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Clinical Transplantation

Calcineurin Inhibitor Nephrotoxicity: Longitudinal Assessment by Protocol Histology

Nankivell, Brian J.1,5; Borrows, Richard J.1,2; Fung, Caroline L.-S.3; O'Connell, Philip J.1; Chapman, Jeremy R.1; Allen, Richard D. M.4

Author Information
doi: 10.1097/01.TP.0000128636.70499.6E

Abstract

The introduction of cyclosporine (CsA) has revolutionized solid organ transplantation, with 1-year cadaveric graft survival now exceeding 90%, allowing transplantation of nonrenal organs and making calcineurin inhibitors (CNI) the backbone of modern immunosuppression. Nevertheless, reservations have been consistently raised about their potential long-term nephrotoxicity in kidney transplantation (1–3). The initial gains of reduced rejection and improved early graft survival with CsA therapy appear to have dissipated with longer follow-up (4,5), and elimination of CsA with sirolimus regimens has resulted in improved allograft function and structure.

Chronic allograft nephropathy (CAN) causes the majority of kidney graft losses and probably represents a nonspecific end-pathway of tubulointerstitial and glomerular damage resulting from a variety of insults to the transplanted kidney (6). The contributions of CsA or tacrolimus nephrotoxicity, relative to other immunologic or nonimmunologic risk factors, are poorly documented. The clinical approach taken to a kidney transplant recipient whose serum creatinine is progressively rising is dependent on whether true immune-mediated chronic rejection, calcineurin nephrotoxicity, or some other factor is the cause of functional decline. Confusion arises from the wide disparity in estimated prevalence of CsA nephrotoxicity (7–9) and the limited number of histologic studies. A single biopsy from a failing kidney transplant may miss transient diagnostic features such as arteriolar hyalinosis, especially if performed late after CsA dose reduction (10–12).

The classic histologic features of CsA nephrotoxicity seen on biopsy specimens early after transplantation have been well described. Recognized lesions include de novo arteriolar hyalinosis, striped cortical fibrosis, tubular microcalcification, peritubular and glomerular capillary congestion, diffuse interstitial fibrosis, toxic tubulopathy, juxtaglomerular hyperplasia, and isometric tubular vacuolization (13–18). Much less is known about the pattern of chronic nephrotoxicity, and a comprehensive histologic description of this entity is lacking. An appropriate approach to understanding the histologic evolution of CsA nephrotoxicity is to examine protocol kidney biopsy specimens in a longitudinal manner.

This article describes the natural history of CsA nephrotoxicity from a subanalysis of CsA-treated, renal-pancreas transplant recipients (6) who underwent regular, prospective protocol kidney transplant biopsies up to 10 years after transplantation. The histologic diagnosis of CsA nephrotoxicity was correlated with putative risk factors and CsA doses and levels. With the advantage of repeated histology and longitudinal analysis, we have determined the onset of structural CsA nephrotoxicity, defined the evolution of its histologic features, and identified two distinct phases of structural nephrotoxicity according to the persistence of lesions in sequential biopsy specimens.

PATIENTS AND METHODS

Study Population and Clinical Data

The study group was derived from 98 consecutive diabetic recipients of combined kidney-pancreas transplants and one kidney transplant alone, in whom CsA was used as the primary calcineurin inhibitor. Protocol biopsy specimens were taken at defined intervals including implantation; at 1 and 2 weeks; at 1, 3, 6, and 12 months; and then annually for 10 years, as previously described (6). Diabetes mellitus was present for 24.9±6.9 years before combined transplantation, but patients had sustained euglycemia after transplantation, with a mean hemoglobin A1C of 5.6±1.3%.

