Histological reversibility of diabetic nephropathy after kidney transplantation from diabetic donor to non-diabetic recipient

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1 bs_bs_banner Nephrology 20, Suppl. 2 (2015) Original Article Histological reversibility of diabetic nephropathy after kidney transplantation from diabetic donor to non-diabetic recipient SHUMPEI HARADA, 1 HIDETAKA USHIGOME, 1 AYAKO NISHIMURA, 2 TOSHIMASA NAKAO, 1 TSUKASA NAKAMURA, 1 KATSUHIRO KOSHINO, 1 TOMOYUKI SUZUKI, 1 TAKASHI ITOH, 1 SHUJI NOBORI 1 and NORIO YOSHIMURA 1 1 Department of Organ Transplantation and General Surgery, Kyoto Prefectural University of Medicine, Kajii-cho 465, Kamigyo-ku, Kyoto-prefecture, , Japan; and 2 Department of Clinical Pathology, Kyoto Prefectural University of Medicine, Kajii-cho 465, Kamigyo-ku, Kyoto-prefecture, , Japan KEY WORDS: Diabetic nephropathy, donor, kidney transplantation, reversibility. Correspondence: Mr Hidetaka Ushigome, Department of Organ Transplantation and General Surgery, Kyoto Prefectural University of Medicine, Postal code: , Kajii-cho 465, Kamigyo-ku, Kyoto-prefecture, Japan. ushi@koto.kpu-m.ac.jp Accepted for publication 14 March doi: /nep Conflict of interest: The authors have no conflict of interest to declare. ABSTRACT: Aim: Given the recent increase in the prevalence of diabetes mellitus, it is not uncommon for kidney transplantation donors to have diabetes. We perform kidney transplantation in our hospital if the diabetic donors are receiving oral hypoglycaemic agents, but not insulin, and their haemoglobin A1C (HbA1C) is below 6.5%. There are few reports about histological changes to diabetic nephropathy after transplantation of kidney grafts from donors with diabetes mellitus to non-diabetic recipients. Therefore, we studied the histological diabetic changes in grafts from diabetic donors at protocol biopsies (1 hour, 1 month, 1 year), and evaluated whether they improved under the recipient s good glycaemic control. Methods: Three cases of kidney transplantation from donors with diabetes mellitus to non-diabetic recipients were selected. We used a pathological classification established by the Renal Pathology Society for evaluating histological improvements in diabetic nephropathy. Results: The results revealed that early diabetic changes found at the 1-hour and 1-month protocol biopsies were reversed and improved at the 1-year biopsy. Conclusion: We concluded that early diabetic changes in grafts from diabetic donors may improve if the graft recipient has good glycaemic control after kidney transplantation. Given the recent increase in the number of diabetic patients in Japan, it is not uncommon for kidney transplantation (KTx) donors to have diabetes mellitus (DM). A guideline for living kidney, marginal DM donors in Japan recommends that donors should not be receiving injected insulin, and should have a haemoglobin A1C (HbA1C) below 6.5% (NGSP) and microalbuminuria below 30 mg/g creatinine (CRE). We perform living KTx according to these criteria in our hospital. Whether diabetic nephropathy (DN) is reversible or not has long been debated. Several researchers have concluded that strict glycaemic control can reverse and improve renal function changes, and reduce microalbuminuria in the early stages of DM. 1 3 Histologically, Fioretto et al. have reported that diabetic changes in the kidneys of type 1 diabetes patients are reversible after pancreatic transplantation alone (PTA). 4 6 However, in the setting of kidney transplantation, there has been only one report in Abouna et al. 7 reported that the diabetic changes, including diffuse glomerulosclerosis and thickening of mesangial matrix and capillary basement membranes, in a graft from a cadaveric donor with a 17-year history of type 1 diabetes had reversed almost completely in the renal biopsy taken 7 months after KTx. Moreover, few reports about pathological reversibility of DN in kidneys of type 2 diabetes patients exist, despite some reports about this in type 1 diabetes patients. 