—  SPECIALTY CONFERENCE  —

Renal Pathology

Case 1 - Rapamycin-associated Nephrotoxicity and Cast Nephropathy

Kelly D. Smith
University of Washington Medical Center
Seattle, WA


Click on each slide thumbnail image for an enlarged view
Clinical History
This is a 52-year-old female, who is status post cadaveric renal transplant for polycystic kidney disease and end stage renal disease (one and a half years status post bilateral nephrectomies). Total ischemic time was 13 hours and 32 minutes, and cold ischemic time was 12 hours and 58 minutes. The patient made a small amount of urine immediately postoperatively, but soon became anuric. Several renal ultrasounds obtained in the post-operative period demonstrated excellent perfusion of the kidney.

She has been followed in transplant clinic for her delayed graft function, and is now 8 weeks status post cadaveric kidney transplant. She continues to complain of marked fatigue and weakness. She has been receiving intermittent transfusions of packed red blood cells, and regular hemodialysis. She has very minimal urine output, and the urine has been relatively dark. Today, she has made 6 cc, and her typical urine output ranges from 10-50 cc per day. She has not experienced dysuria, or noted any change in her urine. She denies any fevers or chills, and has not had any headaches.

Her medications are tacrolimus (2 mg bid), sirolimus (5 mg per day), ganciclovir (500 mg per day), clotrimazole (10 mg qid), Zantac (150 mg per day), Docusate (250 mg bid), Multivitamin (one per day), Temazepam (30 mg hs), Trazodone (12.5 mg per day), Dilaudid (2 mg q 2-4 h prn), Fentanyl (patch 50 mcg every 3 days), and PhosLo (two tablets tid).

On physical exam the patient is afebrile with a blood pressure of 110/80 mmHg. Her abdomen is markedly distended with pain in several areas (secondary to her polycystic liver disease), but her graft is relatively non-tender. Her ankles show symmetrical edema without any effusion in the joints or erythema.

Laboratory studies are notable for a tacrolimus level of 4.4 (target 10), BUN 30, creatinine 3.8, and glucose 154, white blood cell count 3.77, hematocrit 33, and platelet count 106,000. Sirolimus level 8.9 (target 10-15).

Clinical impression
Primary graft non-function. The patient's immunosuppressive levels have been kept relatively low over the last several weeks with tacrolimus levels between 2.1 and 10, but mostly in the 2-3 range. Sirolimus levels have been under the goal of 10-15, often times 8.9 to 9.1. She has not had any evidence of obstruction. She is being dialyzed.

Renal biopsies were done at 2 weeks (SU-01-430), 5 weeks (SU-01-1808), and 8 weeks (SU-01-3502).


Case 1 - Figure 1 - SU-01-430 LM1 - PAS stain at 2 weeks - PAS stain of the 2 week biopsy demonstrates features of acute tubular injury/"acute tubular necrosis," characterized by tubular epithelial cell loss of brush border, accumulation of debris in tubular lumina, and the additional feature of prominent intratubular cast formation. Many casts have irregular sharply demarcated contours, and occasional apparent fracture lines.
Case 1 - Figure 2 - SU-01-430 LM2 - PAS stain at 2 weeks - PAS stain of the 2 week biopsy demonstrates findings similar to those seen in Figure 1.
Case 1 - Figure 3 - SU-01-430 EM1 - electron micrograph at 2 weeks - Electron micrograph of the 2 week biopsy demonstrates tubular epithelial cell injury, with loss of brush border, simplification of basolateral membrane interdigitations, and prominent accumulation of heterogenous lysosomes (tertiary lysosomes).


Case 1 - Figure 4 - SU-01-430 EM2 - electron micrograph at 2 weeks - Electron micrograph of the 2 week biopsy demonstrates tubular epithelial cell injury and cast material within the tubular lumen. The central portion of the lumen contains cells that have accumulated osmiophilic material along membranes and on organelles. These cells are surrounded by a dense ring of osmiophilic cast material, which in some fields is associated with organoid cellular debris.
Case 1 - Figure 5 - SU-01-1808 LM1 - Jones stain at 5 weeks - Jones stain of the 5 week biopsy demonstrates persistent intratubular cast formation, as seen in the 2 week biopsy. At this time there is also an apparent increase in interstitial collagen.
Case 1 - Figure 6 - SU-01-1808 LM2 - Jones stain at 5 weeks - Jones stain of the 5 week biopsy demonstrates persistent tubular injury and intratubular cast formation. Some tubules contain eosinophilic bodies and membranous rings, which correspond to the degenerating cells laden with osmiophilic material, as detected by electron microscopy.