Triple-therapy immunosuppression incorporated oil-based CsA (Sandimmune [S]; Novartis, Basel, Switzerland) initiated at 12.5 mg/kg/day or, after 1996, microemulsion (ME) CsA (Neoral; Novartis, Basel, Switzerland) at 8 mg/kg/day, with doses adjusted according to trough drug levels. Prednisolone was also administered with azathioprine at 1.5 mg/kg/day or mycophenolate mofetil (MMF) (Cellcept; Roche, Nutley, NJ) starting at 3 g/day from 1999. The primary study group consisted of 99 patients treated with CsA. An additional comparison group (n=21) using tacrolimus (Prograf; Fujisawa, Osaka, Japan) initiated at 0.2 mg/kg/day was also evaluated.

Definitions of CsA Nephrotoxicity

Acute clinical CsA nephrotoxicity was defined as an acute increase in serum creatinine greater than 25% above baseline, for which acute rejection had been excluded by biopsy or restoration of serum creatinine with CsA dose reduction. Needle-core protocol biopsy specimens were evaluated by two blinded observers and classified using the Banff schema (19) as previously described (6,20). Structural CsA nephrotoxicity was defined a priori by the presence of any of the following: de novo or progressive arteriolar hyalinosis (not caused by preexisting donor hyalinosis), striped cortical fibrosis, or severe tubular microcalcification (seen by both observers in the absence of preceding acute tubular necrosis) (15,16). The presence of nodularity within the arteriolar hyalinosis score was not used as a diagnostic criterion for nephrotoxicity because of the variations of vascular cross-section appearance according to the plane of section; the lack of definition as to what actually constitutes “nodularity”; and our early preliminary findings that early, mild, CsA-related arteriolar hyalinosis was circumferential (and that nodularity only became more apparent with increasing severity of the lesion). Because of these problems with definition, we did not systematically score nodularity. Glomerular congestion and diffuse cortical interstitial fibrosis were not used to define CsA nephrotoxicity. The same definition for nephrotoxicity was used for both tacrolimus and CsA because of their comparable histopathology (9), described generically as calcineurin inhibitor nephrotoxicity. Because striped fibrosis, microcalcification, and arteriolar hyalinosis are focal lesions that may not be seen on all sections, the true incidence of calcineurin nephrotoxicity was likely to be underestimated by this definition.

Structural (as opposed to functional) CsA nephrotoxicity was defined as the first occurrence of any CsA-mediated histologic lesions on any biopsy specimen. Subclassification of structural CsA nephrotoxicity was undertaken on the basis of its duration in sequential histologic biopsy specimens. Acute CsA nephrotoxicity was arbitrarily defined by the occurrence of any transient CsA lesion (≤6 months' duration). Chronic CsA nephrotoxicity was defined by persistence on sequential histology for 2 or more years, presence in 50% or more of biopsy specimens from a minimum of three biopsy specimens or the last two consecutive biopsy specimens, or by any three or more biopsy specimens showing CsA nephrotoxicity.

Statistical Analysis

Because this was a longitudinal study, all biopsy specimens were scored and included for statistical analysis where possible. If the sample did not have an artery present for evaluation, vascular qualifiers were treated as missing data (and omitted from that analysis) and the remaining Banff qualifiers were scored and included when appropriate. Cox regression was used for survival analysis, logistic regression for dichotomous data, and multiple linear regression for nominal multivariate analysis, preceded by backward elimination. Because repeated measurements in one patient are not statistically independent, a generalized estimating equation and logistic regression adjusted for repeated measurements were used in these instances. Data are expressed as mean±SD unless otherwise stated, and a value of P<0.05 was considered significant.

RESULTS

Study Group

CsA-treated patients (n=99) were 58.6% male patients and 38.2±7.0 years of age. Kidney cold ischemia time was 11.9±3.4 hr, acute tubular necrosis was detected in 22.7% of time-zero (implantation until 1 week after transplantation) biopsy specimens, but posttransplant hemodialysis was required in only two patients. CAN combined with CsA nephrotoxicity caused three kidney failures, and the median patient follow-up was 7.0 years.