8 This is because, for the most part, sequential renal biopsies are not performed in type 2 diabetes patients except in KTx recipients. The diagnosis of DN is made according to clinical manifestations in most cases, and a kidney biopsy is performed only for atypical presentations. Thus, whether DN caused by type 2 diabetes is reversible or not is yet to be elucidated. Early diabetic changes in renal grafts from donors with type 2 diabetes are often found at the 1-hour biopsy in KTx operations. We hypothesized that such early diabetic changes in renal grafts from diabetic donors are reversible if recipients are not diabetic and glycaemic control is good after the KTx. 40

2 Reversibility of diabetic nephropathy The purpose of the present study is to investigate nondiabetic KTx recipients from DM donors, and study how early diabetic histological lesions in grafts change in protocol biopsies after KTx under good glycaemic control. METHODS Recipients Since 1970, 954 kidney transplantations have been performed at Kyoto Prefectural University of Medicine, including 25 KTx from diabetic donors. We excluded cases in which recipients had had DM before KTx, had developed post-transplant DM (PTDM), including new-onset diabetes after transplantation (NODAT), cases of obvious rejection in renal biopsies, and those that did not have at least three renal biopsy results. Finally, we selected three cases. All of the patients received immunosuppressive therapy with basiliximab (Simulect, Novartis Pharma, Basel, Switzerland), prednisolone, cyclosporine (Sandimmun Neoral, Novartis Pharma) or tacrolimus (Prograf, Astellas Pharma) and mycophenolate mofetil (Cellcept, Chugai Pharma) or mizoribine (Bredinin, Asahikasei Pharma) in accordance with our institutional protocol. In case 1 and 2, a blood sugar level was transiently high after KTx due to steroid, and temporal insulin injection was administrated. The glycaemic control of both cases at 1 month after KTx was good. In case 3, the glycaemic control was good after KTx. The demographics of the recipients are shown in Table 1. Donors The three donors had had DM before KTx. In accordance with the Japanese guidelines for living KTx donors, we had confirmed that their HbA1c levels were <6.5%, microalbuminuria was below 30 mg/g CRE and no insulin injections had been administered. The demographics of the donors are shown in Table 1. None of the donors had diabetic retinopathy before KTx. Histological classification of diabetic nephropathy Light microscopy and electron microscopy were performed for each biopsy specimen. Haematoxylin eosin stain, periodic acid-schiff stain, Masson s trichrome stain and Periodic acid-schiff stain were performed for light microscopy sections. Three glomeruli were randomly selected in each biopsy and three electron micrographs of GBM at magnification were taken. GBM width was measured at 5 points in each electron micrograph and the average width of GBM was calculated. We used a pathological classification scheme of DN described by Tervaert et al. 9 to evaluate histological improvements in DN because there is no internationally recognised classification for DN, unlike for other kidney diseases such as lupus nephritis, focal segmental glomerulosclerosis, and IgA nephropathy. In this classification, diabetic nephropathy is classified into four hierarchical glomerular lesions as follows: Class I, glomerular basement membrane thickening; Class IIa, mild mesangial expansion; Class IIb, severe mesangial expansion; Class III, nodular sclerosis (Kimmelstiel Wilson lesions); Class IV, advanced diabetic glomerulosclerosis (Table 2). In addition, we defined cases that did not fit into any class as class 0. In Class I, on average, GBM is thicker than 430 nm in males and 395 nm in females aged 9 years and older. For the patient in case 3, who was younger than 9 years of age, we referred to normal ranges ( nm) from the paper by Haas. 10 DN has