathy. 58, 61 Arterial thrombi have been described in a number of organs in severe COVID-19, including the kidney, in keeping with the clinical finding that patients with severe COVID-19 have features of a distinctive coagulopathy, with a combination of incomplete features from both disseminated intravascular coagulation and TMA. 62 The thrombi likely contribute to some degree to organ dysfunction. Thrombi containing fibrin or platelets were noted in kidneys in several studies, but, in contrast to autopsies from patients with disseminated intravascular coagulation, were usually very focal, only affecting some arteries (Figure 4) and some glomeruli (Figure 5) . 63 Endothelialitis affected the arteries in one study, leading to the suggestion that local endothelial cell injury might play a role in the pathogenesis of the intravascular thrombi. Platelet thrombi are also noted in microvasculature. 54, 59 Arteries often show features of pre-existing arteriosclerosis, with fibrous intimal thickening, as would be expected in an older age group with frequent hypertension. The peritubular capillary network may contain red blood cell "rolls." 55 Glomeruli most often show nonspecific features such as ischemic tuft shrinkage, which is often seen in association with severe ATI. Glomeruli in some cases show evidence of preexisting renal disease, in the form of global glomerulosclerosis, focal and segmental glomerulosclerosis and diffuse or nodular mesangial sclerosis, in many cases due to diabetic nephropathy. 2, 43, 54, 56, 58, 59, 61 In one case series, a single patient presented with features of collapsing focal and segmental glomerulosclerosis. 61 In a single case in one report, a single glomerulus showed microaneurysmal change indicative of endothelial injury. 58 In summary, postmortem examinations have revealed the predominant causes of AKI in patients deceased with COVID-19 to be ATI, with a possible contribution of focal macro-and microvascular thrombi. Important negative findings for patient management and prognosis are the near-complete absence of virus-related collapsing FSGS, immune complex glomerulonephritis, and active tubulointerstitial nephritis. Although a few cases showed very focal vascular inflammation (one study), glomerular microaneurysmal change (Figure 6 ) (one study), and glomerular or arterial thrombosis, in no report were the changes widespread. The findings are in most respects the same as those reported in sepsis-associated AKI. 63 Histological evidence of underlying chronic kidney disease is frequent and mostly related to hypertensive arterionephrosclerosis and diabetes.

Attempts to assign any of these postmortem findings specifically to the effects of COVID-19 would benefit from inclusion of adequate control (non-COVID-19-related) intensive care deaths for comparison, and the use of ancillary studies to identify virus and investigate pathophysiological pathways. Findings to date using immunohistochemistry, in situ hybridization and EM in the autopsy series are covered below. A major limitation of such ancillary studies is the often-advanced state of autolysis present in postmortem kidneys. Electron microscopy done on autopsy tissue shows profound alteration of cell membranes and cytoplasm, making identification of normal cellular structures or virus difficult.

Electron microscopy is an important tool in detecting novel viruses and establishing their tissue tropism. Human coronavirus was first described by virologists Almeida and Tyrell 64 in 1967, using negative staining EM on human nasal and tracheal epithelial cells grown in culture and infected with "nasal washings from a patient with a cold." 8 Almeida and Tyrell 64 named the new virus coronavirus due to the presence of surface spikes, which on negative staining EM photographs "recalled the solar corona."