Case 1 - Figure 7 - SU-01-3502 LM1 - Jones stain at 8 weeks - Jones stain of the 8 week biopsy demonstrates persistent tubular injury and atypical intratubular casts. In this biopsy, there is also a further increase in interstitial collagen and tubular atrophy.
Case 1 - Figure 8 - SU-01-3502 LM2 - Jones stain at 8 weeks - Jones stain of the 8 week biopsy demonstrates focal multinucleate giant cell formation within the tubular lumen.

Renal Biopsy Findings

Light Microscopy
The series of biopsies demonstrated histological features of acute tubular injury, characterized by increased diameter of the tubular lumina, loss of brush border, accumulation of luminal debris, and prominent intratubular cast formation. Many of the casts consisted of amorphous eosinophilic material, which had sharply defined irregular contours, occasional fracture lines and in some instances were associated with a cellular reaction that extended to focal multinucleated giant cell formation. Some casts were less well developed and consisted of eosinophilic bodies and membranous rings, which were histologically consistent with cells in various stages of degeneration. These changes persisted throughout the biopsy series, and were accompanied by a gradual increase in interstitial fibrosis.

Immunofluorescence Microscopy
Immunofluorescence microscopy demonstrated nonspecific staining of tubular casts for IgA, and kappa and lambda light chains.

Electron Microscopy
Electron microscopy demonstrated the presence of degenerating cells within the tubular lumina with varying degrees of accumulation of osmiophilic material. The histologically observed membranous rings were seen to represent cellular membranes with prominent accumulation of osmiophilic material. Further accumulation and coalescence of the degenerating cell and osmiophilic material accounted for the well-defined atypical cast material. Cells and cell fragments within the tubular lumens contained lysosomes with heterogeneous inclusions (tertiary lysosomes). Similar tertiary lysosomes were present in the intact tubular epithelium. Tubular epithelial cells also demonstrated simplification of the basolateral membranes.

Additional studies
Urine and serum immunoelectrophoresis studies were negative.

Interpretation
Differential diagnosis for the pathologic findings included myeloma cast nephropathy. There was no clinical history or significant suspicion of a B cell neoplasm. However, given the unusual histopathology, SPEP and UPEP studies were performed as noted above and were negative, excluding myeloma cast nephropathy as a diagnostic possibility.

Final Diagnosis
Rapamycin-associated nephrotoxicity and cast nephropathy.

Discussion
Delayed graft function (DGF) occurs in renal allografts due to ischemic injury prior to or after procurement, or due to exposure to nephrotoxic medications in the peri-transplant period. Donor, recipient and procedural factors are known to contribute to the development of delayed DGF (Table 1). At our institution, we noted a dramatic increase in the incidence of delayed graft function from 8% to 25% and the appearance of a unique cast nephropathy in renal transplant patients [1]. Both of these events coincided with the introduction of rapamycin (sirolimus) as an immunosuppressive agent in new steroid free immunosuppressive protocols. Rapamycin had not been previously associated with delayed graft function, and its use has been advocated in the setting of DGF [2, 3] . We analyzed the contribution of rapamycin to delayed graft function, and found that patients receiving rapamycin had a significantly increased risk of developing DGF (p = 0.02) [1]. Multivariate analysis demonstrated that DGF was highly significantly associated with rapamycin dose (odds ratio = 1.13 per mg sirolimus, p = 0.04). We also saw a positive correlation between the use of rapamycin and the duration of delayed graft function, but this finding did not reach statistical significance. Since our initial report [1], there was an additional retrospective study [4] that demonstrated a significant association between rapamycin use and prolonged DGF (hazards ratio = 0.49, p = 0.0009).