varying histological findings glomerular lesions, tubulointerstitial lesions and vascular lesions. But we remarked on only glomerular lesions, because they are considered the best reflection of the natural course of progressive DN and because interobserver agreement is good. All sections were observed by two pathologists in order to assess reliability. This study was approved by the Ethics Committee of Kyoto Prefectural University and conducted in accordance with the Declaration of Helsinki. RESULTS Pathological classification The results of the pathological classification are shown in Table 3, and the GBM widths measured by electron microscopy are shown in Figure 2. In case 1, a mild mesangial expansion (IIa) was found in a glomeruli at the 1-hour and Table 1 Demographics of living kidney, non-diabetic (DM) transplant recipients and diabetic (DM) donors IS, immunosuppressant; KTx, kidney transplantation; CGN, chronic glomerular nephritis; CyA, cyclosporine; FK, tacrolimus; MMF, mycophenolate mofetil; Mz, mizoribine; CCr, creatinine clearance; αgi, α-glucosidase inhibitor; SU, sulfonylurea Case (Recipient) Age (y) Sex Primary disease IS Date of KTx Body weight (kg)/ BMI HbA1c (NGSP,%) Pre KTx 1year Pre KTx 1year 1 57 F IgA nephropathy CyA/MMF / / F CGN CyA/Mz / / M Hypoplastic kidney FK/Mz / / Case (Donor) Age (y) Sex Duration of DM (y) Drugs CCr (ml/min) Microalbuminuria (mg/g CRE) Body weight (kg)/ BMI HbA1c (NGSP,%) Pre KTx 1year Pre KTx 1year Pre KTx 1year Pre KTx 1year 1 54 M 2 αgi/su 83.6 not followed <30 not followed 75.8/27.4 not followed 5.7 not followed 2 54 F 2 αgi <30 < / / M 5 SU <30 < / /

3 S Harada et al. Table 2 Pathological classification of diabetic nephropathy (DN) We defined a finding that did not apply to any class as class 0. *For the 7-year-old patient in case 3, we referred to normal ranges ( nm) in the paper by Haas. 9 LM, light microscopy; EM, electron microscopy; GBM, glomerular basement membrane (Tervaert TWC et al. Pathologic classification of diabetic nephropathy. J. Am. Soc. Nephrol. 2010; 21: ) Class Description Inclusion Criteria 0 No significant findings Biopsy does not meet I IV I Mild or nonspecific LM changes and EM-proven GBM thickening Biopsy does not meet any of the criteria mentioned below for class II, III, or IV GBM 395 nm in female and 430 nm in male individuals 9 years of age and older* IIa Mild mesangial expansion Biopsy does not meet criteria for class III or IV Mild mesangial expansion in 25% of the observed mesangium IIb Severe mesangial expansion Biopsy does not meet criteria for class III or IV Severe mesangial expansion in 25% of the observed mesangium III Nodular sclerosis (Kimmelstiel Wilson lesion) Biopsy does not meet criteria for class IV At least one convincing Kimmelstiel Wilson lesion IV Advanced diabetic glomerulosclerosis Global glomerular sclerosis in 50% of glomeruli Lesions from classes I through III A B C Fig. 1 Light microscopic section of glomeruli stained in Periodic acid-schiff stain at 1 hour, 1 month and 1 year after kidney transplantation (KTx) in case 1. A: 1 hour, B: 1 month, C: 1 year Mild mesangial expansion found at 1 hour (A) and 1 month (B) had improved at 1 year (C). Table 3 Results of the pathological classification of diabetic nephropathy (DN) Case Protocol biopsy 1 hour 1 month 1 year 1 IIa IIa 0 2 I I 0 3 I I 0 1-month biopsies on light microscopy, and those findings had improved at the 1-year biopsy (Fig. 1). GBM widths greater than 430 nm were not found in all biopsies (Fig. 2). In case 2, nonspecific light microscopic changes were found in all biopsies and GBM width was above 430 nm (I) at 1 hour and 1 month, but below 430 nm (0) at 1 year (Fig. 2,3). In case 3, nonspecific light microscopic changes were found in all biopsies and GBM width was above 391 nm (I) at 1 hour and 1 month, but was below 391 nm (0) at 1 year (Fig. 2). DISCUSSION In this study, we evaluated the reversibility of diabetic changes in kidney grafts from type 2 diabetic donors after KTx. The results revealed that early diabetic changes (ClassI, IIa) found at the 1-hour biopsy can reverse and improve by the 1-year biopsy under conditions of good glycaemic control. The lack of significant differentiation between the pathological findings at 1 hour and 1 month suggests that this was too short a period for improvement to occur. Thickening of GBM and mesangial expansion are the most important and characteristic findings of DN. 11 GBM thickness improved in case 2 and 3 at the 1-year biopsy. Mesangial 42