Transmission EM has been used to document the ultrastructural features of active coronavirus replication in infected cell cultures. 65, 66 Infected cells contain an often conspicuous viral replicative organelle. The replicative organelle is induced in infected cells by viral nonstructural proteins, and comprises convoluted membranes, double-membrane vesicles and small open double-membrane spherules, all derived from, and remaining connected to, the endoplasmic reticulum (ER). 67, 68 Virions assemble in and acquire their membrane from the ER-Golgi intermediate complex, resulting in virions within cisternal spaces derived from this complex. 69 The virions are transported to the cell membrane within these vesicles, using the usual secretory route of the ER-Golgi complex, and are released by exocytosis. In transmission EM images of infected cells, SARS-CoV-2 virions measure 60-140 nm in diameter, with spikes 9-12 nm length and are found in membrane-bound vesicles within the cytoplasm or outside the cell, near the plasma membrane (Figure 7) . 70 The helical viral nucleocapsid produces characteristic black dots within the virion. [71] [72] [73] Attempts have been made to apply transmission EM to human tissue samples, from both live and deceased patients to document active viral replication within tissues. As could be expected, lower airway samples have yielded the most convincing images of virions, in type II pneumocytes. 57 Tubular epithelial cells express the ACE2 receptor used for viral entry into cells, and in vitro investigations using kidney organoids illustrate the potential for viral replication in the human kidney. 74 A growing body of literature reports on corona virus-like particles in the kidneys of patients with COVID-19. 2, 41, 45, [54] [55] [56] However, these observations appear to not represent virions, with most of the intracellular structures depicted more likely representing subcellular structures that are part of the endosomal pathway, such as clathrin-coated vesicles and multivesicular bodies, whereas extracellular structures are likely exocytosed elements of this same pathway. 71, [75] [76] [77] [78] With respect to telling virions apart from other subcellular structures, intracytoplasmic structures with a "corona" directly projecting into the cytoplasm (rather than into a cisternal space) are likely clathrin-coated vesicles, derived from either endocytosis, from the trans-Golgi network or from endosomes/endolysosomes (Figure 8) . 76 Membrane-bound structures containing virus-sized structures but without internal dots, or of variable size, are more likely multivesicular bodies or other structures from the endosomal pathway.

It is notable that most of the observations of alleged virions have been made in tissue samples from postmortem examinations ( Table 2) . A major limitation of such investigations is the often-advanced state of autolysis, which leads to alterations of both membranes and cytoplasm, resulting in indistinct subcellular structures. This makes it particularly difficult in postmortem samples to distinguish virions from intracellular structures to which virions and their replicative organelles bear some resemblance. Tissue samples from live patients are better preserved, and with the exception of one study, virions were not identified in these samples. 45 On the other hand, it could be argued that live patient samples are typically from patients showing less severe viral infection. This controversy has illustrated the fundamental requirement in histopathological studies for both positive and negative biologic controls, together with "blinded" histological scoring.

Incontrovertible ultrastructural evidence of direct viral infection of renal parenchymal cells in vivo in humans is lacking, and it is possible that standard ultrastructural examination will be insufficient, and that immunoelectron microscopy and ultrastructural in situ hybridization will be needed to provide definitive evidence. However, these techniques are complicated and not usually applicable to routine samples obtained in a diagnostic histopathology department.

Detection of SARS-CoV-2 viral antigen in formalin-fixed paraffin-embedded tissue would facilitate not only clinical diagnosis of viral infection but would also allow studies to 44 1 Collapsing FSGS No Not identified Larsen et al. 42 1 Collapsing FSGS No Not identified Kissling et al. 45 1 Collapsing FSGS Yes Podocyte Kudose et al. 36 17

