Calcineurin inhibitors

Calcineurin inhibitor (CI) (cyclosporine, tacolimus) nephrotoxicity/nephropathy (CIN) was first noticed by Calne and colleagues (1978) . Within the following years, seemingly contradictory morphologic patterns of cyclosporine A toxicity were reported in humans.

An international workshop of pathologists formulated a descripive classification system. Following the nephrotoxicity workshop in Puerto Rico, a clinical classification of acute and chronic CIN was suggested. This time-related classification of CIN, however, proved to be unsatisfactory. Based on a better understanding of the significance of the pathogenic mechanisms underlying CIN the original descriptive classification was widely accepted.

CIN may be found early as well as late after the beginning of therapy. CIN is well documented in patients with bone marrow or solid organ transplants and in patients with autoimmune diseases.

CIN is dose-dependent. Factors that modulate CIN include different individual sensitivities and various additional risk factors. CIN may occur at lower dose or trough levels if individual sensitivity is high or if additional risk factors (e.g.ischaemia) are present.

We suggest two major forms of CIN:

A) Functional toxicity

B, Morphological toxicity (i.e. functional changes accompanied by morphological lesions)

1. Tubulopathy
2. Microangiopathy
3. Tubulo-interstitial lesions

Functional toxicity

Whenever a patient is treated with CI, a decrease of GFR (with or without increase of serum creatinine) and variable signs of tubular dysfunction are noted. The renal biopsy in such cases shows mostly no significant abnormalities (“Hemodynamic ATN”). Tubulonecrotic ATN may be present without any special morphological characteristics in renal transplants early after transplantation due to pre-existing ischemic damage.

Morphological toxicity: tubulopathy

Low-power magnification often shows more or less normal renal tissue. The morphologic changes seen in tubulopathy include inclusion bodies in tubular epithelial cells (corresponding to giant mitochondria), isometric tubular vacuolization, and microcalcification. The different lesions of tubulopathy are most often found in a single biopsy. They may occur, however, in any combination, and even giant mitochondria or isometric vacuolization or microcalcifications may be found alone. Vascular, glomerular and interstitial lesions are absent.

Giant mitochondria occur predominantly in the convoluted part (Sl and S2 segments) of the proximal tubule, usually one per cell. Multiple tubular inclusion bodies must be considered as phagolysosomes. Giant mitochondria are distributed inhomogeneously and are rare, even in cases of severe toxicity. Whereas some tubular cross sections may contain many giant mitochondria, adjacent ones may contain none. Giant mitochondria reach about half the size of a nucleus, are mostly round or oval, are rarely cigar-shaped, and lie adjacent to the nuclei. The morphology of giant mitochondria lacks any specificity. They usually have very few cristae and frequently contain paracrystalline inclusions, large matrix granules, and lipid droplets. Because of the rarity of cristae and their polymorphism, they are easily mistaken for lysosomes. The other mitochondria vary slightly in size and shape. In rare cases, auto-phagolysosomes contain fragments of mitochondria. Giant mitochondria are often found in the tubule lumen.

Isometric vacuolization  (“Vacuolar ATN”) is found almost exclusively in the straight part of the proximal tubule, or S3 segment, of a few nephrons. The light microscopic findings are identical to those of osmotic nephrosis. Most of the tubular cells in a cross section contain densely packed vacuoles of equal size that are free of lipids. Vacuolization is at least partly the result of dilatation of the smooth endoplasmic reticulum. Giant mitochondria and isometric vacuolization are never observed in the same cell. Sometimes the nuclei look pyknotic, and the brush border of the affected cells may be missing.

Microcalcifications of a single tubular cell or a group of them are found in various parts of the nephron. They are round, crescentic, or polycyclic in shape. Microcalcifications often contain Tamm-Horsfall protein and may also be the result of necrotic tubular cells. They are an infrequent finding by light or electron microscopy.

Toxic tubulopathy can easily be reproduced in laboratory animals. It is clearly dose dependent. In humans it is usually found with very high tough levels over a prolonged period of time. It may develop quickly (within hours) and vanish without residues within days.