Calcineurin Inhibitor Doses and Levels

The mean dose of CsA for the entire study period was 5.1±1.7 mg/kg/day (330±108 mg/day) and the mean dose of tacrolimus was 0.11±0.01 mg/kg/day (8.2±3.1 mg/day), yielding averaged trough levels of 204±117 ng/mL (Cyclo-Trac SP whole blood radioimmunoassay; DiaSorin, Stillwater, MN) and 12.7±4.4 ng/L, respectively. CsA doses were reduced in instances of early acute functional and clinically apparent nephrotoxicity and progressively in all patients (Fig. 1). As CsA nephrotoxicity became increasingly apparent, further modest dose reductions were undertaken, although CsA therapy was continued in all study patients. Calcineurin-sparing agents such as diltiazem were avoided.

FIGURE 1.
FIGURE 1.:
(upper) CsA daily dose (bars) and trough levels (line) according to time after transplantation (mean±SEM). (lower) Point prevalence of CsA nephrotoxicity showing the transient early acute CsA nephrotoxicity (stippled bars) and the later and more universal chronic nephrotoxicity (filled bars).

Acute renal dysfunction caused by CsA nephrotoxicity within the first 3 months after transplantation occurred in 35.5% of patients treated with CsA-S, in 10.8% treated with CsA-ME, and in 19.0% treated with tacrolimus (both P<0.01 vs. CsA-S). The reduced incidence of early acute clinical CsA nephrotoxicity with CsA-ME occurred despite higher 3-month CsA doses (449±130 vs. 384±145 mg/day, P<0.05) and trough levels (260±76 vs. 211±136 ng/mL, P<0.05) compared with CsA-S. There were also fewer episodes of acute cellular rejection with microemulsion CsA-ME compared with CsA-S (0.87±0.92 vs. 1.51±1.12, P<0.01) and fewer acute vascular rejection episodes (0.20±0.48 vs. 0.05±0.23, P=0.08).

Isotopic glomerular filtration rate (GFR) increased from at 59.6±16.8 mL/min at 3 months after transplantation to 61.2±17.2 mL/min at 1 year and gradually fell to 47.7±12.7 mL/min by 10 years. Isotopic GFR was reduced in patients with renal protocol biopsy specimens showing the pattern of chronic CsA nephrotoxicity as compared with those patients with early acute CsA nephrotoxicity or no nephrotoxicity (P<0.01) (Table 1). When assessed by multivariate analysis, isotopic GFR was predicted by chronic tubular and glomerular damage (but not CsA nephrotoxicity on biopsy), when corrected for the estimated donor GFR and time after transplantation.

TABLE 1
TABLE 1:
Histologic features of biopsy specimens (taken at 3 mo and beyond) with and without CsA nephrotoxicity (mean±SD)

Renal Transplant Biopsies

A total of 888 study biopsies in 99 CsA-treated recipients yielded 7.9±3.5 biopsy specimens per patient (range, 3–18). The mean number of glomeruli and arteries were 14.3±9.7 and 2.4±1.2 per biopsy specimen, respectively. Inadequate samples, defined as less than seven glomeruli or no artery (9), occurred in 15.6%. The interobserver variability was good to excellent, with a kappa statistic for the presence of CAN, calcineurin nephrotoxicity, chronic interstitial fibrosis, and arteriolar hyalinosis being 0.61, 0.48, 0.80, and 0.46, respectively. The intraobserver kappa statistic for arteriolar hyalinosis was excellent at 0.82. The median biopsy follow-up time for CsA-treated patients was longer compared with the tacrolimus-treated comparison group (4.7 years vs. 1.3 years, respectively; P<0.001). Donors were 25.5±9.8 years old, and on time-zero biopsy specimens (taken at implantation or within the first week, n=121), mild and focal arteriolar hyalinosis was discernible in 14.3%, the mean chronic fibrosis score was 0.08±0.27, and 1.9% of glomeruli were sclerosed, consistent with minimal or absent chronic preexisting damage in transplanted kidneys.