4 Reversibility of diabetic nephropathy expansion found in case 1 at 1 hour and 1 month had improved at 1 year. A main mechanism of DN is the accumulation of extracellular matrix (ECM) in the mesangium, glomerular and tubular basement membranes and the interstitium under hyperglycaemic conditions. 12 Byran et al. 13 reported that tight blood glycaemic control reduced ECM production in experimental DN. Mauer et al. 8 showed that in patients with pancreas transplantation (PTA), kidney GBM width (nm) hour 1 month 1 year case 1 case 2 case3 Fig. 2 Glomerular basement membrane (GBM) width (nm) measured by electron microscopy. In case 1, GBM widths greater than 430 nm were not found in any of the biopsies. In case 2, GBM width was above 430 nm at 1 hour and 1 month, but below 430 nm at 1 year. In case 3, GBM width was above 391 nm at 1 hour and 1 month (for the 7-year-old patient in case 3, we referred to normal ranges nm in the paper by Haas. 9 ), but was below 391 nm at 1 year. Case 1: (±5.63), (±4.78), (±6.32) Case 2: (±4.23), (±5.20), (±7.32) Case 3: (±5.46), (±6.21), (±6.41) GBM width decreased 10 years after PTA compared with baseline and 5 years. Furthermore, Fioretto et al. 6 reported that the glomerular lesions, even Kimmelstiel-Wilson nodules, of most PTA recipients were markedly improved after 10 years. Therefore, it stands to reason that normoglycaemia after KTx can improve the early diabetic changes seen in grafts at the 1-hour biopsy within 1 year. We used a classification of DN described by Tervaert et al., 10 focussing on only glomerular lesions in this study. Several studies have shown that glomerular lesions are the most characteristic histological changes in DN, 14 and there is a significant correlation between the severity of glomerular lesions and decreased renal function. 15,16 Oh et al. 17 reported this classification was a good predictor of renal prognosis in type 2 DM. Moreover, all KTx recipients received a calcineurin inhibitor (CNI), cyclosporine and tacrolimus, as part of their immunosuppressive regimen. CNI are nephrotoxic, 18,19 including interstitial fibrosis, arteriolopathy and tubular atrophy. 20,21 Therefore, this classification is very useful and makes various pathological findings of DN after KTx simple and easy to understand. However, it has not been established whether this classification accurately reflects renal function and outcome after KTx. It will require further and longer investigations to find a significant correlation between this classification and renal function after KTx. This study has a limitation in that the numbers of patients is too small to prove our hypothesis. We excluded cases showing signs of rejection in renal biopsies after KTx, because it was very difficult to distinguish between glomerular changes caused by DN and rejection. Moreover, we also excluded recipients who did not have at least three protocol biopsies. More patients and further study are needed to confirm our hypothesis. A B C Fig. 3 Electron microscopy section of glomeruli at 1 hour, 1 month and 1 year after kidney transplantation (KTx) in case 2 at magnification A: 1 hour, B: 1 month, C: 1 year Thickness of the glomerular basement membrane (GBM) at A and B had improved at C. 43

