Primer: histopathology of calcineurin-inhibitor toxicity in renal allografts Peter Liptak and Bela Ivanyi* SUMMARY Calcineurin inhibitors (ciclosporin and tacrolimus) can cause acute and chronic nephrotoxicity. The serum levels of these drugs do not correlate well with the extent of renal damage caused, and the clinical manifestation is nonspecific. Renal biopsy is a reliable tool with which to diagnose calcineurin-inhibitor-induced nephrotoxicity. Ciclosporin and tacrolimus produce identical lesions, which are focal in nature and can be overlooked, necessitating the evaluation of serial tissue sections. Acute toxicity is characterized histologically by necrosis and early hyalinosis of individual smooth muscle cells in the afferent arterioles, and/or isometric vacuolation of the proximal straight tubules; thrombotic microangiopathy is a rare manifestation. In chronic toxicity, the damaged media smooth muscle cells in afferent arterioles are replaced by beaded medial hyaline deposits that bulge into the adventitia; the interstitium displays striped fibrosis and tubular atrophy. As maintenance doses of calcineurin inhibitors in renal transplant recipients have been lowered during the past decade, the incidence of acute toxicity has decreased markedly. Chronic toxicity, however, is still prevalent, and causes chronic allograft damage. KEYWORDS arteriolopathy, calcineurin inhibitor, ciclosporin, nephrotoxicity, tacrolimus REVIEW CRITERIA Terms used for Pubmed searches were as follows: arteriolopathy, arteriolar hyalinosis, calcineurin inhibitor nephrotoxicity, ciclosporin, haemolytic uraemic syndrome, histology, morphology, renal allograft, tacrolimus, thrombotic microangiopathy, tubulopathy. CME P Liptak is currently a trainee at the Glasgow School of Pathology, Glasgow, UK, and B Ivanyi is Professor of Pathology in the Department of Pathology at the University of Szeged, Szeged, Hungary. Correspondence *Department of Pathology, University of Szeged, Allomás utca 2, Szeged H-6720, Hungary ivanyi@patho.szote.u-szeged.hu Received 5 September 2005 Accepted 8 May 2006 www.nature.com/clinicalpractice doi:10.1038/ncpneph0225 This article offers the opportunity to earn one Category 1 credit toward the AMA Physician s Recognition Award. INTRODUCTION The introduction in 1983 of a new class of immuno suppressive agents, the calcineurin inhibitors (CNIs), has dramatically improved renal allograft survival rates. The two agents in this class, ciclosporin and tacrolimus, prevent T-cell activation by inhibiting calcineurin. Calcineurin is a cytoplasmic phosphatase required for the transcription of interleukin-2 and other cytokines that are upregulated when antigen is presented to T cells. Unfortunately, the CNIs also have adverse effects, including nephro toxicity, hypertension, hyperlipidemia, glucose intolerance, hirsutism and gum hypertrophy. Combination therapy has been used to achieve adequate immunosuppression and to lower the risk of drug-related toxicity. The most frequently administered regimens include a corticosteroid plus either ciclosporin or tacrolimus plus a lymphocyte-proliferation inhibitor (e.g. mycophenolate mofetil). As CNI nephrotoxicity can occur even when the serum concentration of the drug is within the therapeutic range, toxicity-induced renal impairment can be diagnosed only if a renal biopsy is performed. During standard evalua tion (of two tissue cores whenever possible) special light microscopic staining of serial sections and immunostaining for complement (C)4d are used. To extract optimum interpretative power from histological changes, comparison with a time-zero biopsy is strongly advised. 1 3 Some pathologists also perform elastin staining with the full immuno fluorescence panel (IgG, IgA, IgM, C3, C1q, and fibrinogen), and investigate upregula tion of tubular human leukocyte antigen (HLA)-DR. 4 5 These additional stains sometimes provide extra diagnostic information. Sampling of tissue for electron microscopy is recommended if there is a clini cal suspicion of de novo or recurrent glomerular 398 NATURE CLINICAL PRACTICE NEPHROLOGY JULY 2006 VOL 2 NO 7