CsA Nephrotoxicity and Histology

The point prevalence of CsA-associated lesions increased with time after transplantation (Figs. 1 and 2). The 1-, 5-, and 10-year cumulative Kaplan-Meier prevalences were 61.3%, 90.5%, and 100% for de novo or progressive arteriolar hyalinosis; 35.1%, 69.9%, and 88.0% for striped fibrosis; and 44.0%, 68.2%, and 79.2% for any tubular microcalcification seen by either observer, respectively (Fig. 2). In individual biopsy specimens taken between 3 months and 10 years with histologically defined CsA nephrotoxicity (both acute and chronic patterns, n=375 biopsy specimens), 80.0% had arteriolar hyalinosis, 32.9% had striped fibrosis, 36.2% had microcalcification, and 62.4% had combinations of these lesions. When repeated biopsy specimens were taken 1 year after transplantation from patients with the diagnosis of chronic CsA nephrotoxicity, 53.9% of transplanted kidneys had two or more histologic features that were diagnostic of nephrotoxicity. Therefore, the diagnosis of CsA nephrotoxicity based on a single hallmark lesion was usually confirmed by another diagnostic lesions present in subsequent biopsy specimens within that same kidney.

FIGURE 2.
FIGURE 2.:
(upper) Point prevalence of defining lesions of CsA nephrotoxicity from sequential protocol histology, including de novo arteriolar hyalinosis (gray), striped cortical fibrosis (striped), and tubular microcalcification without preceding acute tubular necrosis (filled bars). (lower) Cumulative prevalence of the lesions of CsA nephrotoxicity lesions (1−Kaplan-Meier survival) on repeated biopsy up to 10 years after transplantation.

The median onset of the first lesion of CNI nephrotoxicity was 0.5 year (interquartile range, 0.25–2 years). The histologic onset of nephrotoxicity between CsA (n=888) and tacrolimus biopsy specimens (n=71), or between the microemulsion (Neoral) formulation compared with the older oil-based Sandimmune formulations of CsA, were statistically indistinguishable, with overlapping of the Kaplan-Meier prevalence curves (data not shown). The cumulative 1- and 10-year Kaplan-Meier prevalence of CsA nephrotoxicity was 75.9% and 96.9%, respectively, and it was present in all 10-year biopsy specimens.

Striped fibrosis increased with time to a 10-year cumulative incidence of 88.0%, and its instantaneous hazard rates were largely constant over the study period (data not shown). Statistical analysis of striped fibrosis was confounded by its relatively low and variable prevalence rate on any individual biopsy specimen, and so these data should be interpreted with caution. Using Cox regression, it was predicted by the requirement for posttransplant hemodialysis with induction antilymphocyte globulin providing a protective effect (both P<0.05). Striped fibrosis was predictive of subsequent chronic persistent CsA nephrotoxicity.

Arteriolar Hyalinosis and CsA Nephrotoxicity

De novo arteriolar hyalinosis exhibited an early onset peak from 3 to 12 months after transplantation (Figs. 2 and 3), corresponding to early high-dose CsA exposure, followed by later occurrence or reoccurrence of gradual and progressive arteriolar narrowing, associated with increasing glomerulosclerosis (Fig. 3). Predictive factors for early arteriolar hyalinosis using multivariate Cox regression included a 3-month trough CsA level greater than 200 ng/mL (hazard ratio, 1.68; 95% confidence interval [CI], 1.02–2.80; P<0.05), preceding acute clinical nephrotoxicity (hazard ratio, 2.55, 95% CI, 1.44–4.52; P<0.001), and a trend with CsA-ME use (hazard ratio, 1.67; 95% CI, 0.96–2.91; P=0.07 vs. CsA-S). CsA dose was not significant when CsA trough level was included in the multivariate analysis. Arteriolar hyalinosis was not affected by the type of calcineurin inhibitor (tacrolimus vs. CsA). Thus, early arteriolar hyalinosis could be statistically linked to CsA by direct correlation of CsA trough levels and clinical acute CsA dysfunction, independent of other histologic features of nephrotoxicity.

