Renal donation from pediatric donors is an effective means to expand the organ donor pool for adult patients with end-stage renal disease (ESRD).1,2 Studies on prognosis of adults receiving small pediatric-donor kidneys (PDKs) compared with those receiving elder pediatric or adult donor kidneys (ADKs) are controversial. Studies have shown that the intermediated-term and long-term graft survival of adult patients receiving PDKs is comparable to, or better than that of patients receiving standard-criteria adult deceased donor kidneys (ADKs).3-5 However, a disparity of recipient/donor body surface area > 1.3 m2, recipients weighted more 30 kg than the donor and a kidney/recipient weight ratio < 2.3g/kg were reported to be associated with higher risk of graft loss.6-8 These studies indicated that the outcomes of adults receiving PDKs are inferior to those of adults receiving ADKs.
The cause of renal allograft loss is multifactorial. Acute T-cell-mediated rejection (TCMR), antibody-mediated rejection, transplant glomerulopathy, de novo or recurrent glomerular pathologies, polyomavirus nephropathy, and chronic histological damage without specific causes are factors related to graft survival.9,10 Glomerular diseases including recurrent/de novo glomerulonephritis and transplant glomerulopathy are common reasons for graft failure.11
The incidence of posttransplant proteinuria is higher in adults who received small PDK compared to those who received ADK.12-14 Choung et al15 found that adults who received kidneys from pediatric donors < 10 years old had a higher incidence of a special glomerulopathy, pediatric donor glomerulopathy (PDG), which is characterized by spitting and lamination of the glomerular basement membrane (GBM), similar to the lesions seen in kidney of Alport syndrome (AS).
The aim of this study is to determine whether PDG is a cause of abnormal urinalysis (proteinuria and hematuria) in adults receiving small PDKs, to identify the specific pathological changes and to find predictors of occurrence of PDG.
PATIENTS AND METHODS
We retrospectively reviewed the renal transplant list from the First Affiliated Hospital of Sun Yat-sen University to identify adults who received single or en bloc kidney transplantation from pediatric donors < 10 years from July 1, 2011, to June 30, 2018, with follow-up through June 30, 2019. Because the thickness of GBM can grow to adult level of 300 ± 42 µm at around 10 years old, and will not increase thereafter,16 we selected pediatric donors < 10 years in this study. The renal biopsy database was reviewed to identify patients who have renal allograft biopsies after transplantation for any cause during the follow-up period. Clinical and pathological data were collected from the databases.
This study was approved by the Institutional Review Board of our hospital, and because of the retrospective nature the requirement of informed patient consent was waived.
At our institution, renal allograft biopsies were typically performed in case of increased serum creatinine (Scr), proteinuria (urine protein positive or 24 h urinary protein >150 mg), or hematuria (>3 RBC/high power field or >5 RBC/μl). Biopsies were routinely examined by light, immunofluorescent, and electron microscopy. Light microscopy included hematoxylin and eosin, periodic acid-Schiff (PAS), Masson’s trichrome, and silver methenamine stained specimens. Immunostaining for IgG, IgA, IgM, C3, C1q, Fib, and C4d was performed on frozen sections. Testing with monoclonal antibodies to the alpha 3 and alpha 5 chains of type IV collagen was performed when AS was suspected. Each kidney biopsy sample was subjected to electron microscope examination. At least one nonsclerotic glomerulus was examined if available for each patient, and the thickness of GBM was measured. GBM thickness defined as distance from endothelial to podocytic plasma membrane was measured on electronic microscope using the method reported by Hass.17 Pathological diagnosis of renal allograft biopsy specimens were in accord with the published diagnostic categories of Banff criteria.18
In this study, considering no basic diseases in pediatric grafted kidney, time-zero biopsy was not performed. In addition, given that the recipients with stable disease (no proteinuria and hematuria) after transplantation did not agree to receive biopsy, we only performed indication biopsies but not protocol biopsy, which might affect the outcome.