disease. Ultrastructural examination of glomerular and peritubular capillaries facilitates more definitive identification of lesions of chronic rejection, 6 but it is not routinely performed because of cost benefit concerns. This introductory survey will discuss the histopathology, clinicopathological aspects and differential diagnostics of CNI nephrotoxicity. Ciclosporin and tacrolimus nephrotoxicity occur in acute and chronic forms that can affect arterioles, glomeruli, tubules and interstitium. These drugs apparently produce identical lesions. 7 The pathogenetic pathways underlying CNI nephrotoxicity are complex, and beyond the scope of this paper. ACUTE CNI-INDUCED NEPHROTOXICITY Acute CNI-related nephrotoxicity can take the form of functional toxicity, delayed recovery from post-transplantation acute tubular necrosis (ATN), toxic tubulopathy or vascular toxicity. 4,8,9 Functional toxicity CNIs administered at pharmacological doses can cause vasoconstriction, a drop in glomerular filtration rate and elevation of serum creatinine level, in the absence of structural abnormalities in the biopsy specimen. This condition is most common in the early post-transplantation period. Clinical correlation: functional toxicity is reversible in response to a lowering of the dose of the drug. Delayed recovery from post-transplantation ATN Prolonged warm or cold ischemia causes ATN in renal allografts, and manifests as oligoanuria in the immediate postoperative period. This condition is a common complication of cadaveric renal transplantation. Morphological features include dilation and flattening of tubular profiles, loss of brush borders, necrosis of individual tubular epithelial cells, cytoplasmic basophilia and enlarged regenerative nuclei. Desquamation of tubular epithelial cells into the tubular lumen might be observed. Tubular changes are accompanied by interstitial edema and sparse lymphocytic infiltrates. 4 Allografts with any degree of ATN are markedly sensitive to CNIs, which can prolong recovery from the ATN. Biopsy findings of CNI-induced nephrotoxicity are the same as those of post-transplantation ATN (above). Clinical correlation: patients are maintained on dialysis until urine output has been restored and renal function recovered. Because of the potential for CNIs to exaggerate ischemic tubular damage, a drug of this class is introduced only when allograft function has been established. Toxic tubulopathy Patients with CNI-induced toxic tubulopathy present with acute allograft dysfunction, usually associated with an elevated serum drug level. As a consequence of the lowering of maintenance CNI doses over the past decade, the incidence of tubulo pathy has fallen markedly. Morphologically, isometric tubular vacuolation is observed focally, predominantly affecting the proximal straight tubules (Figure 1). The vacuoles are small, clear and evenly distributed throughout the cytoplasm. Ultrastructurally, the vacuoles correspond to dilations of the endoplasmic reticulum. 10 Focal tubular calcification might also be present. The features of this type of tubulopathy are indistinguishable from those of radiocontrast nephrotoxicity and osmotic nephrosis, conditions to be considered when making the diagnosis. Clinical correlation: toxic tubulopathy can be reversed by lowering the CNI dose. Vascular toxicity CNIs can have a direct toxic effect on endothelial cells, with or without the involvement of platelet aggregation. Two types of vascular toxicity have been identified, acute arteriolopathy and thrombotic microangiopathy. Acute arteriolopathy Patients present with acute allograft dysfunction. The serum level of CNI is usually elevated. On biopsy, lesions are confined to afferent arteriolar profiles. There is swelling and vacuolation of endothelial cells, vacuolation, necrosis and/or apoptosis and sometimes dropout of individual myocytes, and early replacement of damaged myocytes with rounded plasma protein insudates (hyalinization). Immunohistochemically, the insudates are positive for IgM and C3. Acute arteriolo pathy and toxic tubulopathy can co-exist. Clinical correlation: the clinical outcome of CNI-induced acute arteriolopathy varies. Some patients recover renal function after the dose of CNI has been reduced, while in others renal damage is irreversible. In clinical practice, the diagnosis of arteriolopathy frequently leads to cessation of CNI administration. JULY 2006 VOL 2 NO 7 LIPTAK AND IVANYI NATURE CLINICAL PRACTICE NEPHROLOGY 399
