2023 Volume 30 Issue 7 Pages 720-732
Advances in immunosuppressive therapy; posttransplant management of allograft rejection; and measures against infectious diseases, cardiovascular diseases, and malignancy dramatically improved graft and patient survival after kidney transplantation (KT). Among them, kidney allograft biopsy is an important tool and the gold standard for the diagnosis of various kidney allograft injuries, including allograft rejection, virus-induced nephropathy, calcineurin inhibitor toxicity, and posttransplant glomerular diseases. The Banff Conference on Allograft Pathology has contributed to establishing the diagnostic criteria for kidney allograft rejection and polyomavirus-associated nephropathy that are used as a common standard worldwide. In addition to the for-cause biopsy, many transplant centers perform protocol biopsies in the early and late posttransplant periods to detect and treat allograft injury earlier. Preimplantation biopsy in deceased-donor KT has also been performed, especially in the marginal donor, and attempts have been made to predict the prognosis in combination with clinical information and the renal resistance of hypothermic machine perfusion. Regarding the preimplantation biopsy from a living kidney donor, it can provide useful information on aging and/or early changes in lifestyle diseases, such as glomerulosclerosis, tubulointerstitial changes, and arterial and arteriolar sclerosis, and be used as a reference for the subsequent management of living donors. In this review, morphologic features of important kidney allograft pathology, such as allograft rejection and polyomavirus-associated nephropathy, according to the latest Banff classification and additional information derived from protocol biopsy, and future perspectives with recently developed technologies are discussed.
It is well-known that patients with chronic kidney disease (CKD) and its terminal state, end-stage kidney disease, are strongly associated with arteriosclerotic disease1, 2) and poor patient survival3). Patients who reach end-stage kidney disease usually receive renal replacement therapies, including hemodialysis, peritoneal dialysis, and kidney transplantation (KT). Regarding modern KT, advances in immunosuppressive treatment have reduced the incidence and severity of early acute rejection, and posttransplant management of rejection, infectious diseases, cardiovascular diseases, and malignancies has also improved graft and patient survival. Currently, KT is considered the first choice for renal replacement therapy worldwide.
Kidney allograft biopsy is an important tool for posttransplant management and is the gold standard for the diagnosis of various allograft injuries, including acute or chronic active rejection, BK polyomavirus-associated nephropathy (BKPyVAN), calcineurin inhibitor toxicity, and recurrent or de novo glomerular diseases4-6). The Banff Conference on Allograft Pathology has established the diagnostic criteria for allograft rejection and other KT-related pathologies that are used as a common standard. In addition to the for-cause biopsy for allograft dysfunction and/or urinary abnormalities, many institutes perform protocol biopsies at 3, 6, and 12 months or later mainly to detect subclinical rejection7, 8). A baseline biopsy is performed preimplant (0-hour biopsy) or 1-hour postreperfusion (1-hour biopsy), which can also provide useful information about the donor. In addition, a baseline biopsy from a living kidney donor is a valuable opportunity to observe the effects of aging and/or lifestyle diseases without CKD on early renal pathological changes and can be used as a reference for the subsequent management of living kidney donors.
In this review, morphologic features of important kidney allograft pathology, such as acute or chronic active rejections and BKPyVAN, according to the latest Banff classification and additional information derived from protocol biopsy, and future perspectives are discussed.
Diagnosis of Kidney Allograft Rejection: Contribution of the Banff Conference on Allograft PathologyHistopathological changes associated with kidney allograft rejection have been reported since the 1960s9, 10). However, no standardized diagnostic criteria were present at that time. The Banff Conference on Allograft Pathology was founded in 1991 by a group of renal pathologists, nephrologists, and transplant surgeons11), and the conference has been held biannually thereafter. In the Banff Conference, they have discussed mainly allograft rejection by the following methods: creating working groups, proposing diagnostic criteria, verifying their validity, revising the criteria, and publishing the meeting reports.