Morphological toxicity: microangiopathy and tubulo-interstitial lesions

Vascular-interstitial lesions may affect either the small vessels - arterioles eventually together with the glomeruli - or the tubular-interstitial space alone or both. Vascular interstitial lesions may be classified as follows:

1. CI-associated microangiopathy
   1. CI -associated arteriolopathy: early lesions, thrombotic lesion, typical  lesion (CI-associated arteriolopathy)
   2. CI -associated glomerulopathy: thrombotic lesion, typical HUS-like lesion, segmental focal glomerular sclerosis
2. Tubular atrophy with concomitant interstitial fibrosis (striped form).

CI-associated microangiopathy predominates in the peripheral vascular tree, including the arterioles (afferent vessels) and arteries with up to two layers of smooth muscle cells. The vascular lesions sometimes extend downstream into the glomerulus and upstream into arteries close to where they branch into arterioles. Proliferative arteriopathy of interlobular or arcuate arteries or intimal mononuclear cell infiltrates are not a feature of CIN in our experience. Intimal fibrosis in arteries, however, is quite common in patients with severe and longstanding CI-associated arteriolopathy.

CI-associated arteriolopathy

Early minor lesions include vacuolization (at least partly attributable to a dilatation of the endoplasmatic reticulum), inclusion bodies resembling giant  mitochondria or giant lysosomes, single cell necrosis of endothelial or smooth muscle cells, and most often a clear cell transformation of smooth muscle cells. Except for giant vacuoles in smooth muscle cells and the clear cell transformation, the early minor lesions are hardly ever seen by light microscopy.

Thrombotic lesions-arteriolar and/or glomerular fibrin or platelet thrombi usually affect only a few arterioles or glomeruli. In rare cases all glomeruli and arterioles may be involved.

Typical CI-associated arteriolopathy occurs in two forms which may coexist. In the first form, circular nodular protein deposits may permeate the arteriolar wall and may narrow or even occlude the vascular lumen. Often, the protein deposits are arranged in a pattern resembling a pearl necklace or cloverleaf. Electron microscopy reveals that the protein deposits replace necrotic myocytes. The protein deposits consist of IgM or complement  (C3, C1q, 5b-9), or both. Fibrin, demonstrated by immunofluorescence microscopy, is present in up to 20 percent of affected patients as well. This lesion is often rather similar to severe arteriolar hyalinosis or necrosis by light microscopy. The second form, which is very rare, consists of a mucoid thickening of the intima that results in narrowing of the vascular lumen. Electron microscopy reveals thickening of the intimal layer by a loose, amorphous material. Necrotic myocytes are also present as indicated by positive immunofluorescence microscopic findings.

1.2 CI-associated glomerulopathy

In general, glomerular lesions are probably always associated with CI-associated arteriolopathy. Thus, CI-associated glomerulopathy may be interpreted as the extension of arteriolopathy into the glomerulus. The frequency of CI-associated glomerulopathy decreases in parallel with the severtity of arteriolopathy.

The morphologic pattern of cyclosporine A-associated glomerulopathy is variable:

a) fibrin or platelet thrombi;

b) pouch-lesion i.e. thrombotic and proliferative lesion at the vascular pole of the glomerulus;

c) hemolytic uremic syndrome-like lesion;

d) mesangiolysis;

e) segmental focal glomerular sclerosis (including collapsing variant);

f) lesions not encompassed by the term, cyclosporine A-associated glomerulopathy: glomerular collapse, glomerular obsolescence, thickening and multilayering of Bowman's capsular basement membrane.

The glomerular lesions (a-e) are usually focal. In some cases only a single glomerulus may be affected. The involved glomeruli are supplied by an arteriole showing CI-associated arteriolopathy. This finding may be helpful in the distinction of CI-associated glomerulopathy from glomerular lesions similar to it e.g. transplant glomerulopathy or segmental focal glomerular sclerosis as a recurrent basic disease.