Box 1 Summary of lesions of acute calcineurininhibitor-induced nephrotoxicity, acute rejection and post-transplantation acute tubular necrosis. Acute calcineurin-inhibitor-induced nephrotoxicity Acute afferent arteriolopathy: endothelial swelling/vacuolation; necrosis and early-stage hyaline replacement of individual myocytes Tubulopathy: small, evenly distributed vacuoles mainly in the proximal straight tubules Thrombotic microangiopathy (rare) Acute cellular rejection Interstitial infiltrates of activated lymphocytes Lymphocytic tubulitis; HLA-DR expression in tubules Figure 1 Acute calcineurin-inhibitor-induced nephrotoxicity. Isometric vacuolation of proximal straight tubular profiles (arrows). Trichrome; 200. Lymphocytic intimal arteritis Acute humoral rejection Fibrinoid necrosis of small arteries and/or afferent arterioles Glomerular microthrombi Neutrophils in peritubular capillaries Ischemic tubular damage Complement 4d localized along peritubular capillaries Acute tubular necrosis Dilation and flattening of tubules Loss of brush border Necrosis and desquamation of individual tubular epithelial cells Figure 2 Ciclosporin-induced thrombotic microangiopathy. Hyaline thrombi in the lumen of glomerular capillary loops (arrows). Hematoxylin eosin; 400. Enlarged regenerative nuclei Interstitial edema Thrombotic microangiopathy A rare manifestion of CNI-related vascular nephrotoxicity is a clinical picture resembling the hemolytic uremic syndrome. Biopsy reveals thrombotic microangiopathy (TMA), characterized by thrombi in the lumina of arterioles and glomerular capillaries (Figure 2). Pronounced arterial intimal changes are not typical of CNIinduced TMA. It is important to bear in mind, however, that CNI-induced TMA cannot be differentiated from other forms of TMA on the basis of morphology alone. The following possibilities should therefore be considered: acute humoral rejection (C4d positivity), bacterial or viral infection, recurrent hemolytic uremic syndrome, and anticardiolipin antibody-induced TMA. Clinical correlation: diagnosis of CNI-induced TMA should prompt withdrawal of the drug because, if lesions are widespread and associated with extensive arterial mural necrosis and luminal thrombosis, graft loss develops. Differential diagnosis The main clinical conditions to be differ entiated from acute CNI-induced nephrotoxicity are acute rejection, post-transplantation ATN (Box 1) 400 NATURE CLINICAL PRACTICE NEPHROLOGY LIPTAK AND IVANYI JULY 2006 VOL 2 NO 7
Figure 5 Interstitial fibrosis and tubular atrophy in a band-like pattern indicative of chronic calcineurin-inhibitor-induced nephrotoxicity (area between the arrows). Hematoxylin eosin; 100. Figure 3 Chronic calcineurin-inhibitor-induced nephrotoxicity. Beaded medial hyalinosis in afferent arteriolar profiles (arrow). Periodic acid- Schiff; 400. H E and, on occasion, acute pyelonephritis. Carefully derived clinical pathological correlations are required to ensure correct diagnosis. Acute rejection Acute rejection of a grafted kidney can be cellular and/or humoral. Cellular rejection is characterized by the presence of interstitial infiltrates predominantly composed of activated lymphocytes, tubulitis (lymphocytes infiltrate the tubular wall) and upregulation of HLA-DR by tubular epithelial cells. Lymphocytic intimal arteritis, also termed endothelialitis, might be A H A 5μm Figure 4 Hyaline arteriolopathy on electron microscopy. Endothelial cells are swollen. Hyaline material is present subendothelially and at sites where media smooth muscle cells have dropped out previously. Hyaline material bulges into the adventitia (asterisk). 