From 1991 through 1997, the conference focused on the evaluation of individual pathological lesions and the establishment of the lesion scoring system (score 0–3), which can be used across countries and specialties: tubulitis (t), intimal arteritis (v), mononuclear cell interstitial inflammation (i), glomerulitis (g), interstitial fibrosis (ci), tubular atrophy (ct), allograft glomerulopathy (cg), mesangial matrix increase (mm), arterial fibrous intimal thickening (cv), arteriolar hyaline thickening (ah)12). They also outlined allograft pathology in six categories: category 1, normal; category 2, antibody-mediated rejection (ABMR; hyperacute and accelerate acute); category 3, borderline changes “suspicious” for acute rejection; category 4, acute/active rejection; category 5, chronic/sclerosing allograft nephropathy; and category 6, others. The Banff 97 Working Classification forms the basis of the subsequent revisions12).
Acute/active rejection in the Banff 97 classification mainly indicated acute T-cell-mediated rejection (TCMR) (Fig.1A–C), and an ABMR component should be suspected if polymorphonuclear leukocytes are confirmed in peritubular and glomerular capillaries or a type III vascular lesion (v3, fibrinoid change/transmural arteritis) is found. Diagnostic criteria for pure acute/active antibody-mediated rejection had been discussed in the 2001 Banff Conference, in which peritubular capillary staining with split C4 complement component (C4d) was accepted as a useful marker of ABMR, and classification of ABMR was established as follows: Type I: C4d+, acute tubular necrosis-like change with minimal inflammation; Type II: C4d+, capillary margination and/or thrombosis; and Type III: C4d+, transmural arteritis (v3) (Fig.2A–C)13). Until the Banff 2015 Conference, evaluation of C4d staining (immunohistochemical staining and immunofluorescence), diagnostic criteria for ABMR without C4d deposition in peritubular capillaries, and inclusion of increased expression of gene transcripts in the biopsy tissue indicative of endothelial injury were discussed and validated14). Diagnostic criteria of chronic active ABMR were also discussed and are characterized by persistent capillary endothelial cell injury resulting in a double contour of the glomerular basement membrane (by light or electron microscopic observation) and peritubular capillary basement membrane multilayering on electron microscopy (Fig.3A and 3B).
A. Severe interstitial mononuclear cell infiltration was found in more than half of the cortical area (i3 score) (PAS stain).
B. Severe tubulitis with ＞10 mononuclear cells in the tubulus (t3 score) (PAS stain).
C. Severe intimal arteritis with at least 25% narrowing of the luminal area by subendothelial edema and infiltrating mononuclear cells (arrow) (v2 score) (PAS stain).
A. Transplant glomerulitis with mononuclear infiltrate and enlargement of endothelial cells (PAS stain).
B. Severe peritubular capillaritis with ＞10 mononuclear cell infiltration (ptc3 score) (PAS stain).
C. Diffuse C4d staining in peritubular capillaries, as demonstrated by immunofluorescence microscopy (c4d3 score).
A. Double contours affecting more than 50% of peripheral capillary loops in the non-sclerotic glomerulus (cg3 score) (PAM stain).
B. Peritubular capillary basement membrane multilayering on electron microscopy (arrow) (ptcml1 score).
C. Active interstitial inflammation in the scarred area in the cortical area (i-IFTA 3 score) (PAS stain).
D. Fibrous arterial intimal thickening with mononuclear infiltrate (arrow) (cv3 score) (Masson Trichrome stain).
Since short- to medium-term kidney allograft survival has improved, the diagnosis and treatment of chronic active rejection have become more important for long-term graft survival. The Banff 2015 and 2017 Conferences mainly concentrated on the clinical outcomes of inflammation in the areas of interstitial fibrosis and tubular atrophy (i-IFTA) and its association with TCMR (Fig.3C). Inflammation involving ＞25% of areas of cortex with IFTA, corresponding to Banff 2015 i-IFTA scores 2 and 3, was associated with a high risk of graft loss15, 16). Thus, the Banff 2017 Conference revised the classification and added moderate i-IFTA plus moderate or severe tubulitis as diagnostic of chronic active TCMR17). Chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell inflammation in the fibrosis and formation of neointima) might be found in a more severe form of chronic active TCMR (Fig.3D). This arterial lesion may also be a manifestation of chronic active ABMR or mixed ABMR/TCMR. The impact of chronic active TCMR at 1-year protocol biopsy was investigated using the large retrospective cohort of two Japanese centers, and it was demonstrated that 8% of biopsies were diagnosed as chronic active TCMR. Determinants of the diagnosis were cyclosporin use, previous acute rejection, and previous BKPyVAN. Longitudinal observation revealed that chronic active TCMR had a higher risk of graft dysfunction than normal tissue, and the incidence of graft dysfunction was comparable with ABMR, BKPyVAN, and glomerulonephritis8). A summary of the Banff lesion score and the latest Banff classification is summarized in Tables 1 and 2 4).