Tubular atrophy and interstitial fibrosis (striped form)

Irregular foci or stripes of tubular atrophy accompanied by fibrosis are observed in the renal cortex. The atrophic tubules exhibit mostly basement membrane thickening but basement membrane thinning may also be present. Tubules in other areas appear to be essentially normal. A sparse mononuclear cell infiltrate is often seen in the fibrotic area, not only in patients with kidney transplants but also in those with autoimmune disease without primary renal involvement. Tubular atrophy and interstitial fibrosis may be associated with any of the morphologic lesions described above or may be found alone which is most often the case in patients with autoimmune diseases. Intimal fibrosis may be seen in the arteries. Glomerular changes are usually not prominent but become more common in patients with advanced interstitial fibrosis and tubular atrophy, mainly completely obsolescent glomeruli.  Hand in hand with progressive glomerular obsolescence an increase in the glomerular cross sectional area of the remaining patent glomeruli is found. Completely scarred arterioles or non-characteristic arteriolar lesions may also be found.

Differential Diagnosis and Specificity

The morphologic lesions seen in vessels, glomeruli, and tubular-interstitial space are nonspecific. The changes of CI-associated arteriolopathy are very similar or even identical to those that may be found in advanced cases of thrombotic microangiopathy, independent of CI treatment (e.g. mitomycin therapy, octreotide, radiation, etc.). The differentiation of CI-associated arteriolopathy from hypertensive or diabetic arteriolar hyalinosis is usually easy owing to the presence of focal nodular deposits that are situated inside of intact smooth muscle cells in the latter, in comparison to the deposits in CI-associated arteriolopathy, which are circular and replace necrotic smooth muscle cells. However, in advanced cases of hypertensive or diabetic arteriolopathy, or in late stages of CI-associated arteriolopathy, differentiation by light microscopy may be difficult or even impossible. Because of the rarity of CI-associated arteriolopathy in patients with autoimmune diseases and the increased frequency of unspecific arteriolar hyalinosis, the latter may be considered to be a forme fruste of the more typical lesions.

The correct interpretation of CI-associated glomerulopathy is usually easy because of the consistent correlation with CI-associated arteriolopathy. In questionable cases, serial sections may be helpful to show the co-occurrence of CI-associated arteriolopathy in individual glomeruli. In cases where the primary renal disease is a hemolytic-uremic syndrome or segmental focal glomerular sclerosis, correct interpretation of the glomerular lesions may be difficult or even impossible.

Differentiation of CI-induced vascular-interstitial lesions from vascular lesions seen in transplant rejection may be extremely difficult. Kidney transplant biopsies showing fibrin thrombi limited to the arterioles and/or glomeruli without any arterial lesions pose a special problem. In the case of concomitant arterial involvement or interstitial rejection, the thrombi are interpreted to be most likely due to rejection, in the absence of arterial involvement CI toxicity is considered to be most likely present. As a rule, predominant involvement of the arteries in patients with kidney transplants is usually attributable to rejection, whereas predominant involvement of the arterioles is more likely to be the result of CI toxicity. Interstitial fibrosis with tubular atrophy is also a non-specific renal lesion. Numerous pathogenic causes result in this type of interstitial fibrosis. It should not be attributed to CI therapy unless other pathogenic factors are excluded.

Reversibility of CIN

Concern about CI-toxicity has distressed clinicians from the beginning. Increasing experience with the use of CI in patients with non-life threatening autoimmune diseases shows that only a minority is at risk of progressive renal failure. In patients with functional toxicity  (including those with minor structural renal abnormalities possibly related to CI) the symptoms usually disappear within a few weeks after stopping CI. Complete clinical reversibility is therefore suggested as test for the absence of significant morphological abnormalities.

With respect to morphological toxicity, tubular lesions vanish rapidly as demonstrated by repeat biopsies in man and in studies of experimental animals.

Vascular lesions, however, with interstitial fibrosis and tubular atrophy were primarily considered to be irreversible. This holds true for tubular atrophy with interstitial fibrosis but not for CI-associated arteriolopathy. Repeat biopsies in patients with CI-associated arteriolopathy have shown that, after stopping or reducing the dose of CI,  the arteriolopathy may either progress to complete vascular occlusion or remodelling of the vascular lesion may take place. If complete severe circular involvement of the arteriole is present, the arteriole may either become completely scarred or eventually merge and finally vanish within the interstitial fibrosis. In the case of incomplete involvement of the arteriole, remodelling of the vascular wall may develop with progressive decrease of protein deposits, so that in the end patent arterioles are found with an increase in basement membrane material throughout the vessel wall. 

The remodelling of the vascular wall prevents the progression of tubular atrophy and interstitial fibrosis.