FIGURE 3.
FIGURE 3.:
Progressive glomerulosclerosis (filled circles) accompanied by arteriolar hyalinosis (open squares) defined by protocol histology and increasing with time (mean±SEM).

We excluded alternative explanations for arteriolar hyalinosis including preexisting donor arteriolar hyalinosis, ischemic arteriolar injury, dyslipidemia, diabetes, and hypertension (below). Significant donor arteriolar hyalinosis was relatively uncommon, focal, and mild in our study (14.3% seen by either observer or 7.1% prevalence confirmed by both observers) because of the young donor kidneys used. CsA nephrotoxicity was not diagnosed in these kidneys unless arteriolar hyalinosis increased by one or more Banff ah score in subsequent biopsy specimens. Ischemic microvascular injury (which some have speculated to cause hyalinosis) was unrelated since arteriolar hyalinosis and was not correlated with kidney anastomosis time (r=0.00, P=NS) or total renal ischemic times (r=0.034, P=NS) in biopsy specimens taken up to 1 year after transplantation. Arteriolar hyalinosis was not associated with contemporaneous oral glucose tolerance tests (P=not significant [NS]) (Fig. 4), hemoglobin A1C levels (5.7±0.13% and 5.5±0.07%, P=NS), fasting serum cholesterol (5.8±1.4 and 5.9±1.2 mM, P=NS), or triglycerides (1.6±0.9 and 1.6±0.8 mM, P=NS) compared with biopsy specimens without arteriolar hyalinosis. Similarly, the arteriolar hyalinosis scores in patients with sustained euglycemia and a functioning pancreas transplant were comparable to the small number of renal biopsy specimens (n=59) from patients with early pancreas thrombosis (0.57±0.65 vs. 0.52±0.74, P=NS). The small number of patients who admitted that they smoked regularly after transplantation (n=3) precluded analysis of this effect. Of interest, the use of MMF in subgroup analysis reduced the development of progressive arteriolar hyalinosis (coefficient=−0.25±0.12 when compared to azathioprine therapy, P<0.05), which remained significant after correction for time after transplantation and CsA dose and level.

FIGURE 4.
FIGURE 4.:
(upper) No relationship between hyperglycemia and the presence of arteriolar hyalinosis assessed by oral glucose tolerance tests in patients with (n=185) and without (n=270) arteriolar hyalinosis (P=NS for differences) (mean±SEM). (lower) Onset of arteriolar hyalinosis preceded the occurrence of hypertension as shown by Kaplan-Meier survival curves.

In addition, there was no significant correlation between the presence of arteriolar hyalinosis and hypertension (r=0.15, P=NS), where hypertension was defined as sustained blood pressure exceeding 140/90 mm Hg or by the requirement of antihypertensive therapy. The Kaplan-Meier curves for patients with graft CsA nephrotoxicity and hypertension overlapped for the first 6 months after transplantation and then separated, with the onset of arteriolar hyalinosis preceding the onset of hypertension (P<0.001) (Fig. 4). Arteriolar hyalinosis was present in 68% of hypertensive and 60% of normotensive patients (P=0.11). There was no effect of hypertension in predicting the presence or severity of arteriolar hyalinosis using multivariate analysis. Therefore, the development of arteriolar hyalinosis was unrelated to blood pressure, dyslipidemia, hyperglycemia, ischemia, and donor disease, and appeared highly specific for the diagnosis of CsA nephrotoxicity in our study population.

Patterns of Structural CsA Nephrotoxicity

CsA nephrotoxicity occurred in two distinct phases, each with a different histologic picture and clinical outcome (Table 2 and Fig. 1). The first phase of acute CsA nephrotoxicity occurred early after transplantation, usually within the first year (median onset at 1 year), with a 1-year point-prevalence of 12.6%. Early-onset acute CsA nephrotoxicity was predicted by a CsA trough level greater than 200 ng/mL at 3 months (hazard ratio, 1.67; 95% CI, 1.03–2.71; P<0.05) and preceding CsA-induced renal dysfunction (hazard ratio, 1.68; 95% CI, 1.03–2.76; P<0.05) using Cox regression analysis. CsA nephrotoxicity within the first 3 months after transplantation was diagnosed primarily by the presence of arteriolar hyalinosis (90.3% of biopsy specimens) and resolved or was seen intermittently on subsequent biopsy specimens in 50.4% of cases.