Acute Banff scores (score 0, 1, 2, 3) |
Chronic Banff scores (score 0, 1, 2, 3) |
Acute and Chronic Banff scores (score 0, 1, 2, 3) |
|---|---|---|
| i | ci | ti |
| t | ct | i-IFTA |
| v | cv | t-IFTA |
| g | cg | |
| ptc | ptcml | |
| c4d |
i: Interstitial inflammation, t: Tubulitis, v: Intimal arteritis, g: Glomerulitis, ptc: Peritubular capillaritis, c4d: c4d deposition in peritubular capillary, ci: Interstitial fibrosis in cortex, ct: Tubular atrophy in cortex, cv: Arterial intimal fibrosis, ptcml: Peritubular capillary basement membrane multilayering, ti: Total cortical inflammation, i-IFTA: Inflammation in scarred cortex, t-IFTA Tubulitis in tubules within scarred cortex.
| Category 1: Normal biopsy or nonspecific changes |
| Category 2: Antibody-mediated changes |
| Active ABMR; all 3 criteria must be met for diagnosis |
| 1. Histologic evidence of acute tissue injury, including 1 or more of the following: |
| - Microvascular inflammation (g ＞0 and/or ptc ＞0), in the absence of recurrent or de novo glomerulonephritis, although in the presence of acute TCMR, borderline infiltrate, or infection, ptc ＞1 alone is not sufficient and g must be ＞1 |
| - Intimal or transmural arteritis (v ＞0) - Acute thrombotic microangiopathy, in the absence of any other cause |
| - Acute tubular injury, in the absence of any other apparent cause |
| 2. Evidence of current/recent antibody interaction with vascular endothelium, including 1 or more of the following: |
| - Linear C4d staining in peritubular capillaries or medullary vasa recta (C4d2 or C4d3 by IF on frozen sections, or C4d ＞0 by IHC on paraffin sections) |
| - At least moderate microvascular inflammation ([g＋ptc] ＞2) in the absence of recurrent or de novo glomerulonephritis, although in the presence of acute TCMR, borderline infiltrate, or infection, ptc ＞2 alone is not sufficient and g must be ＞1 |
| - Increased expression of gene transcripts/classifiers in the biopsy tissue strongly associated with ABMR, if thoroughly validated |
| 3. Serologic evidence of circulating donor-specific antibodies (DSA to HLA or other antigens). C4d staining or expression of validated transcripts/classifiers as noted above in criterion 2 may substitute for DSA; however thorough DSA testing, including testing for non-HLA antibodies if HLA antibody testing is negative, is strongly advised whenever criteria 1 and 2 are met |
| Chronic active ABMR; all 3 criteria must be met for diagnosis |
| 1. Morphologic evidence of chronic tissue injury, including 1 or more of the following: |
|
- Transplant glomerulopathy (cg ＞0) if no evidence of chronic TMA or chronic recurrent/de novo glomerulonephritis; includes changes evident by electron microscopy (EM) alone (cg1a) |
| - Severe peritubular capillary basement membrane multilayering (ptcml1; requires EM) |
| - Arterial intimal fibrosis of new onset, excluding other causes; leukocytes within the sclerotic intima favour chronic ABMR if there is no prior history of TCMR, but are not required |
| 2. Identical to criterion 2 for active ABMR, above |
| 3. Identical to criterion 3 for active ABMR, above, including strong recommendation for DSA testing whenever criteria 1 and 2 are met. Biopsies meeting criterion 1 but not criterion 2 with current or prior evidence of DSA (post-transplant) may be stated as showing chronic ABMR, however remote DSA should not be considered for diagnosis of chronic active or active ABMR |
| Chronic (inactive) ABMR |
| 1. cg ＞0 and/or severe ptcml (ptcml1) |
| 2. Absence of criterion 2 of current/recent antibody interaction with the endothelium |
| 3. Prior documented diagnosis of active or chronic active ABMR and/or documented prior evidence of DSA |
| C4d staining without evidence of rejection; all 4 features must be present for diagnosis |
| 1. Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or C4d ＞0 by IHC on paraffin sections) |
| 2. Criterion 1 for active or chronic active ABMR not met |
| 3. No molecular evidence for ABMR as in criterion 2 for active and chronic active ABMR |
| 4. No acute or chronic active TCMR, or borderline changes |
| Category 3: Borderline (Suspicious) for acute TCMR |
| Foci of tubulitis (t1, t2, or t3) with mild interstitial inflammation (i1), or mild (t1) tubulitis with moderate-severe interstitial inflammation (i2 or i3) No intimal or transmural arteritis (v= 0) |