Descriptive statistics were expressed as mean ± SD, median values were presented for continuous variables, and percentages or ratios for categorical data. Comparisons between continuous variables were performed with Student’s t-test or Mann-Whitney U-test for, as appropriate, while the chi-square test was used for categorical variables. For categorical variables, the predictive factor was performed with univariate logistic regression, multivariate logistic regression, and multinomial logistic regression. A 2-tailed value of P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS version 23.0 software for Windows.
During the study period, 121 adults received kidneys from pediatric donors < 10 years of age. Of the 121 adults, a total of 29 biopsies and nephrectomies were performed on 23 patients between 6 and 896 days posttransplantation. The number of biopsy was one in 17 cases and 2 in 5 cases. One case with en-bloc transplantation underwent nephrectomy. All biopsies were examined by light and immunofluorescent microscope, while only 20 biopsies were examined successfully by electron microscopy, 7 biopsies failed due to no glomeruli or only fibrous tissue was examined and 2 nephrectomy cases did not applied for EM. In 7 of the 23 cases, segmental or diffuse lamination of the GBM was noted, among which 3 allografts were from infants. Examination with monoclonal antibodies to the alpha 3 and alpha 5 chains of type IV collagen was performed in all of these 7 cases. Recipients and donors’ characteristics of the 23 patients are presented in Table 1.
Each of the 29 allografts had at least 1 pathological diagnosis. Eight biopsies had rejections including 2 borderline rejections and 6 acute TCMR among which 3 combined with antibody-mediated rejection. Four cases were diagnosed with BK virus-associated nephropathy (BKVAN), 2 with acute tubular injury, 2 with IgA nephritis, 2 with focal segmental glomerulosclerosis (FSGS), 2 with graft vein rupture, 2 with nonspecific renal tubulointerstitial lesions, and 4 with nonspecific minor lesions.
In 7 of the 23 patients (30.4%), we identified PDG developed from 113 to 615 days posttransplantation. In these 7 patients, 2 had concurrent BKVAN, 1 had TCMR, 1 had FSGS, and 1 had IgA nephropathy which could not be determined recurrent or de novo because renal biopsy had not been performed in the recipients’ native kidney before renal transplantation.
A total of 18 (78.3%) recipients developed posttransplantation proteinuria and/or hematuria, which were present before or after the biopsy. At the end of the follow-up, 23 recipients were followed regularly, 2 allograft lost function, 4 lost following up, and 2 died.
Outcomes of PDG Cases
Clinical data of the 7 PDG patients were summarized in Table 2. Six of the recipients were adults ranging from 23 to 38 years of age and one (case 5) was 13 years old. Native renal disease included IgA nephritis, FSGS not otherwise specified, focal glomerulonephritis, lupus nephritis, and chronic nephritis without a specific diagnosis. The donor’s ages were from 22 days to 5 years, including 3 infants < 1 year old. Single kidney transplantation was performed in 6 cases and one case (case 4) was en bloc transplantation. Kidney length ranged from 2.5 to 8.0 cm. Kidney weight ranged from 15 to 88 g.
Six recipients showed urinary protein positive and 6 cases showed persistent hematuria until the end of follow-up or until the patient died. The death of case 2 was caused by pneumonia at 14 months posttransplantation. In addition to a pathological diagnosis of PDG, 3 patients were diagnosed with concurrent BKVAN or TCMR.
Each of the PDG cases had at least one glomerulus examined by electron microscopy. The most characteristic electron microscopy change in PDG cases was irregular lamination and splintering of GBM, leading to the increased thickness of GBM and irregular appearance of both outside and inside couture which resembled the morphologic changes in patients of AS (Figure 1). The GBM lesions were segmental or diffuse, involving mostly the lamina rara interna and externa, while the lamina densa was less affected. The lamination of lamina rara interna and externa resulted in increased distance from endothelial to podocytic plasma membrane, that is, GBM thickness, which varied from 900 to 2100 nm, while the relatively intact portion of GBM had a thickness of 120–390 nm. Segmental podocyte foot process effacement was found. No electronic dense deposits were noted within the GBM, while segments with lamination exhibited electron lucency. However, in 2 patients (case 3 and 7), electronic dense deposits were found in the mesangial area, suggesting an additional diagnosis of immune complex-mediated glomerulonephritis (Table 3).