TABLE 2
TABLE 2:
Clinical and histologic features of acute CsA and persistent CsA nephrotoxicity (n=406 nonimplantation biopsy specimens) (mean±SD)

Chronic CsA nephrotoxicity occurred with a point prevalence of 67.3% by 5 years and 100% by 10 years after transplantation. By definition, it was persistent, and CsA lesions were present in several biopsy specimens over 2 or more years. It was accompanied by more severe arteriolar hyalinosis and progressive glomerulosclerosis that occurred later after transplantation (Table 2 and Figs. 1 and 3). Partial glomerulosclerosis and periglomerular fibrosis were also increasingly present in chronic CsA nephrotoxicity (P<0.001). Although small arterial myxoid change was also occasionally observed, the vascular lumen of smaller muscular arteries was not greatly compromised, with corresponding chronic fibrointimal thickening (Banff cv) scores showing minimal increases with time. Chronic CsA nephrotoxicity was associated with a greater time-averaged CsA dose over the duration of the study (hazard ratio, 1.71; 95% CI, 1.21–2.42; P<0.001) compared with control kidneys without CsA nephrotoxicity (Fig. 5).

FIGURE 5.
FIGURE 5.:
(upper) Higher CsA dose in those with chronic CsA nephrotoxicity (filled circles) compared with those without CsA nephrotoxicity (open circles) (*P<0.05). (lower) CsA dose increases the progression of arteriolar hyalinosis on sequential biopsies from 1 to 10 years after transplantation (P<0.05 for group). Delta arteriolar hyalinosis is the difference in Banff scores between sequential biopsy specimen pairs (mean±SEM).

In biopsy specimen pairs sampled from 1 to 10 years after transplantation, higher doses of CsA resulted in a greater progressive accumulation of arteriolar hyalinosis (P<0.05, expressed as delta change and calculated between the difference of arteriolar hyalinosis scores between two sequential biopsy specimen pairs); these are displayed as a nonlinear dose-response relationship (Fig. 5). A threshold CsA dose for progressive arteriolar injury of 5 mg/kg/day was inferred from this analysis. Beyond 1 year after transplantation, arteriolar hyalinosis tended to progress and was associated with higher CsA trough levels exceeding the median of 180 ng/mL (P<0.05).

DISCUSSION

This longitudinal analysis provides new insights into the natural history of structural CsA nephrotoxicity, which comprises two distinct histologic phases. Acute histologic changes occurring soon after transplantation have been described previously (9,14–16,18,21), and in our study they were associated with early high-dose CsA exposure and resulted in patchy, mild, and potentially reversible arteriolar hyalinosis. A more chronic phase of persistent CsA nephrotoxicity occurred later after transplantation, associated with relatively lower CsA doses given over a longer duration, progressive and severe arteriolar hyalinosis, luminal narrowing, and ischemic glomerulosclerosis. The high prevalence of nephrotoxicity with both types of calcineurin inhibitors, their contribution to progressive histologic damage and chronic allograft nephropathy, and the lack of minimal histologic reversibility raise serious doubts about their suitability for long-term use in kidney transplantation.

The reported prevalence of CsA nephrotoxicity varies substantially because of differing methods of detection and variable study follow-up times. When an insensitive surrogate marker such as serum creatinine or GFR is used (22), the apparent prevalence is less common (7,8,23). In nonrenal solid organ transplantation (2,21,24) or with CsA treatment for autoimmune diseases not involving the kidney (25), declining renal function attributable to CsA nephrotoxicity is becoming an increasingly recognized and common problem. The 5-year prevalence of end-stage renal failure in nonrenal transplant recipients varies from 6.9% to 28.3%, exacerbated by CNI nephrotoxicity and markers of recipient renal impairment including older age, diabetes mellitus, hypertension, and posttransplant acute tubular necrosis (24). In histologic studies of kidney transplantation (arguably the most sensitive method of detection), CsA nephrotoxicity was found to be common (9,14) and essentially universal by 10 years after transplantation in our study. Although our patients received minimally damaged kidneys, it is likely that the clinical impact of CsA nephrotoxicity would be much greater with a marginal kidney. CsA nephrotoxicity was the predominant cause of ongoing microvascular and glomerular damage beyond 1 year.