| Category 4: TCMR |
| Acute TCMR |
| Grade IA: Interstitial inflammation involving ＞25% of non-sclerotic cortical parenchyma (i2 or i3) with moderate tubulitis (t2) involving 1 or more tubules, not including tubules that are severely atrophic |
| Grade IB: Interstitial inflammation involving ＞25% of non-sclerotic cortical parenchyma (i2 or i3) with severe tubulitis (t3) involving 1 or more tubules, not including tubules that are severely atrophic |
| Grade IIA: Mild to moderate intimal arteritis (v1), with or without interstitial inflammation and/or tubulitis |
| Grade IIB: Severe intimal arteritis (v2), with or without interstitial inflammation and/or tubulitis |
| Grade III: Transmural arteritis and/or arterial fibrinoid necrosis involving medial smooth muscle with accompanying mononuclear cell intimal arteritis (v3), with or without interstitial inflammation and/or tubulitis |
| Chronic active TCMR |
| Grade IA: Interstitial inflammation involving ＞25% of sclerotic cortical parenchyma (i-IFTA2 or i-IFTA3) AND ＞25% of total cortical parenchyma (ti2 or ti3) with moderate tubulitis (t2 or t-IFTA2) involving 1 or more tubules, not including severely atrophic tubules; other known causes of i-IFTA should be ruled out |
| Grade IB: Interstitial inflammation involving ＞25% of sclerotic cortical parenchyma (i-IFTA2 or i-IFTA3) AND ＞25% of total cortical parenchyma (ti2 or ti3) with severe tubulitis (t3 or t-IFTA3) involving 1 or more tubules, not including severely atrophic tubules; other known causes of i-IFTA should be ruled out |
| Grade II: Chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell inflammation in fibrosis and formation of neointima). This may also be a manifestation of chronic active or chronic ABMR or mixed ABMR/TCMR |
| Category 5: polyomavirus nephropathy |
| PVN Class 1: pvl 1 and ci 0-1 |
| PVN Class 2: pvl 1 and ci 2-3 OR pvl 2 and ci 0-3 OR pvl 3 and ci 0-1 |
| PVN Class 3: pvl 3 and ci 2-3 |
ABMR: Antibody mediated rejection, TCMR: T-cell mediated rejection, TMA: Thrombotic microangiopathy, DSA: Donor-specific antibody, HLA: Human leukocyte antigen, PVN: Polyomavirus nephropathy
Importantly, molecular diagnostics using microarray technology have significantly contributed to the validation of the diagnosis of ABMR, TCMR, viral infection, acute kidney injury, and the corresponding pathological lesions. The utility of gene expression analysis has been discussed since the 2001 Banff Conference and has been an important part of the scientific program at every meeting thereafter. The ultimate purpose of molecular diagnostics is to improve diagnostic precision, and the 2013 Banff classification officially included molecular diagnostics in the criteria of ABMR as an equivalent to C4d deposition in peritubular capillaries, based on the results that the overexpression of endothelial cell-associated transcript in the presence of donor-specific antibodies was associated with graft loss even in the absence of C4d deposition in peritubular capillaries18). Although the use of molecular diagnostics for the analysis of kidney allograft biopsies remains limited to a small number of centers, mainly in North America and Western Europe, standardization of molecular diagnostics across laboratories and multicenter validation studies, including the definition of diagnostic and clinically relevant thresholds for molecular analyses, is progressing. Most of the published studies of molecular diagnostics in the biopsy samples used microarray techniques performed on an extra biopsy core in addition to routine pathological diagnosis. More recently, the results derived from formalin-fixed paraffin-embedded section analysis have emerged, which revealed the expression of the gene sets overlapping with previously reported microarray analyses, demonstrating that significant associations between molecular and pathologic phenotypes are reported19, 20). The advantage of this technique for formalin-fixed paraffin-embedded sections is that the analysis can be performed on the same sample that is used for routine light microscopic observation. In addition, it can be used in large-scale retrospective and prospective studies with multicenter collaboration so that the associations between gene expression and clinical endpoints such as allograft loss can be investigated.
