1. Calcineurin Inhibitor Toxicity In Kidney Allograft Protocol Biopsies Neeraja Kambham M.D. Stanford University

2. Calcineurin Inhibitor Toxicity (CNIT) CNI toxicity is a very important cause of chronic allograft nephropathy (CAN) Later phase of CAN (i.e. > 1 year post txp) is likely due to CNIT, and it’s contribution progressively increases ( Nankivell et al.) Acute phase of CNI toxicity is reversible, but chronic phase is probably irreversible

3. CNI Toxicity Functional Structural: –Acute: tubulopathy (proximal tubules), endothelial injury (thrombotic microangiopathy) –Chronic: arteriolopathy, tubular atrophy, striped fibrosis, glomerulosclerosis

4. Calcineurin Inhibitor (CNI) Toxicity Can it it be scored objectively? Is it clinically useful? Does it correlate with subsequent graft function? Is it better than Banff Chronicity score?

5. Study Design 50 consecutive pediatric renal transplant patients (November 1999- December 2004) Patients on Steroid free immunosuppression protocol* Immunosuppression: Extended Daclizumab induction; Tacrolimus and Mycophenolate mofetil maintenance Biopsies: Protocol 3, 6, 12 and 24 months (P); also as indicated clinically (NP) ( Sarwal MM et al: Transplantation. 76 (9): 2003)

7. Study Design… 231 biopsies (P+NP) scored in a blinded fashion –27 were inadequate (diagnosis rendered on 5) CNI toxicity (CNIT) score in biopsies with histological evidence of CNI toxicity Banff chronicity score (BChS): cg, ct, ci, cv Modified Banff chronicity score (MBChS): gs, ct, ci, cv Chronic Allograft Damage Index (CADI) C4d Stains on paraffin embedded tissue

8. Diagnostic Categories CNI Toxicity Acute Rejection –graded by Banff criteria Chronic Allograft Nephropathy –Unclear etiology of chronic damage –Any tubular atrophy or interstitial fibrosis > 5% No Significant Abnormality –No tubular atrophy; interstitial fibrosis < 5% Other: ATN, glomerulonephritis, reflux

9. Acute Rejection (n=29) Non-protocol Biopsies: 21 (9 %) –Borderline: 13 –IA: 6 –IB: 2 Protocol Biopsies: 8 (4.8 %)* –Borderline: 4 –IA: 3 –IB: 1 * Includes clinical & subclinical acute rejections

10. Features of CNI Toxicity Tubular isometric vacuolization Arteriolar medial/peripheral hyaline Striped pattern of tubular atrophy and interstitial fibrosis * Ischemic collapse of glomeruli, Tubular dystrophic calcifications, juxtaglomerular apparatus hyperplasia

16. CNI Toxicity Scoring Histological featureCriteriaScore % Glomerulosclerosis0; 1-25%, 26-50%; >50%0-3 Mesangial matrix expansion0; 1-25%, 26-50%; >50%0-3 Isometric tubular vacuolization 0; 1-25%, 26-50%; >50%0-3 Tubular atrophy0; 1-25%, 26-50%; >50%0-3 Interstitial fibrosis0-5%; 6-25%, 26-50%; >50%0-3 Arteriolar hyaline 0; mild-moderate (1 arteriole); moderate-severe (1-2 arteriole); severe (many arterioles) 0-3 Total score18

17. Results (P+NP Biopsies) DiagnosisN (%) CNI toxicity77 (37%) CNIT + AR8 (4%) Acute Rejection (AR)21 (10%) Chronic allograft nephropathy75 (36%) No significant abnormality16 (7%) Other (ATN, GN, reflux etc)12 (6%) Total (P+NP)209 *C4d +ve in 1 of 189 biopsies (NP, AR IB)

18. Protocol Biopsies Diagnosis 3 mo6 mo12 mo24 moN (%) CNI toxicity1718 1467 (41%) CNIT + AR11013 (1.8%) Acute Rejection02305 (3%) CAN1713 1053 (32%) NSA721212 (7%) Other + inadequate487524 (15%) Total46444232164

19. Diagnostic features of CNIT (n=70) TV: tubular vacuoles; AH: arteriolar hyaline; SF: striped fibrosis

20. End points for graft function CNIT, BChS, MBChS and CADI correlated with –Creatinine Clearance (by Schwartz method) –Hypertension: # of anti-HTN agents to normalize blood pressure –Proteinuria CNIT score also correlated with Tacrolimus trough levels (ng/ml) and dosage (mg/kg)

21. Follow up Mean follow up period: 25.7 months (range 24-44 months) 2 patients died with functioning grafts None had urine protein/creatinine ratio > 1 Mean Creatinine Clearance at 24 months: 88.2 ml/min (range: 46-135) Mean # anti-HTN agents: 0.27 (range 0-2)

22. Results By Pearson parametric correlation (one side test) –CNI Toxicity Score at 3 months significantly correlates with 12 mo CrCl (p=0.021, r 2 =-0.54) and 24 mo CrCl (p=0.03, r 2 =-0.58)

23. Results… –No correlation with hypertension, Tacrolimus dose or levels –CADI, BChS and MBChS did not correlate with outcome –CNIT and MBChS seem to correlate with each other* * gs, ct and ci are common parameters in both

24. Scoring of Protocol Biopsies

25. Parameters of CNIT Score Gs:glomerulosclerosis; ct:tubular atrophy; ci:interstitial fibrosis; ah:arteriolar hyaline; tv:tubular vacuolization

26. Can we create a CNIT scoring model ? We reduced the # of parameters to create the model for CNIT score: = -0.16+1.05 gs+ 2.05 ct + 0.94 ah +1.03 tv Gs: glomerulosclerosis; ct: tubular atrophy; ah: arteriolar hyaline; tv: tubular vacuolization (P<0.001; r2=-0.95)

27. Testing the validity of the model Identified 14 patients with CNI toxicity on 3 month protocol biopsy with at least 12 months follow up Patients on steroid based (3) and steroid free (11) immunosuppression 11 patients had 24 mo post- txp follow up

28. Validity.. Mean CNIT score (calculated using model): 4.08 (range 1.97-9.28) 3 month CNIT score correlated significantly –12 mo CrCl (p= 0.02; r 2 =-0.54) –24 mo CrCl (p= 0.004; r 2 =-0.75)

29. CNIT Score Correlation with 12 mo CrCl (p=0.02)

30. CNIT Score Correlation with 24 mo CrCl (p=0.004)

31. Scoring System Is linear scoring of parameters better? Image analysis may be helpful Need to validate the data with more protocol biopsies (steroid free and steroid based regimens) We are probably underestimating the incidence of CNI toxicity

32. Conclusions CNIT score: helpful in objective grading A diagnosis of CNIT requires aggressive monitoring of CNI therapy Need to modify maintenance immunosuppression Arteriolar hyaline: most important factor and likely irreversible cause of progressive loss of graft function

33. Minnie Sarwal M.D., Ph.D. – Suja Nagarajan, M.D. – Sheryl Shah – Li Li Acknowledgements