Light microscopy of the PDG biopsy showed most of the glomeruli were small, some even immature. Segmental or global glomerulosclerosis was present in 2 biopsies, one of which was diagnosed with FSGS not otherwise specified. Mild mesangial expansion and mesangial hypercellularity was present in 2 biopsy specimens (Figure 2). Overall, mild chronic changes (<25%) including interstitial fibrosis and tubular atrophy were common in the biopsy. Focal Inflammation of the interstitium and peritubular capillaries was observed in 4 specimens. Changes suggestive of BKVAN were present in 2 cases (cases 3 and 4), and TCMR in 1 case (case 6). Other glomerular lesions including glomerulomegaly, glomerular hyalinosis, glomerular obsolescence, crescents, endothelial proliferation, basement membrane double couture, and arteriolar hyalinosis were rare in the PDG cases.
In most of the cases, immunofluorescent staining was negative for IgG, IgA, IgM, C3, C1q except for case 7 showing IgA dominant staining in mesangial area and IgM, C3 plus C1q positive in case 3 indicating immune complex-associated glomerulonephritis. C4d deposition in the peritubular capillaries was negative in all PDG cases. Immunofluorescence of alpha 3 and alpha 5 chains of type IV collagen was performed in all PDG cases, and strong staining along GBM and some TBM in PDG (Figure 3), which excluded the possibility of AS. Immunohistochemistry staining for SV40-T was performed in each biopsy and tubular epithelial cells were positive in cases 3 and 4, indicating an additional diagnosis of BKVAN.
For further analysis, recipients were divided into PDG group and non-PDG group (Table 4). Time from the day of transplantation to biopsy in PDG group was longer than that in non-PDG group (P < 0.05). The incidence of proteinuria and hematuria at biopsy time were significantly higher in PDG group (P < 0.05). Donor age, donor weight, donor kidney length, and serum creatinine at biopsy time were significantly less in PDG group compared with non-PDG group (P < 0.05), while the recipient weight in non-PDG group was significantly greater than that of PDG group (P < 0.05). Single kidney transplantation, recipient age, and recipient gender were not different between the 2 groups (all, P > 0.05). The presence of FSGS, IgA nephropathy, rejection, and BKVAN were not associated with PDG development.
The predictor of PDG incidence was analyzed by univariate and multivariable logistic regression. The donor age, donor weight, recipient age and weight, 24-hour urinary protein, and urinary RBC count until follow-up were analyzed in the logistic regression model. The data showed that increased urinary RBC count during follow-up was an independent predictor for the occurrence of PDG (Table 5), while proteinuria was not an independent predictor. Furthermore, the increasing donor age and donor weight were protective factors for PDG incidence (Table 5), which further suggested that PDG mainly occurred in pediatric donor kidney transplantation.
Finally, risk factors for increasing Scr in 23 cases of PDK and 7 cases of PDG were analyzed by multivariate logistic regression and multinomial logistic regression. Age difference between recipient and donor, posttransplant rejection, immune complex-mediated glomerulonephritis, BKVAN, and PDG was analyzed in the logistic regression model. Increasing Scr post kidney transplantation was defined as increased by >30% as compared with posttransplant baseline. As shown in Table 6, for all PDK cases, age difference itself was not a risk factor of increasing Scr, but it had an interaction effect with rejection, suggesting that the age difference becomes a significant risk factor when a rejection occurred (P < 0.05). Notably, PDG did not contribute to increasing Scr of PDKs (P = 0.941). As for the PDG cases, when there were no accompanied complications, such as posttransplant rejection, immune complex-mediated glomerulonephritis or BKVAN, recipients were less prone to increasing Scr (P < 0.001). PDG itself did not contribute to increasing Scr. One possible explanation was that the injury of GBM was reversible to some extent and proteinuria or hematuria disappeared with the growth of the allograft. However, once PDG was accompanied with rejection and immune complex-mediated glomerulonephritis, the immature allograft was relatively more susceptible to these factors, thus leading to increasing Scr.