BKPyVAN: Pathology and Risk Prediction of Graft DysfunctionModern immunosuppressive regimens are more potent and T-cell-specific and have reduced the incidence of early graft loss caused by acute rejection. Conversely, infectious complications are still an important issue. Adenovirus and BKPyV are major pathogens that could directly involve kidney allograft injury. Of those, adenovirus can cause hemorrhagic cystitis and tubulointerstitial nephritis of the kidney allograft. While patients show apparent clinical symptoms such as fever, dysuria, gross hematuria, and frequency and urgency of urination, and most patients show acute allograft dysfunction, these symptoms and graft dysfunction are generally reversible21). As compared with adenovirus infection, BKPyV infection in kidney transplant patients rarely shows clinical symptoms, but the incidence of viruria, viremia, and tissue invasive nephropathy (BKPyVAN) is higher than that observed in adenovirus infection22). Kidney Disease: Improving Global Outcomes and the American Society of Transplantation (AST) Infectious Disease Community of Practice published guidelines that recommend screening for viral replication by quantitative PCR and preemptive reduction of immunosuppression in patients with viremia23, 24).
Although the detection of viremia by the PCR method is helpful, the gold standard for diagnosing BKPyVAN is a kidney allograft biopsy. On light microscopic observation, various degrees of interstitial inflammation with mononuclear cells and occasionally with plasma cells are evident. This interstitial inflammation is difficult to distinguish from acute TCMR by light microscopic observation alone. More specific findings of BKPyVAN are cytopathic changes in tubular epithelial cells caused by viral replication. These changes are associated with intranuclear inclusion bodies, tubular cell necrosis resulting in the cell shedding into the tubular lumen and denudation of the tubular basement membrane (Fig.4A and 4B). Typical cytopathic changes in tubular cells are focally observed and may cause misdiagnosis through sampling error, especially in the early stages of the disease. In addition to the light microscopic observation, most centers add immunohistochemical staining for Simian Virus 40 large T-antigen (Fig.4C), which is a more sensitive staining than the one using anticapsid protein VP1 antibody25).
A. Intranuclear inclusion bodies in the tubular epithelial cells (arrow) (H&E stain).
B. Tubular cell shedding into the lumen and denudation of tubular basement membrane (arrow) (H&E stain).
C. Positive immunohistochemical staining for SV40 large T-antigen in the infected tubular epithelial cells.
D. Electron microscopy to demonstrate intranuclear viral particles measuring 45–55 nm in diameter.
Currently, no safe and effective antiviral therapy has been established, and modification of immunosuppressive regimens remains the mainstay of treatment for BKPyVAN. As BKPyVAN is a pathological diagnosis, there has been an interest in exploring the effects of pathological findings on response to treatment and graft outcome. A composite system to stage BKPyVAN based on viral cytopathic effect, severity of inflammation, and fibrosis was first proposed by Drachenberg et al.26), and subsequently, the AST Infectious Disease Community of Practice published modifications of this scheme24). The Banff Working Group on Polyomavirus Nephropathy also proposed their first staging system in 2009 (Banff Working Proposal 2009), emphasizing the degree of virus-induced tubular epithelial injury, measured by necrosis, cell lysis, shedding into the tubular lumen, and denudation of tubular basement membranes27). Both staging systems categorize severe IFTA as stage C, which is associated with poor graft function reversal. However, regarding stages A and B, the discriminating power for serum creatinine reversal was low in both systems28), and it was necessary to reconsider the staging system.
Further statistical analysis by the Banff Working Group using a retrospective cohort of 192 patients identified two independent histological factors associated with clinical presentation: intrarenal viral load (Banff pvl score) and the extent of interstitial fibrosis (ci score). They proposed a new classification using those parameters in 2013 (Banff Working Proposal 2013, Fig.5)18) and published the final results of a multicenter study in 2018 29). They demonstrated that changes in serum creatinine levels from baseline after 12 and 24 months were significantly higher in class 3 than in classes 1 and 2. Importantly, those values were also significantly different between classes 1 and 2. Graft failure rates within 24 months were 16% in class 1, 31% in class 2, and 50% in class 3, suggesting that the Banff Working Proposal 2013 has a strong discriminating power for graft outcome in BKPyVAN30). Eventually, the proposal was incorporated into the latest Banff classification as a new category 5 (Table 2)3).