Typical histological findings in kidney transplant biopsies from patients with DGF, include dilation of tubular lumina, loss of proximal tubular epithelial cell brush border, epithelial cell necrosis/apoptosis,and cellular casts. The patients receiving rapamycin with DGF had similar findings, but a subset of those patients progressed to a state where numerous atypical eosinophilic casts filled tubular lumina. The histological similarity of this rapamycin-associated cast nephropathy to myeloma cast nephropathy was in some instances striking. In general, however, the leukocytic infiltration of the interstitium and the leukocytic reaction to the cast material was less pronounced in rapamycin-associated cast nephropathy. This may be the result of the increased level of immunosuppression in renal transplant patients or differences in the nature of the casts. In addition, rapamycin-associated and myeloma cast nephropathies demonstrated differences at the ultrastructural level. Rapamycin-associated casts consisted of a prominent epithelial cell component. Degenerate epithelial cells were a central component in cast formation and were a nucleation site for precipitates that accumulated and coalesced to eventually obscure the underlying cellular component. Whereas in myeloma casts, the paraprotein (monoclonal immunoglobulin chain(s) ) is prerequisite for cast formation and a major component of the cast material. The pathologic alterations in rapamycin-associated nephrotoxicity and cast nephropathy indicate that there is enhanced tubular epithelial cell death, reduced epithelial cell regeneration, and perhaps an alteration of macrophage clearance of apoptotic cells. Together the pathologic findings and patient data support the premise that renal allograft injury may be augmented, and recovery impeded by the actions of rapamycin.

Rapamycin is being used increasingly as a maintenance immunosuppressive agent especially with designs to decrease steroid and calcineurin inhibitor exposure. It has been most commonly demonstrated to spare renal function when used without a calcineurin inhibitor, although in combination with calcineurin inhibitors, serum creatinine values may be elevated [3]. As both cyclosporine and rapamycin are dependent upon metabolism by cytochrome P450 3A4 and influenced by P-glycoprotein activity, when the drugs are used in combination there is an increase in blood and tissue concentrations. The increased incidence of delayed graft function seen in our patient population was attributed solely to rapamycin, since the use of cyclosporine inhibitors was delayed for two weeks in these patients. However, administration of both calcineurin inhibitor and rapamycin resulted in prolonged post-transplant dialysis times with 5 (23%) of 22 requiring dialysis for over 30 days post-transplant. Furthermore, all patients requiring prolonged dialysis had renal biopsies demonstrating cast nephropathy. This is in contrast to the average reported duration of DGF of 10 to 15 days, and the DGF prevalence of 14% at 2 weeks, 9.5% at 3 weeks, and 1.7% at 1 month post-transplant.

The immunosuppressive effects of rapamycin are derived from the inhibition of cytokine- and growth factor-mediated signal transduction in T and B lymphocytes, resulting in inhibition of lymphocyte proliferation. Rapamycin acts through the FKBP12 protein to inhibit the mammalian targets of rapamycin (mTOR) a central controller of cell growth and survival [5]. When mitogenic stimuli such as IL-2, IL-4, IL-15, insulin or other growth factors are present, mTOR phosphorylates proteins, including p70S6 and PHAS-I. The P70S6 kinase phosphorylates the 40S ribosomal protein, S6, and causes an increase in the translation of mRNA's, thus increasing protein synthesis. When PHAS-I is phosphorylated, it detaches from an initiation factor allowing the start of protein synthesis. mTOR also phosphorylates and inactivates the retinoblastoma protein, permitting cellular transition from G1 to S phase and proliferation.