PDG characterized by GBM lamination has only been reported in a few studies, and the first case was reported in 1991.19 The earliest case showing a 24-year-old female received a kidney from an 8-year-old boy and developed proteinuria of 8.5 g/24 hours 33 months after transplantation. The graft biopsy revealed diffuse GBM lamination. Nadasdy et al20 reported 6 cases of diffuse GBM lamination in 44 kidneys transplanted into adults from pediatric donors < 10 years old during a 10-year period from 1986 to 1995. The series showing GBM lamination was presented from 10 weeks to 36 months posttransplantation. The donor age ranged from 1 month to 6 years, and the recipient age ranged from 28 to 59 years. Four of the 6 grafts developed dysfunction after 3 months to 3 years posttransplantation, and in all cases AS was excluded. In 2014, Choung and Meleg-Smith15 reported a unique lesion of the glomerular in pediatric grafts transplanted into adults identifiable on electron microscopy, and named the lesion PDG.
To some extent, the ultrastructural lesions of the GBM in patients with PDG are similar to those seen in AS.21 However, in AS lamination of the GBM is severe, widespread, and the lamina densa is often affected, while in PDG the AS-like change mostly affected the lamina rara interna and externa. The lesion could be segmental or diffuse. Compared to diffuse AS-like GBM change reported by Nadasdy et al,20 only 3 cases in our series showed diffuse lesion, while the other 4 only segmental. Choung and Meleg-Smith15 described that PDG affected GBM mainly on the epithelial side, external lamina rara, while in our series both the internal and external lamina rara were thickened.
Most PDG cases developed proteinuria after transplantation. Up to now, there was still no threshold of proteinuria in the kidney allograft. Living kidney donors with only one functional kidney left, often have increased urinary protein after donation.22 Similarly, the recipients usually have only one functional kidney, and as such protein excretion may differ from the normal condition. The incidence of proteinuria in renal transplant patients ranges from 7.5% to 45%, depending on the definition of proteinuria.23-25 For renal transplant patients, proteinuria is the most common indicator of kidney injury, and is an independent risk factor for reduced graft and patient survival,23,24 especially when creeping persistent proteinuria is present.26 Possible reasons for posttransplant proteinuria include glomerulonephritis, chronic or acute rejection, chronic transplant glomerulopathy, and cyclosporine nephrotoxicity.27
The degree of proteinuria in PDG patients varies considerably, which may be related to the severity of GBM lesions. In our series, 6 of 7 PDG patients had mild-to-moderate proteinuria. Cases 2, 5, and 7 which developed diffused GBM change had a higher level of proteinuria, ranging from 1.210 to 2.089 g/24 hours compared to those with only segmental GBM lesion. Some reports have suggested that en bloc kidney transplantation may reduce the incidence of proteinuria, and had better long-term function if the allograft survives the early postoperative period.28 In our series, case 4 received en bloc transplantation and developed proteinuria at 615 days posttransplantation which is the latest, suggesting an en bloc transplantation might delay the occurrence of PDG.
Donor/recipient age and body weight mismatch contribute to posttransplantation complications.6,7,29 In our study, we found differences in some clinical factors between the PDG and non-PDG patients. A donor/recipient body weight mismatch was closely related with PDG incidence. PDG patients had younger PDKs compared with non-PDG. The decreasing age or weight of the pediatric donor caused increasing incidence of PDG. Persistent hematuria in recipients of PDK could be a predictor of PDG incidence. In our continuous follow-up, proteinuria in 4 PDG cases at biopsy time could gradually decrease and finally disappeared (mean: 458 d, range from 113 to 774 d) at the end of follow-up time, while hematuria persisted.
The persistent hematuria in PDG patients was not related to declined renal function made us think more about the events occurred after PDK transplantation. Kawaguchi et al30 reported that in the body weight mismatch group, the mean GBM was significantly thinner at 1-hour posttransplantation, while at 1-year posttransplantation, the mean GBM thickness has no significant difference compared with the control group. Hirukawa et al31 reported that the size of PDK grafts in adult recipient increased to adult size by 3 months posttransplant, but the glomerular area and volume were less than half of adult size at 3.5 years posttransplant, while at this time, podocytes was mature. Sureshkumar et al32 found that the glomerular filtration rate was significantly higher in PDK recipients, and Mueller-Deile et al33 demonstrated that hyperfiltration is the key trigger inducing the rapid growth response of glomerulus. All these research studies proved that hyperfusion did exist in PDK recipients and played an important role in the rapidly grow of the glomeruli and thickening of GBM, while these 2 processes might not parallel with each other. In our study, PDG was found between 3 months and 2 year (113–615 d, mean 241 d) after kidney transplantation during which the glomeruli were hyper-fused and gradually expanded together with the thickening of GBM, while podocyte was not yet mature. We also tracked the cases of PDG and found that in some cases proteinuria gradually decreased to negative in the later stage, which might represent the phenomenon of self-healing of GBM in some patients.