Banff classification of BKPyVAN (references 3, 27)
In modern KT, immunosuppressive protocols commonly consist of antibody induction (rabbit-derived antithymocyte globulin or IL-2 receptor antagonist) followed by a triple drug regimen consisting of calcineurin inhibitors, antimetabolites, and corticosteroids. As a result, the onset of acute rejection is delayed, and the severity is reduced. The rate of subclinical rejection without elevated serum creatinine levels is also increased. Mehta et al.30) and Nankivell and Chapman31) described literature review for subclinical rejection, which revealed that the incidence of subclinical rejection reported by different centers and eras was widely distributed from 2.6% to 60.8%, but the incidence was relatively lower in individuals receiving tacrolimus and mycophenolate mofetil-based regimens with or without corticosteroids30, 31). The incidence of subclinical rejection appeared to be multifactorial and was influenced by several clinical characteristics, such as recipient demographics, HLA mismatch counts, ABO incompatibility, timing of protocol biopsies, use or non-use of T-cell depleting antibodies, maintenance immunosuppression, and steroid therapy.
The author and coinvestigators reviewed the results of protocol biopsies performed 3 and 12 months after KT, separately for ABO blood-type compatible (N=226) and incompatible transplants (N=101) without preformed donor-specific antibodies7). At our institute, approximately 85% of KT patients underwent protocol biopsy, and more than 93% of the patients underwent induction immunosuppression with basiliximab, followed by tacrolimus, mycophenolate mofetil, and methylprednisolone for maintenance, and desensitization with a single dose (200 mg/body) and plasmapheresis for ABO-incompatible KT. The Banff 2009 classification was used for pathological interpretation. Under those uniform policies, subclinical rejection defined by Banff grades IA or higher in acute TCMR was found in 6.9% and 9.9% of patients in the ABO-compatible and ABO-incompatible KT groups at 3 months (P=0.4) and in 12.4% and 10.1% at 12 months, respectively (P=0.5). ABMR mixed with TCMR was found in only one patient who underwent ABO-incompatible KT. Those results suggested comparable allograft pathology and medium-term graft survival between ABO-compatible and ABO-incompatible KTs under desensitization with low-dose rituximab and plasmapheresis. However, subsequent Banff classifications have changed the diagnostic criteria for chronic active ABMR and chronic active TCMR and have also changed the diagnostic threshold for borderline changes, so the pathology of protocol biopsies needs to be revisited.
There have been a few studies to demonstrate the beneficial effect of subclinical TCMR on kidney allograft function. An early randomized controlled study reported by Rush et al. investigated the 72 patients who underwent either repeated protocol biopsy at 1, 2, 3, 6, and 12 months (biopsy arm) or at 6 and 12 months (control arm) and treated subclinical rejection based on the biopsy findings. A 2-year follow-up revealed better allograft function in the biopsy arm32). Another randomized study conducted by Kurtkoti et al. investigated 102 living-donor kidney transplant patients randomly assigned to receive protocol biopsies or for-cause biopsies only. Although the incidence of clinically evident rejection episodes was similar between the two groups, the biopsy group showed a lower serum creatinine level at 6 months and 1 year33). Szederkényi et al. also reported the results of their single-center randomized trial, consisting of 113 patients in the protocol biopsy group and 51 in the control group. The protocol biopsy group revealed significantly better graft function at 3 years and better graft survival at 5 years than the control group34).