Even after stopping cyclosporine a progressive disease presumably related to hyperfiltration of remnant glomeruli has been observed but not well documented. In patients requiring for continuous CI therapy despite microangiopathy, progressive deterioration of renal function up to the stage of hemodialysis has been observed. Terminal renal insufficiency is usually the consequence of severe, overt haemolytic uremic syndrome-like complications of CI therapy. In heart transplant patients, repeat biopsies have shown that nephron loss progresses and the remaining glomeruli become hypertrophic. Terminal renal insufficiency is expected in 10 percent of the heart graft recipients

In summary, CI-associated arteriolopathy  is a relatively benign lesion, which is acceptable in patients with life threatening diseases. The use of the lowest possible dose of cyclosporine will prevent the development of terminal renal insufficiency in the vast majority of patients. In patients with non-life threatening autoimmune diseases, strict adherence to the recommended doses and laboratory monitoring will  prevent terminal renal insufficiency from developing.

Etiology and Pathogenesis

Molecular mechanism leading to nephrotoxicity might be shared to some extent with those pathways resulting in immunosuppression. CIN is a new type of drug toxicity which is characterized by both, vascular and tubular morphologic changes. Cyclosporine leads to a dose dependent reduction of glomerular filtration rate and renal plasma flow (functional toxicity); superimposed on functional toxicity morphological changes in the vascular-interstitial space (small vessels) and tubules may occur.

The present knowledge on the pathogenesis of cyclosporine nephrotoxicity suggests that glomerular filtration rate is mainly reduced due to a preglomerular vasoconstriction. The origin of arteriolar constriction is multifactorial: direct effect of the drug on smooth muscle cells, release of endothelial cell derived prostaglandin metabolites, PAF and endothelin as well as release of renin from the juxtaglomerular apparatus; all of these mediators result in smooth muscle contraction and possibly lead to mesangial cell contraction. Finally CI might exert at high concentration a direct cytotoxic effect on tubular cells and more importantly to endothelial and smooth muscle cells; the latter effect results in thrombocyte aggregation (hemolytic uremic syndrome) and alterations of the arteriolar wall with obliteration of the vascular lumen.

Focal tubular atrophy accompanied by interstitial fibrosis in a striped pattern is, in our opinion, the consequence of ischemic damage to the tubular interstitial space. Owing to the fact that the blood supply of the tubular interstitial space depends upon the integrity of the afferent arteriole, narrowing of the vascular lumen, and even a more complete occlusion result in ischemic damage of the corresponding nephrons.

The assumption that interstitial fibrosis develops independently of vascular lesions is based mainly on animal experiments in normotensive rats, and above all on biopsy findings in patients with heart transplants or autoimmune disease. In these studies, the  biopsies were performed late in the course of disease when florid vascular and thrombotic lesions were no longer present. In patients with kidney transplants, the sequential development of vascular lesions and interstitial fibrosis can be demonstrated in repeat biopsies. The relationship between lesions can also be inferred from the time of presentation of CI-associated arteriolopathy and interstitial fibrosis. Furthermore, this is confirmed in our material by statistical analysis showing that interstitial fibrosis (striped form) correlates best with CI-associated arteriolopathy, vascular rejection, arteriolar hyalinosis, glomerulonephritis, and segmental focal glomerular sclerosis, all of which are vascular lesions which may give rise to hypoperfusion of the tubular interstitial space.

Microangiopathy and accompanying tubular-interstitial lesions are related to CI trough levels and doses. Essential risk factors for their development include episodes of acute renal impairment (acute renal failure, increase of serum creatinine of more than 30 percent above baseline value) owing to toxic CI doses; co-medication with nephrotoxic drugs; rejection episodes; and ischemia e.g. kidney transplant recipients.The important role of risk factors in the development of vascular-interstitial toxicity may explain why the full clinical picture of hemolytic-uremic syndrome is found mainly in patients with bone marrow transplants who are exposed to multiple risk factors, including total body irradiation, cytostatic drugs, nephrotoxic co-medication or infections.

Some individuals, however, develop vascular-interstitial toxicity despite low CI blood levels and an apparent lack of other risk factors. The reasons for this variable individual sensitivity are not yet understood.