On the basis of previous studies and our findings, we proposed a possible pathogenesis of PDG. At the time of transplantation, GBM was initially thin. Small PDKs had a smaller glomerular volume and less total filtration area and the nephron units were relatively insufficient for adults. With the increasing glomerular filtration rate in PDK grafts, the sizes of kidney kept growing and reached adult size at about 3 months. The glomerular basement membrane began to splitting and forming GBM lamination due to high filtration pressure. At the same time, the glomerular inner cells including podocytes kept growing and differentiating. The filtration barrier became mature and reached adult level over several years after transplantation. Our findings suggest that PDG is a potential cause of abnormal urinalysis in adults receiving small PDKs, and clinically, it may be confused with recurrent or de novo glomerulonephritis without biopsy. The predominant change of PDG is GBM splitting and lamination ultrastructurally. Persistent hematuria after transplantation in recipients of PDK is a predictor of PDG development.
1. Wang Y, Lv J, Xie W, et al. Outcome of single kidney transplant from pediatric donors younger than 5 years of age after cardiac death in China: a single center experience. Exp Clin Transplant. 2015; 13:239–242
2. Li Y, Li J, Fu Q, et al. En bloc dual kidney transplantation from pediatric donors after cardiac death: initial experience in China. Urol Int. 2014; 93:482–486
3. Bhayana S, Kuo YF, Madan P, et al. Pediatric en bloc kidney transplantation to adult recipients: more than suboptimal? Transplantation. 2010; 90:248–254
4. Kayler LK, Magliocca J, Kim RD, et al. Single kidney transplantation from young pediatric donors in the United States. Am J Transplant. 2009; 9:2745–2751
5. Sharma A, Ramanathan R, Behnke M, et al. Single pediatric kidney transplantation in adult recipients: comparable outcomes with standard-criteria deceased-donor kidney transplantation. Transplantation. 2013; 95:1354–1359
6. Goldberg RJ, Smits G, Wiseman AC. Long-term impact of donor-recipient size mismatching in deceased donor kidney transplantation and in expanded criteria donor recipients. Transplantation. 2010; 90:867–874
7. Miller AJ, Kiberd BA, Alwayn IP, et al. Donor-recipient weight and sex mismatch and the risk of graft loss in renal transplantation. Clin J Am Soc Nephrol. 2017; 12:669–676
8. Giral M, Foucher Y, Karam G, et al. Kidney and recipient weight incompatibility reduces long-term graft survival. J Am Soc Nephrol. 2010; 21:1022–1029
9. Naesens M, Kuypers DR, De Vusser K, et al. The histology of kidney transplant failure: a long-term follow-up study. Transplantation. 2014; 98:427–435
10. Sellarés J, de Freitas DG, Mengel M, et al. Inflammation lesions in kidney transplant biopsies: association with survival is due to the underlying diseases. Am J Transplant. 2011; 11:489–499
11. El-Zoghby ZM, Stegall MD, Lager DJ, et al. Identifying specific causes of kidney allograft loss. Am J Transplant. 2009; 9:527–535
12. Modlin C, Novick AC, Goormastic M, et al. Long-term results with single pediatric donor kidney transplants in adult recipients. J Urol. 1996; 156:890–895
13. Hayes JM, Steinmuller DR, Streem SB, et al. The development of proteinuria and focal-segmental glomerulosclerosis in recipients of pediatric donor kidneys. Transplantation. 1991; 52:813–817
14. Chen CH, Shu KH, Yang CR, et al. Long-term results with pediatric kidney transplants in adult recipients. J Formos Med Assoc. 1999; 98:807–813