Those findings suggest that intensive protocol biopsy and treatment of subclinical TCMR might be beneficial. However, those randomized trials have several limitations, such as a small sample size, a lack of long-term follow-up, and differences in immunosuppressive therapy depending on the era. Mehta et al. simulated various potential scenarios (incidence of clinical TCMR, incidence of subclinical TCMR, and reduction rate of event with an intervention) and sample size calculations and estimated that at least 294 and possibly as many as 3,213 cases will be required based on 80% power and 5% type I error30). The incidence of subclinical ABMR is relatively rare compared to that of subclinical TCMR and is found in patients with ABO- and HLA-incompatible KTs. Although the study of the treatment of subclinical ABMR is limited, Parajuli et al. conducted a single-center retrospective study and demonstrated that patients with subclinical ABMR who are treated with bolus steroids, plasmaphereses, and intravenous immunoglobulins show good graft survival that is similar to that of donor-specific antibody-positive and no rejection patients35). Conversely, the prevalence of borderline changes is higher, and the significance of antirejection treatment for subclinical borderline changes is also to be clarified. Currently, the Treatment of Early Borderline Lesions in Low Immunological Risk Kidney Transplant Patients (TRAINING) trial (clinicaltrials.gov: NCT04936282) is undergoing36).
Significance of Preimplantation Biopsy in Deceased- and Living-Donor KTsShortage of kidney donors is a serious problem worldwide, and expanded criteria donors (ECDs, including those aged ＞60 years or 50–59 years and meeting at least two of the following criteria: cerebrovascular death, history of hypertension, or last serum creatinine of ＞1.5 mg/dL) and kidney donor profile index (KDPI, evaluating 10 donor-related factors including age, height, weight, history of diabetes and hypertension, serum creatinine, hepatitis C status, ethnicity, cause of death, and donation after cardiac death) are mainly used as criteria for predicting the prognosis after KT and evaluating whether the deceased-donor is suitable for transplantation or not37). Preimplantation biopsy is performed in many kidney transplant centers as an important predictive tool, as well as renal resistance to hypothermic machine perfusion38). In deceased-donor KT, preimplantation biopsy is recommended when the deceased-donor meets the criteria of ECD or a high KDPI. There are many studies investigating the correlation between preimplantation biopsy findings, clinical features of the donor, and outcomes after KT. Those studies mainly focused on global glomerulosclerosis, IFTA, arteriolar hyalinosis, and arterial fibrous intimal thickening, and some of them created composite scoring systems and demonstrated their predictive value on graft survival39-41). Preimplantation biopsy was also discussed at the Banff Conference. It created a preimplantation biopsy working group and published their discussion of various issues and limitations, such as tissue sampling, interobserver reproducibility of individual lesions, histopathological factors associated with graft function, comprehensive clinical evaluation using KDPI, urinary biomarkers, training of general pathologists to read donor biopsies, and adoption of rapid formalin-fixation and paraffin-embedding protocols42).
In the living-donor KT, kidneys are donated by non-CKD, healthy individuals who undergo detailed preoperative evaluation43). Thus, a preimplantation biopsy is not conducted to determine whether to proceed with transplantation. Instead, living kidney donors will survive for a long time after donation, so we consider the donors as patients with newly developed CKD due to kidney donation, assess possible complications based on the implantation biopsy, and continue a careful follow-up. A baseline biopsy may be performed as a preimplantation (0-hour biopsy) or postreperfusion (1-hour biopsy), depending on the institute. In the latter, ischemia–reperfusion injury and, rarely, early changes in ABMR can be observed. Tissue sampling is performed by either wedge biopsy or needle core biopsy. Different from the deceased-donor KT, there is no urgent need for evaluation, so formalin-fixed paraffin-embedded sections are evaluated, and the quality of the specimen is good. Histopathological evaluation also includes global glomerulosclerosis, interstitial inflammation, IFTA, arterial fibrous intimal thickening, arteriolar wall thickening, and hyaline changes possibly associated with aging, hypertension, and other lifestyle diseases of the living-donor. Although rare, subclinical glomerular diseases might be diagnosed on baseline biopsy44), and latent glomerular IgA and C3 deposition are often observed45). As in the deceased-donor KT, there have been several observational studies that examined the relationship between pathological lesions in the donor kidney and allograft function46) and recipients’ posttransplant anemia47).