The high frequency and widespread CsA-induced damage may explain the paradox of modern calcineurin-based immunosuppression, where the substantial improvements in early acute rejection rates have not been matched by commensurate improvements in long-term outcome (4,5,26). We hypothesize that the early benefit from control of immune injury by calcineurin inhibitors may be subsequently lost from long-term nephrotoxicity. Both CsA and tacrolimus are established nephrotoxins (3,9,15,16,18), and their chronic exposure to a kidney transplant is likely to incur nephron damage from nephrotoxicity. Our inferential CsA threshold dose for progressive nephrotoxicity of 5 mg/kg/day is remarkably close to the widely quoted optimal dose range of 4 to 5 mg/kg/day to prevent immunologic graft failure (27). This suggests that the dose ranges for therapeutic effect and histologic nephrotoxicity of CsA are overlapping.

Structural CsA nephrotoxicity is usually thought of as a single entity, as distinct from the acute functional impairment of vasospastic origin. Our study demonstrated two phases of anatomic CsA nephrotoxicity, with distinct histologic and clinical characteristics occurring at different times after transplantation and with different outcomes. Early CsA nephrotoxicity was characterized by mild and patchy arteriolar hyalinosis within the first 2 years after transplantation. It was distinct from the acute renal dysfunction induced by CsA, although the two conditions were interrelated. It was intermittently observed on early sequential biopsy specimens, being present on one biopsy specimen and absent on the following biopsy specimen and not apparently because of sampling or observer variability. It was usually reversible with the routine reduction of high initial CsA doses. Previous studies of repeat biopsies of CsA-related arteriolopathy have been contradictory, with some showing vascular remodeling and improvement with reduction or cessation of CsA therapy (11,20,28) and others demonstrating progression to complete vascular occlusion (1,16,18,29). Reversibility of early arteriolar hyalinosis in our study depended on the intensity and cumulative CsA exposure and may reflect exhausted microvascular repair (30).

CsA therapy causes vacuolation of arteriolar smooth muscle and endothelial cells, endothelial cell necrosis, and insudation of protein deposits to form nodular arteriolar hyalinosis, with consequent narrowing of the vascular lumen (13). Arteriolar hyalinosis was specific for CsA-induced injury in our study and etiologically supported by the associations with acute clinical nephrotoxicity; CsA dose and level; and exclusion of alternative explanations including donor arteriolar hyalinosis, ischemic arteriolar injury, dyslipidemia, hyperglycemia, and hypertension. When arteriolar hyalinosis is found in a failing kidney transplant, the diagnosis of CNI nephrotoxicity is strengthened by evidence of histologic progression from comparison with an implantation or earlier biopsy specimen (12), a nodular pattern rather than diffuse hyalinosis (15), and by exclusion of other diagnoses such as hypertensive nephrosclerosis, which can be distinguished by elastic lamina reduplication and medial hyperplasia in larger arteries (15,16,21). Interestingly, we have demonstrated an independent protective effect of MMF in retarding progressive arteriolar hyalinosis, comparable to experimental data and possibly reflecting its nonimmune antiproliferative properties (31,32).