15. Choung HY, Meleg-Smith S. Glomerulopathy in adult recipients of pediatric kidneys. Ultrastruct Pathol. 2014; 38:141–149
16. Ramage IJ, Howatson AG, McColl JH, et al. Glomerular basement membrane thickness in children: a stereologic assessment. Kidney Int. 2002; 62:895–900
17. Haas M, Sis B, Racusen LC, et al.; Banff Meeting Report Writing CommitteeBanff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am J Transplant. 2014; 14:272–283
18. Loupy A, Haas M, Solez K, et al. The banff 2015 kidney meeting report: current challenges in rejection classification and prospects for adopting molecular pathology. Am J Transplant. 2017; 17:28–41
19. Truong LD, Yoshikawa Y, Mawad J, et al. Electron microscopic study of an unusual posttransplant glomerular lesion. Arch Pathol Lab Med. 1991; 115:382–385
20. Nadasdy T, Abdi R, Pitha J, et al. Diffuse glomerular basement membrane lamellation in renal allografts from pediatric donors to adult recipients. Am J Surg Pathol. 1999; 23:437–442
21. Kalluri R, Shield CF, Todd P, et al. Isoform switching of type IV collagen is developmentally arrested in X-linked alport syndrome leading to increased susceptibility of renal basement membranes to endoproteolysis. J Clin Invest. 1997; 99:2470–2478
22. Garg AX, Muirhead N, Knoll G, et al.; Donor Nephrectomy Outcomes Research (DONOR) NetworkProteinuria and reduced kidney function in living kidney donors: a systematic review, meta-analysis, and meta-regression. Kidney Int. 2006; 70:1801–1810
23. Amer H, Fidler ME, Myslak M, et al. Proteinuria after kidney transplantation, relationship to allograft histology and survival. Am J Transplant. 2007; 7:2748–2756
24. Fernández-Fresnedo G, Plaza JJ, Sánchez-Plumed J, et al. Proteinuria: a new marker of long-term graft and patient survival in kidney transplantation. Nephrol Dial Transplant. 2004; 19Suppl 3iii47–iii51
25. Naesens M, Lerut E, Emonds MP, et al. Proteinuria as a noninvasive marker for renal allograft histology and failure: an observational cohort study. J Am Soc Nephrol. 2016; 27:281–292
26. Calabuig AS, Martínez EG, Berga JK, et al. Prognostic significance of creeping proteinuria in the first year after transplantation. Transplantation. 2015; 99:2598–2607
27. Vathsala A, Verani R, Schoenberg L, et al. Proteinuria in cyclosporine-treated renal transplant recipients. Transplantation. 1990; 49:35–41
28. Sureshkumar KK, Reddy CS, Nghiem DD, et al. Superiority of pediatric en bloc renal allografts over living donor kidneys: a long-term functional study. Transplantation. 2006; 82:348–353
29. Yamakawa T, Kobayashi A, Yamamoto I, et al. Clinical and pathological features of donor/recipient body weight mismatch after kidney transplantation. Nephrology (Carlton). 2015; 20 (Suppl 236–39
30. Kawaguchi Y, Oguchi H, Mikami T, et al. Donor-recipient body weight mismatch may affect glomerular basement membrane thinning in electron microscopic examination of 1-hour renal allograft biopsy specimens. Transplant Proc. 2019; 51:1348–1352
31. Hirukawa T, Suzuki H, Niimura F, et al. En bloc cadaver kidney transplantation from a 9-month-old donor to an adult recipient: maturation of glomerular size and podocyte in the recipient. Transplant Direct. 2017; 3:e130
32. Sureshkumar KK, Habbach A, Tang A, et al. Long-term outcomes of pediatric en bloc compared to living donor kidney transplantation: a single-center experience with 25 years follow-up. Transplantation. 2018; 102:e245–e248
33. Müller-Deile J, Bräsen JH, Pollheimer M, et al. Graft growth and podocyte dedifferentiation in donor-recipient size mismatch kidney transplants. Transplant Direct. 2017; 3:e210