Preimplantation biopsy in living-donor KT is a valuable opportunity to estimate predonation clinical settings and early histopathological changes. The author and coinvestigators retrospectively analyzed the specific histopathological findings of preimplantation biopsies from living kidney donors. We identified renal arteriolar hyaline changes in 158 (40.2%) and arteriolar wall thickening in 148 (37.6%) among 393 biopsy samples from living kidney donors and demonstrated that serum uric acid concentration was the independent risk factor of arteriolar hyaline changes after the multivariable adjustment (odds ratio of quartile 4 versus quartile 2 [reference], 2.22; 95% confidence interval, 1.17–4.21). Importantly, the serum uric acid level of quartile 4 corresponds to ＞6.5 mg/dL in males and ＞5.1 mg/dL in females, suggesting that the development of arteriolar injury could be influenced by a high normal range of serum uric acid levels even in non-CKD individuals, beyond conventional atherosclerotic risk factors48). Another study focused on the global glomerulosclerosis and predonation left ventricular hypertrophy of the living kidney donor. We categorized 238 preimplantation biopsies into tertiles according to the percentage of global glomerulosclerosis. The left ventricular mass index was measured by echocardiography. Donors with high tertiles (global glomerulosclerosis ≥ 11.77%) had a sevenfold greater risk of having left ventricular hypertrophy than those with low tertiles (0%–3.45%), even after adjusting for age, gender, systolic blood pressure, history of diabetes, total serum cholesterol, and measured glomerular filtration rate by radioisotopic technique49). Those findings suggest that renal impairment associated with lifestyle diseases and cardiac hypertrophy associated with cardio-renal syndrome could develop early even in individuals without meeting the criteria for CKD. If a baseline kidney biopsy reveals any of those findings, careful and long-standing management of the living kidney donor is necessary.
Future PerspectivesCurrent diagnostics for kidney allograft biopsy have developed over the more than 30-year history of the Banff Conference on Allograft Pathology. In addition to conventional diagnostics using light microscopy, immunofluorescence studies, and electron microscopy, they have discovered molecular diagnostics. In the 2013 classification, increased expression of endothelial gene transcript was incorporated into the criteria of ABMR for the first time, and molecular diagnostics of TCMR, calcineurin inhibitor toxicity, and viral infections (BKPyV, cytomegalovirus, Epstein–Barr virus) have also been investigated. Recently, the Banff NanoString consortium established a consensus-based and commercially available standardized Banff Human Organ Transplant (B-HOT) discovery gene panel that can be reproducibly applied to formalin-fixed and paraffin-embedded samples across organs and in multicenter studies. They also suggested that data integrated platform (DIP) design consists of three elements: 1) data production (histology, molecular, and clinical), 2) DIP (web interface, cloud computing) to centralize, check, and validate data, and 3) results production by participating physicians or scientists using built-in analytical tools50).
In addition to tissue-based diagnostics, the discovery of non-invasive molecular markers for effective diagnosis of kidney allograft rejection is of interest and an active field of research. Several body fluid assays, such as kSORT (kidney solid organ response test, comprising peripheral blood transcriptome assessment), chemokines CXCL9 and CXCL10, and donor-derived cell-free DNA, for diagnosing kidney allograft rejection from peripheral blood or urine sediment, have been launched commercially51). Thus far, the sensitivity and specificity of those assays are insufficient to allow using them as a replacement for allograft biopsy, as none of the suggested markers appear to be sufficiently specific for the phenotypic heterogeneity of transplant pathology.
Digital pathology has become increasingly common in pathology practice, not only in the diagnosis of biopsy samples but also for research purposes. At the 2019 Banff Conference, a digital pathology working group was formed. Digital pathology refers to a broad collection of computerized techniques applied to pathology, including whole slide imaging, algorithms of morphometric analysis, algorithms employing artificial intelligence or machine learning, and natural language processing. According to the survey in the international transplant pathology community conducted by the Banff digital pathology working group, routine scanning of whole slide images was performed in 71% of the centers, a digital pathology image analysis algorithm to examine certain features was available in 24%, and artificial intelligence or machine learning in the analysis of digital pathology images was available in 12%52). They also described their future plans: practice standardization, integrative approaches for study classification, scoring of histologic parameters (e.g., IFTA and inflammation), algorithm classification, and precision diagnosis (e.g., molecular pathways and therapeutics)52). Several studies investigating the utility of immunohistochemical staining, whole-scan images, and an automated image analysis algorithm for individual pathological lesions have already been published53, 54), and further development in this field is expected.
The author thanks Drs. Akihiro Tsuchimoto, Kaneyasu Nakagawa, Yuta Matsukuma, Kenji Ueki and Eri Ataka from Kyushu University for providing pathological images of kidney allograft biopsy in this article.
The author declares to have no relevant financial interests.