2,500. Abbreviations: A, apoptotic myocytes; E, endothelial cells; H, hyaline material. E * evident. Diagnosis of humoral rejection is based on histological features (ATN-type tubular damage, neutrophilic peritubular capillaritis or fibrinoid necrosis of the arteries) and bright, linear C4d-positivity along the peritubular capillaries. 1 4 Post-transplantation ATN See section above. Acute pyelonephritis This condition is characterized by an interstitial infiltrate of neutrophils and monocytes macrophages, and is associated with neutrophilic tubulitis and neutrophilic casts. CHRONIC CNI-INDUCED NEPHROTOXICITY This form of CNI-related toxicity occurs several months after renal transplantation. Characterized by a slow, insidious increase in the serum creatinine level, patients usually have hypertension and sometimes moderate to nephrotic-range proteinuria. Some patients have an elevated serum level of ciclosporin or tacrolimus. The incidence of chronic CNIinduced nephrotoxicity increases with time since transplantation. 11 The histological indicators of chronic CNI-induced nephro toxicity are hyaline arteriolopathy, striped interstitial fibrosis and tubular atrophy. 4,9,12 Hyaline arteriolopathy This lesion is the marker of chronic CNIinduced nephrotoxicity. It affects the afferent arterioles and distal portions of small interlobular arteries. Damaged smooth muscle cells are replaced by beaded hyaline deposits on the outer aspect of the media that bulge into the adventitia (Figures 3 and 4). Beaded JULY 2006 VOL 2 NO 7 LIPTAK AND IVANYI NATURE CLINICAL PRACTICE NEPHROLOGY 401
medial hyalinosis is focal and can be overlooked as a result. Evaluation of two cores of kidney tissue and serial sections overcomes the problem of sampling error. In advanced cases, the entire wall is replaced by the hyaline material and the lumen is severely narrowed. Immunohistochemistry detects IgM and C3 at the sites of insudation. Staining is in a pearl necklace pattern. Hyaline arteriolopathy must be differentiated from the arteriolar hyalinosis that is a feature of aging, hypertension and diabetes. In these three conditions, hyalinosis is primarily subendothelial and rarely extends into the adventitia, and necrosis of myocytes is not observed. Interstitial fibrosis and tubular atrophy In chronic CNI-induced nephrotoxicity, the interstitium shows prominent patchy fibrosis and corresponding tubular atrophy, with preferential and early involvement of the medullary rays. This results in a band-like pattern (Figure 5). This striped pattern is characteristic of, but not specific to, chronic CNI-induced nephro toxicity; hyper tensive kidney disease can produce a similar lesion. Glomerular changes The effects of chronic CNI-induced toxicity on glomeruli tend to be nonspecific. Compensatory glomerular hypertrophy, mesangial matrix expansion, capillary collapse, and focal segmental and/or focal global sclerosis can be observed. Focal segmental glomerulo sclerosis seems to result from hyperfiltration in response to loss of functioning nephrons, rather than being a direct effect of toxicity to podocytes and/or endo thelial cells. Focal segmental glomerulosclerosis related to CNI use should be distinguished from recurrent or de novo idiopathic focal sclerosis. The presence of hyaline arteriolopathy, striped fibrosis and tubular atrophy with or without isometric vacuolation of proximal straight tubules, and absence of fullblown nephrotic syndrome are evidence against idiopathic focal sclerosis. Clinical correlation: chronic CNI-induced kidney damage is irreversible. Appropriate management is dose reduction or discontinuation of ciclosporin or tacrolimus, and administration of alternative immunosuppressive agents. Differential diagnosis The main clinical conditions to be differentiated