Although the effects of rapamycin have been most thoroughly investigated in T lymphocytes, mTOR is ubiquitously expressed, and other cells in the body also are potential targets including fibroblasts, endothelial cells, hepatocytes and smooth muscle cells. Additionally, renal tubular epithelial cells, which express FKBP12 and mTOR may be influenced by rapamycin. Furthermore, the kidney has been shown to concentrate rapamycin especially in the face of cyclosporine treatment. These in vitro data indicate that rapamycin may effect the proliferation of renal epithelium. We have demonstrated the expression of FKBP12 and mTOR within the renal tubular epithelium, with the most prominent expression found in the distal nephron. Thus, in the setting of renal injury, where organ repair depends on tubular cell proliferation, rapamycin may be harmful. These risks of enhanced injury and decreased repair involve not only the immediate post-transplant period, but may also carry over to long-term events causing renal injury, such as volume depletion and urinary tract obstruction. Rapamycin therapy has also been recently associated with delayed surgical wound healing [6], providing further evidence that the growth suppressive effects of rapamycin extend beyond immunosuppression, and involve additional targets in the body.

Animal studies also support the conclusion that rapamycin can contribute to renal failure. Repair from ischemic acute renal failure involves stimulation of cellular proliferation. Acute renal dysfunction has been seen in experimental animal models using combinations of cyclosporine A (CsA) and rapamycin. In the setting of experimental renal injury, rapamycin has recently been demonstrated to inhibit proliferation and increase apoptosis of renal tubular epithelial cells in vitro and in vivo [7]. Furthermore, it appears to directly cause renal injury in salt-depleted and renal ischemic rat models [7, 8] . Pharmacokinetic interactions between rapamycin and calcineurin inhibitors could augment ischemic injury and inhibit tissue repair when used in combination [9, 10] .

This case presents the pathologic correlates for a new risk factor for the development of renal allograft DGF, the early post-transplant use of rapamycin. We recommend delaying the use of rapamycin in the post-transplant period until renal function is established, as is common practice for calcineurin inhibitors. Using this recommendation, we have seen a reduction in the incidence of delayed graft function from 25% to 11%. In addition to the immediate post-transplant period, it is possible that rapamycin may compound or adversely affect the recovery from nephrotoxic insults, such as acute tubular injury secondary to volume depletion. Renal allograft biopsies demonstrating acute tubular injury and, more specifically, the cast nephropathy reported herein should alert practitioners to the possibility of rapamycin toxicity.

References

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  2. MacDonald, A. S., A worldwide, phase III, randomized, controlled, safety and efficacy study of a sirolimus/cyclosporine regimen for prevention of acute rejection in recipients of primary mismatched renal allografts. Transplantation 2001. 71: 271.
  3. Groth, C. G., Backman, L., Morales, J. M., Calne, R., Kreis, H., Lang, P., Touraine, J. L., Claesson, K., Campistol, J. M., Durand, D., Wramner, L., Brattstrom, C. and Charpentier, B., Sirolimus (rapamycin)-based therapy in human renal transplantation: similar efficacy and different toxicity compared with cyclosporine. Sirolimus European Renal Transplant Study Group. Transplantation 1999. 67: 1036.
  4. McTaggart, R. A., Gottlieb, D., Brooks, J., Bacchetti, P., Roberts, J. P., Tomlanovich, S. and Feng, S., Sirolimus prolongs recovery from delayed graft function after cadaveric renal transplantation. Am J Transplant 2003. 3: 416.
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  6. Troppmann, C., Pierce, J. L., Gandhi, M. M., Gallay, B. J., McVicar, J. P. and Perez, R. V., Higher surgical wound complication rates with sirolimus immunosuppression after kidney transplantation: a matched-pair pilot study. Transplantation 2003. 76: 426.
  7. Lieberthal, W., Fuhro, R., Andry, C. C., Rennke, H., Abernathy, V. E., Koh, J. S., Valeri, R. and Levine, J. S., Rapamycin impairs recovery from acute renal failure: role of cell-cycle arrest and apoptosis of tubular cells. Am J Physiol Renal Physiol 2001. 281: F693.
  8. Andoh, T. F., Burdmann, E. A., Fransechini, N., Houghton, D. C. and Bennett, W. M., Comparison of acute rapamycin nephrotoxicity with cyclosporine and FK506. Kidney Int 1996. 50: 1110.
  9. Podder, H., Stepkowski, S. M., Napoli, K. L., Clark, J., Verani, R. R., Chou, T. C. and Kahan, B. D., Pharmacokinetic interactions augment toxicities of sirolimus/cyclosporine combinations. J Am Soc Nephrol 2001. 12: 1059.
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