Collapsing FSGS, ATI, TMA, anti-GBM, IC-GN No Not identified Sharma et al. 40 38 12 Collapsing FSGS, ATI No Not identified Deceased patient autopsy reports Diao et al. 53 2 ATI; interstitial inflammation Yes TEC Varga et al. 54 2 Vasculitis Yes Endothelium Su et al. 55 9 ATI; fibrin thrombi Yes TEC, podocyte Bradley et al. 56 Not reported ATI Yes TEC, podocyte, endothelium Farkash et al. 41 1 ATI Yes TEC Menter et al. 2 2 ATI; thrombi Yes TEC, podocyte, endothelium Martines et al. 57 2 ATI No Not identified Bryce et al. 59 Not reported ATI No Not identified *Corona virus-like particles have been reported in the kidney on EM; however, they appear to not represent virions, but subcellular structures. ANCA, antineutrophil cytoplasmic antibodies; Anti-GBM, antiglomerular basement membrane antibody disease; ATI, acute tubular injury; COVID-19, coronavirus disease 2019; EM, electron microscopy; FSGS, focal segmental glomerulosclerosis; IC-GN, immune complex glomerulonephritis; TEC, tubular epithelial cell; TMA, thrombotic microangiopathy. advance our understanding of the pathogenesis of its associated disease, COVID-19. As discussed, identification of the virus within renal biopsies and postmortem kidney samples by ultrastructure examination is complicated by confounding normal mimics of viral particles; thus, additional methods for viral detection are needed. In a limited study of two autopsies, SARS-CoV-2 RNA was detected from formalin-fixed paraffin-embedded tissue by quantitative reverse transcription-polymerase chain reaction in both cases. 80 SARS-CoV-2 viral genome was also successfully sequenced by next-generation sequencing in one of these cases. Although this study was performed on lung tissue, it demonstrates molecular testing from infected tissue with inactivated virus can detect viral RNA and SARS-CoV-2 genome sequencing can be successfully performed on formalin-fixed paraffin-embedded blocks. Testing methodologies more readily available to clinical pathology practices including immunohistochemistry and in situ hybridization with commercially available probes have been successfully implemented, though detection of the virus in kidney samples has not been demonstrated. 50, 81 In summary, a significant subset of patients with COVID-19 experience AKI. Renal biopsy findings in patients with proteinuria and hematuria have demonstrated a glomerular dominant pattern of injury, most notably a collapsing glomerulopathy reminiscent of findings seen in HIVAN. In contrast, postmortem examination of kidneys from patients who died with COVID-19 show ATI as the main morphologic finding. Notably, detection of direct viral infection of the kidney parenchymal cells has not been definitely demonstrated, although clinical and histologic findings suggest this possibility, and there is no single pathognomonic lesion attributable to COVID-19. 82 Much has been learned about this virus and its effects on the human body within the first months of the current pandemic, but more studies supported by molecular and immunohistochemical tests are needed to fully understand the mechanism of renal injury in this clinical setting.

Johns Hopkins Coronavirus Resource Center

Postmortem examination of COVID-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction

COVID-19 and multiorgan response

China Medical Treatment Expert Group for Covid-19: Clinical characteristics of coronavirus disease 2019 in China

COVID-19 pathophysiology: A review

Advanced pulmonary and cardiac support of COVID-19 patients: Emerging recommendations from ASAIO-A "living working document

Respiratory virus infections: Understanding COVID-19

Coronaviruslike particles in human gastrointestinal disease. Epidemiologic, clinical, and laboratory observations

Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia

Cell entry mechanisms of SARS-CoV-2

Case-fatality rate and characteristics of patients dying in relation to COVID-19 in Italy

Should COVID-19 concern nephrologists? Why and to what extent? The emerging impasse of angiotensin blockade

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Acute kidney injury in critically ill patients with COVID-19

Role of angiotensin-converting enzyme 2 (ACE2) in COVID-19

Renal manifestations of Covid-19: Physiology and pathophysiology

Novel evidence of acute kidney injury in COVID-19

Northwell COVID-19 Research Consortium

Northwell Nephrology COVID-19 Research Consortium: Acute kidney injury in patients hospitalized with COVID-19

Fatal outcomes of COVID-19 in patients with severe acute kidney injury

Associations between blood type and COVID-19 infection, intubation, and death

The clinical implication of dynamic neutrophil to lymphocyte ratio and D-dimer in COVID-19: A retrospective study in Suzhou China

Kidney disease is associated with in-hospital death of patients with COVID-19

AKI in hospitalized patients with COVID-19

Characterization of acute kidney injury in critically ill patients with severe coronavirus disease 2019

COVID-19 and ACE2 in Cardiovascular, Lung, and Kidney Working Group: Acute kidney injury in COVID-19: Emerging evidence of a distinct pathophysiology

Renoprotective postoperative monitoring: What is the best method for computing renal perfusion pressure? An observational, prospective, multicentre study

Management of acute kidney injury in COVID-19

Is the kidney a target of SARS-CoV-2?