5 S Harada et al. Evaluating progression and regression of DN using this classification in protocol biopsies, including electron microscopy, may make it possible to predict the prognosis of the graft. In the case of DN progression, we should achieve stricter control of recipients glycaemic levels. As the donors did not have severe DM, in accordance with the guideline for living KTx donors, the diabetic changes in grafts are usually slight on light microscopy, and it is too difficult to evaluate improvements of DN using only light microscopy. Thus, measuring GBM thickness by electron microscopy is effective for evaluating minor diabetic changes. Using electron microscopy to routinely follow up KTx recipients grafts from DM donors is recommended. CONCLUSIONS In summary, early diabetic changes in grafts from DM donors can improve after KTx if subsequent glycaemic control is good. Following up using electron microscopy is recommended to precisely evaluate the progression and regression of DN. Further investigation is required to show whether there is a significant correlation between this classification and renal function after KTx. REFERENCES 1. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N. Engl. J. Med Sep 30; 329 (14): Turner R, UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet. 1998; 352: Steffes M, Chavers B, Molitch M. Sustained effect of intensive treatment of type 1 diabetes mellitus on development and progression of diabetic nephropathy: the Epidemiology of Diabetes Interventions and Complications (EDIC) study. JAMA Oct 22; 290 (16): Fioretto P, Mauer M. Effects of pancreas transplantation on the prevention and reversal of diabetic nephropathy. Contrib. Nephrol. 2011; 170: Fioretto P, Mauer M. Reversal of diabetic nephropathy: lessons from pancreas transplantation. J. Nephrol. 2012; 25: Fioretto P, Barzon I, Mauer M. Is diabetic nephropathy reversible? Diabetes. Res. Clin. Pract. 2014; 104: Abouna GM, Kremer GD, Daddah SK, Al-Adnani MS, Kumar SA, Kusma G. Reversal of diabetic nephropathy in human cadaveric kidneys after transplantation into non-diabetic recipients. Lancet 1983; 322: Mauer M, Fioretto P. Pancreas transplantation and reversal of diabetic nephropathy lesions. Med. Clin. North. Am. 2013; 97: Tervaert TWC, Mooyaart AL, Amann K, Cohen AH, Cook HT, Drachenberg CB et al. Pathologic classification of diabetic nephropathy. J. Am. Soc. Nephrol. 2010; 21: Haas M. Alport syndrome and thin glomerular basement membrane nephropathy: a practical approach to diagnosis. Arch. Pathol. Lab. Med. 2009; 133: Dalla Vestra M, Saller A, Mauer M, Fioretto P. Role of mesangial expansion in the pathogenesis of diabetic nephropathy. J. Nephrol. 2001; 14 (Suppl 4): S Fioretto P, Mauer M. Histopathology of diabetic nephropathy. Semin. Nephrol. 2007; 27: Conway BR, Betz B, Sheldrake TA, Manning JR, Dunbar DR, Dobyns A et al. Tight blood glycaemic and blood pressure control in experimental diabetic nephropathy reduces extracellular matrix production without regression of fibrosis. Nephrology (Carlton) Sep 4; doi: /nep Alsaad KO, Herzenberg AM. Distinguishing diabetic nephropathy from other causes of glomerulosclerosis: an update. J. Clin. Pathol. 2007; 60: Heaf JG, Løkkegaard H, Larsen S. The relative prognosis of nodular and diffuse diabetic nephropathy. Scand. J. Urol. Nephrol. 2001; 35: Hong D, Zheng T, Jia-qing S, Jian W, Zhi-hong L, Lei-shi L. Nodular glomerular lesion: a later stage of diabetic nephropathy? Diabetes. Res. Clin. Pract. 2007; 78: Oh SW, Kim S, Na KY, Chae D-W, Kim S, Jin DC. Clinical implications of pathologic diagnosis and classification for diabetic nephropathy. Diabetes. Res. Clin. Pract. 2012; 97: Naesens M, Kuypers DRJ, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin. J. Am. Soc. Nephrol. 2009; 4: Issa N, Kukla A, Ibrahim HN. Calcineurin inhibitor nephrotoxicity: a review and perspective of the evidence. Am. J. Nephrol. 2013; 37: Davies DR, Bittmann I, Pardo J. Histopathology of calcineurin inhibitor-induced nephrotoxicity. Transplantation 2000; 69 (12 Suppl): SS Liptak P, Ivanyi B. Primer: Histopathology of calcineurin-inhibitor toxicity in renal allografts. Nat. Clin. Pract. Nephrol. 2006; 2: ; quiz following