Tubular microcalcification is usually caused by localized cell necrosis and was relatively common (79.2% of kidneys by 10 years with exhaustive examination) and predicted subsequent chronic CsA nephrotoxicity. Proximal tubules are susceptible to CsA injury, showing early isometric vacuolation, cellular necrosis, tubular cytoplasmic inclusion bodies (corresponding to abnormal giant mitochondria), and microcalcification (15,16,18). It is possible that the chronic diffuse tubulointerstitial damage observed in our later histology (6) may also have been because of CsA nephrotoxicity, although this is difficult to distinguish from immune-mediated tubular damage. Striped fibrosis represents more severe but localized tubular damage, characterized by a stripe(s) of fibrosis and tubular atrophy adjacent to normal cortex (15). Striped fibrosis is usually regarded as pathognomonic of CsA nephrotoxicity, although its specificity has been questioned (33) and its histologic definition is imprecise and subjective. It often heralded chronic nephrotoxicity in our study, but was difficult to analyze because of its intermittent presence in any one kidney. The only independent clues to its cause were its relationship to the need for posttransplant hemodialysis and reduced frequency after induction antilymphocyte globulin, where the CsA dose was routinely reduced or avoided. It is possible that CsA exposure to a vulnerable graft affected by ischemia-reperfusion injury may initiate striped fibrosis by microvascular watershed infarction. The cumulative incidence of striped fibrosis increased with time and was present in the majority of grafts by 10 years. The combination of a high cumulative prevalence substantially exceeding a lower point prevalence indicates that any single biopsy core may miss the striped pattern, making it a relatively insensitive diagnostic marker of CsA nephrotoxicity. In addition, the appearance of a striped area of fibrosis adjacent to normal cortex becomes increasingly difficult to appreciate in allografts that are engulfed by diffuse and widespread interstitial fibrosis with the margin blurred. This and other studies suggest that striped fibrosis is a suboptimal diagnostic indicator of calcineurin inhibitor nephrotoxicity

CsA nephrotoxicity occurring late after transplantation was characterized by increasing and high-grade arteriolar hyalinosis, progressive glomerulosclerosis, and chronic tubulointerstitial damage in the context of long-term but lower dose exposure of CsA. CsA-mediated arteriolar toxicity appeared to be the dominant cause of ongoing microvascular and glomerular injury from 5 years after transplantation, when demonstrable cellular immunologic activity had largely subsided (6). Chronic and severe arteriolar hyalinosis was generally irreversible once established despite modest CsA dose reductions and resulted in vascular narrowing and ischemic glomerulosclerosis. Renal function is a relatively insensitive method of detecting microvascular transplant damage from CsA nephrotoxicity (21), and its use will consequently underestimate the true prevalence and severity. In our study, widespread ischemic glomerulosclerosis was accompanied by only a modest fall in isotopic GFR late in the natural history of CsA nephrotoxicity, which may explain the lack of impact from late CsA reduction or withdrawal once substantial nephron damage had occurred (1,29).

We recommend that care be exercised with calcineurin dose reduction because inadequate immunosuppression may allow a late reactivation of subclinical rejection. In immunologically high-risk recipients or in patients with lymphocytic activity on biopsy, that experience coexistent calcineurin nephrotoxicity, calcineurin substitution with the strengthening of other components of therapy (such as the conversion of azathioprine to MMF, the addition of MMF per se, or the addition of corticosteroids to dual-therapy regimens) may be more appropriate than dose reduction or cessation. However, these strategies and the optimal use of new agents such as target of rapamycin (TOR) inhibitors have yet to be fully validated by clinical trials.

CONCLUSION

Using a longitudinal protocol biopsy analysis, we have demonstrated a high incidence of CsA nephrotoxicity that presented as two distinct clinical and histologic phases and contributed to chronic allograft nephropathy. CsA nephrotoxicity was associated with striped fibrosis, progressive arteriolar hyalinosis, and ischemic glomerulosclerosis. It was essentially universal by 10 years after transplantation and usually irreversible with ongoing CsA therapy, despite modest dose reductions. Our data suggest that reliance on CsA is inappropriate for long-term immunosuppression of kidney transplant recipients. Strategies to ameliorate or avoid calcineurin inhibitor nephrotoxicity need to be clinically validated, and methods for its routine and early detection need to be used in individual patients.

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