AKI and collapsing glomerulopathy associated with COVID-19 and APOL 1 highrisk genotype

SARS-CoV-2 infection: The role of cytokines in COVID-19 disease

Macrophages: A Trojan horse in COVID-19?

Identification of druggable inhibitory immune checkpoints on natural killer cells in COVID-19

COVID-19-associated acute kidney injury: Consensus report of the 25th Acute Disease Quality Initiative (ADQI) Workgroup

Kidney biopsy findings in patients with COVID-19

COVID-19-associated glomerular disease

Kidney biopsy findings in patients with COVID-19, kidney injury, and proteinuria

Cliniques universitaires Saint-Luc (CUSL) COVID-19 Research Group: SARS-CoV-2 causes a specific dysfunction of the kidney proximal tubule

Northwell Nephrology COVID-19 Research Consortium: COVID-19-Associated kidney injury: A case series of kidney biopsy findings

Ultrastructural evidence for direct renal infection with SARS-CoV-2

Collapsing glomerulopathy in a patient with COVID-19

Acute kidney injury due to collapsing glomerulopathy following COVID-19 infection

Tubuloreticular inclusions in COVID-19-related collapsing glomerulopathy

Collapsing glomerulopathy in a COVID-19 patient

APOL1 genetic variants in focal segmental glomerulosclerosis and HIV-associated nephropathy

APOL1 risk variants are strongly associated with HIV-associated nephropathy in Black South Africans

COVID-19-related collapsing glomerulopathy in a kidney transplant recipient

Kidney disease-associated APOL1 variants have dose-dependent, dominant toxic gain-offunction

Detection of SARS-CoV-2 in formalin-fixed paraffin-embedded tissue sections using commercially available reagents

Molecular mechanisms of injury in HIVassociated nephropathy

Anti-glomerular basement membrane disease during the COVID-19 pandemic

Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 infection

Endothelial cell infection and endotheliitis in COVID-19

Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China

Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: A case series

COVID-19 Pathology Working Group: Pathology and pathogenesis of SARS-CoV-2 associated with fatal coronavirus disease, United States

Histopathological findings and viral tropism in UK patients with severe fatal COVID-19: A post-mortem study

Pathophysiology of SARS-CoV-2: Targeting of endothelial cells renders a complex disease with thrombotic microangiopathy and aberrant immune response. The Mount Sinai COVID-19 autopsy experience. medRxiv

Histopathologic and ultrastructural findings in postmortem kidney biopsy material in 12 patients with AKI and COVID-19

Postmortem kidney pathology findings in patients with COVID-19

COVID-19 coagulopathy vs disseminated intravascular coagulation

The autopsy pathology of sepsis-related death

The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture

SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis

Membrane rearrangements mediated by coronavirus nonstructural proteins

Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex

Human coronavirus: Host-pathogen interaction

China Novel Coronavirus Investigating and Research Team: A novel coronavirus from patients with pneumonia in China

Electron microscopy of SARS-CoV-2: A challenging task

Severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States

Modern uses of electron microscopy for detection of viruses

Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2

Multivesicular bodies mimicking SARS-CoV-2 in patients without COVID-19

Visualization of putative coronavirus in kidney

Electron microscopic investigations in COVID-19: Not all crowns are coronas

Appearances can be deceiving -viral-like inclusions in COVID-19 negative renal biopsies by electron microscopy

Multicenter clinicopathologic correlation of kidney biopsies performed in COVID-19 patients presenting with acute kidney injury or proteinuria

Molecular detection of SARS-CoV-2 infection in FFPE samples and histopathologic findings in fatal SARS-CoV-2 Cases

Molecular detection of SARS-CoV-2 in formalin-fixed, paraffinembedded specimens

COVID-19 and the kidney: What we think we know so far and what we don't