from chronic CNI-induced nephrotoxicity are chronic rejection/chronic allograft nephro pathy (CAN), de novo or recurrent glomerulo nephritis, and polyomavirus nephropathy. The symptoms of chronic CNI-induced nephrotoxicity are similar to those of chronic rejection/can. Chronic rejection involves ongoing, smoldering damage to the allograft, mediated by cellular and/or humoral alloimmune mecha nisms. In addition to chronic allo immune damage to renal parenchyma, a variety of non-immunological factors (e.g. advanced donor age, ischemic injury to the graft during implanta tion, hypertension, chronic CNI-induced nephro toxicity, infection, increased ureteral pressure, hyperfiltrating glomeruli) deplete nephrons. Alloimmune and non-immunological mechanisms act in parallel. Ultimately, these processes cause sclerosing changes in arteries and glomeruli, interstitial fibrosis and tubular atrophy. As chronic rejection, chronic CNI-induced nephrotoxicity, hypertensive vascular disease and chronic infection and/or reflux cannot always be distinguished in the biopsy specimen, the panel that compiled the Banff classification of kidney transplant pathology 1 coined the term CAN to emphasize the multi factorial nature of transplantation-related chronic renal injury. Presently, there is no therapeutic consequence by which chronic rejection can be distinguished from CAN. The usual diagnostic label is either chronic rejection/can or CAN with or without features of chronic rejection. 3,4 Some pathologists believe that use of the term CAN as a histological descriptor should be restricted as much as possible because it generates uncertainty rather than precision. Box 2 summarizes the main features of chronic CNI-induced nephro toxicity, chronic rejection and CAN. Chronic rejection A diagnosis of chronic allograft rejection is made 4,6 if there are light microscopic and/or ultrastructural and/or immunohistochemical signs of chronic alloimmune injury; that is, transplant arteriopathy (new-onset intimal fibrosis with or without foam cells and/or mononuclear cells in the intima), transplant glomerulopathy (thickening and double contours of the glomerular basement membrane), transplant capillaropathy (peritubular capillary profiles with five or more circumferential basement 402 NATURE CLINICAL PRACTICE NEPHROLOGY LIPTAK AND IVANYI JULY 2006 VOL 2 NO 7
Box 2 Summary of lesions of chronic calcineurin-inhibitor-induced nephrotoxicity, chronic rejection and chronic allograft nephropathy. Chronic calcineurin-inhibitor-induced nephrotoxicity Hyaline arteriolopathy: myocytes are replaced by beaded hyaline deposits that bulge into the adventitia Nonspecific segmental or global glomerular sclerosis Striped interstitial fibrosis and tubular atrophy Chronic rejection Transplant arteriopathy: new-onset intimal fibrosis; foam cells and/or mononuclear cells in the intima Transplant glomerulopathy: double-contoured glomerular capillaries Transplant capillaropathy: peritubular capillaries with five or more circumferential basement membrane layers on electron microscopy; complement 4d positivity Interstitial fibrosis and tubular atrophy Chronic allograft nephropathy Intimal fibroelastosis in arteries No change, or subendothelial hyalinosis, in arterioles Nonspecific segmental or global glomerular sclerosis Interstitial fibrosis and tubular atrophy membrane layers detected by electron microscopy), and C4d-positivity along the thickened walls of peritubular capillaries. The interstitium is fibrotic and tubules are atrophic. Chronic rejection is frequently accompanied by active cellular and/or humoral rejection. Chronic CNI-induced nephrotoxicity and rejection can co-exist. Verification of marker lesions facilitates correct reporting of coincidental conditions. CAN CAN is diagnosed 6 if there is intimal fibroelastosis in arteries, arterioles exhibit no or eccentric subendothelial hyalinosis, glomeruli are sclerosed in a nonspecific segmental or global pattern, the interstitium is fibrotic, and tubules in the fibrotic areas are atrophic. Recurrent or de novo glomerulonephritis These forms of glomerulonephritis can be readily detected if a full immunohistochemical panel is applied and electron microscopy performed. Polyomavirus nephropathy This form of renal damage is characterized by viral cytopathic effects in tubules (inclusion bodies and tubular epithelial injury), a varying degree of interstitial inflammation, interstitial fibrosis and tubular atrophy. 4 CONCLUSIONS CNI-induced nephrotoxicity is a clinicopathological entity. The histological features indicative of acute toxicity are necrosis and dropout of individual myocytes, onset of replacement of these myocytes with hyaline insudates in afferent arterioles, and isometric vacuolation, predominantly in the straight proxi mal tubules. A TMA-like presentation is rare. In chronic toxicity, beaded medial hyalinization of afferent arterioles, striped interstitial fibrosis and tubular atrophy are observed. Careful analysis of the morphology of arteriolar lesions is necessary, and comparison with time-zero biopsies is advised. KEY POINTS Immunosuppressants of the calcineurin inhibitor (CNI) class (tacrolimus and ciclosporin) can have acute and chronic deleterious effects on transplanted kidneys Renal biopsy and microscopy of serial tissue sections provide information that is essential to diagnosis of CNI-induced nephrotoxicity Histological features of acute CNI-induced nephrotoxicity include early-stage hyalinization and dropout of individual myocytes in afferent arterioles, and isometric vacuolation of proximal straight tubules Histological features of chronic CNI-induced nephrotoxicity include replacement of myocytes in afferent arterioles with beads of hyaline that bulge into the adventitia, striped interstitial fibrosis and tubular atrophy References 1 Solez K et al. (1993) International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int 44: 411 422 2 Racusen LC et al. (1999) The Banff 97 working classification of renal allograft pathology. Kidney Int 55: 713 723 JULY 2006 VOL 2 NO 7 LIPTAK AND IVANYI NATURE CLINICAL PRACTICE NEPHROLOGY 403
Acknowledgments The nephropathological activities of B Ivanyi are supported by the Hungarian Scientific Research Fund (OTKA) via grant T-038271. Competing interests The authors declared they have no competing interests. 3 Racusen LC et al. (2003) Antibody-mediated rejection criteria an addition to the Banff 97 classification of renal allograft rejection. Am J Transplant 3: 708 714 4 D Agati VD et al. (Eds; 2005) Pathology of renal transplantation. In AFIP Atlas of Non Tumor Pathology: Non-Neoplastic Kidney Diseases, 667 709 Silver Spring: ARP Press 5 Nickeleit V et al. (1998) Histological characteristics of interstitial renal allograft rejection. Kidney Blood Press Res 21: 230 232 6 Ivanyi B (2003) Transplant capillaropathy and transplant glomerulopathy: ultrastructural markers of chronic renal allograft rejection. Nephrol Dial Transplant 18: 655 660 7 Solez K et al. (1998) Histopathologic findings from 2-year protocol biopsies from a US multicenter kidney transplant trial comparing tacrolimus versus cyclosporine: a report of the FK506 Kidney Study Group. Transplantation 66: 1736 1740 8 Mihatsch MJ et al. (1994) Cyclosporine nephropathy. In Renal Pathology With Clinical and Functional Correlations, edn 2, 1555 1586 (Eds Tisher CC and Brenner BM) Philadelphia: JB Lippincott 9 Colvin RB (1998) Renal transplant pathology. In Heptinstall s Pathology of the Kidney, edn 5, 1409 1540 (Eds Jennette JC et al.) Philadelphia: Lippincott-Raven 10 Mihatsch M et al. (1988) Cyclosporine nephrotoxicity. Adv Nephrol Necker Hosp 17: 303 320 11 Nankivell BJ et al. (2004) Calcineurin inhibitor nephrotoxicity: longitudinal assessment by protocol histology. Transplantation 78: 557 565 12 Mihatsch MJ et al. (1994) Cyclosporin A nephropathy: standardization of the evaluation of kidney biopsies. Clin Nephrol 41: 23 32 404 NATURE CLINICAL PRACTICE NEPHROLOGY LIPTAK AND IVANYI JULY 2006 VOL 2 NO 7