Prediction of Renal Function in Living Kidney Donors and Recipients of Living Donor Kidneys Using Quantitative Histology : Transplantation

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Original Clinical Science—General

Prediction of Renal Function in Living Kidney Donors and Recipients of Living Donor Kidneys Using Quantitative Histology

Buus, Niels Henrik MD, PhD1,2,*; Nielsen, Cecilie Mølgaard MD1,3,*; Skov, Karin MD, PhD1; Ibsen, Lotte MD4; Krag, Søren MD, PhD5; Nyengaard, Jens Randel MD, DMSc2,3

Author Information
Transplantation 107(1):p 264-273, January 2023. | DOI: 10.1097/TP.0000000000004266
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Abstract

INTRODUCTION

There is epidemiological evidence that living kidney donors (LKDs) have an increased probability of end-stage kidney disease compared with matched nondonors.1-3 This may be related to risk factors such as hypertension and obesity4-6 but is also more common in donors with low predonation renal function or higher age.1,2 In healthy and hypertensive individuals, a low glomerular number has been associated with development of hypertension and chronic kidney disease (CKD)7,8—a phenomenon possibly related to single-nephron hyperfiltration and accelerated glomerular sclerosis.9,10 Special awareness of donors at increased risk for CKD is, therefore, important, and assessment of glomerular number and other histological features in addition to function of the nondonated kidney may potentially improve prediction of postdonation renal function and risk of CKD development.11

Preexisting histological abnormalities such as interstitial fibrosis and glomerular sclerosis in deceased donor kidneys are known to influence long-term graft function,12-14 and similar findings have been reported in recipients of living donor kidneys.15 However, numerous posttransplant immunological and nonimmunological factors affect the transplanted kidney possibly diminishing the value of histomorphometric parameters assessed at the time of transplantation. This makes it important to compare the value of donor kidney histology assessment on donor and recipient renal outcome in the same methodological setting.

Many studies relating histological features to renal outcome in LKDs and recipients are based on assessment of renal function using estimated glomerular filtration rate (eGFR), which can be imprecise.16,17 Exact determination of GFR and calculation of postdonation hyperfiltration demands measurements of single-kidney GFR (skGFR) determined by a marker truly reflecting glomerular filtration.

The aim of this study was to identify histological parameters predicting renal outcome in terms of measured GFR 1 y after donation in both LKDs and recipients of transplants from LKDs. Using a set of model- and design-based stereological methods, estimates of kidney structural parameters from LKDs and recipients were created from a contrast-enhanced computed tomography (CT) scan and a perioperative kidney biopsy.

MATERIALS AND METHODS

Study Population

The present study is part of a comprehensive project investigating blood pressure (BP) and vascular alterations in LKDs and recipients of living donor kidneys.18 During a 33-mo period, LKDs and their corresponding recipients were considered for participation. Non–Danish-speaking donors and recipients and foreign donors not likely to be available for study-related follow-up were not asked. The standard pretransplantation donor examination program consisted of a renal angiography to evaluate kidney anatomy, an isotope renography, and determination of GFR using a plasma clearance technique (see below). Only healthy individuals with a measured GFR >80 mL/min/1.73 m2 (if ≤60 y) and >60 mL/min/1.73 m2 (if >60 y), even distribution of GFR between the kidneys (±5%), no albuminuria, and normal BP during treatment with maximum one antihypertensive drug are accepted as donors. Of relevance for the current article, donors and recipients were asked to have a GFR measurement 1 y after donation or transplantation. The study was approved by the Central Denmark Region Committees on Health Research Ethics, and the participants signed an approved consent.

Among a total of 69 available donor-recipient pairs, 53 donors and 57 recipients agreed to have the additional 1-y follow-up GFR measurement (Figure 1). However, 4 donors and 5 recipients did not attend the GFR measurement at 1 y, and 1 recipient died 6 mo after the transplantation. However, to minimize the loss of participants, we included predonation standard examination results (cortex volume and skGFR) from 15 non participating donors to participating recipients. Likewise, we used the renal graft morphology data from 11 nonparticipating recipients to evaluate the outcome of their participating donors. This resulted in 49 donors and 51 recipients (of whom 38 were donor-recipient pairs) with 1-y GFR measurement (Figure 1).

F1
Figure 1.:
Flowchart illustrating the number of potential and participating living kidney donors and recipients. See text for further details.

skGFR and Hyperfiltration

Donors had measurements of GFR before and 1 y after donation and the recipients 1 y after transplantation in terms of a 51chrome-EDTA plasma clearance.19 Within a few days of the GFR determination, a 99mTc-MAG3 renography was also performed allowing calculation of skGFR. Hyperfiltration in donors is calculated as GFR at follow-up divided by skGFR of the nondonated kidney before donation, while hyperfiltration in recipients is calculated as the GFR at 1-y follow-up divided by skGFR of the donated kidney before donation.

Renal Angiography

Among the included donors, 49 had a contrast-enhanced renal CT and 2 an MRI. Using systematic uniform random sampling, a minimum of 10 images was chosen per kidney. For each of these images, the kidney cortical area was determined as the difference between the total kidney area and medulla area.20 Cortex volume (Vcortex) was then determined using the Cavalieri estimator based on the slice thickness and number of images between the sampled images as previously explained in detail.21 Determination of cortex volume was performed in the same way in CT- and MRI-based angiographies. The volumes of both the donated and nondonated kidney cortices were calculated with the donated kidney representing the recipient and the nondonated kidney representing the donor at the time of follow-up.

Ambulatory Blood Pressure

Twenty-four-hour ambulatory BP measurements were performed with Spacelab Medical 90217BP monitors (Spacelab Healthcare, Issaquah, WA) every 20 min as explained previously.18 Mean arterial BP was retrieved from the Spacelab output. However, 4 participants (2 donors and 2 recipients) did not have ambulatory recordings at follow-up, and for these, we used unattended office BP, which corresponds to daytime ambulatory BP.22

Histological Examination of Renal Biopsies

As a standard procedure during the transplantation, 1 or 2 biopsies (18G needle) were taken immediately after implantation of the kidney graft. The biopsies were fixed in phosphate-buffered 4% formaldehyde and embedded in paraffin. Approximately 15 serial sections were cut at 2-μm thickness and stained with hematoxylin and eosin, periodic acid-Schiff, and Masson trichrome. The quantitative histological evaluation was used to evaluate the biopsies as described previously in detail21 using an Olympus BX50 light microscope (Olympus Denmark, Ballerup, Denmark) equipped with a prior motorized stage, an Olympus DP70 digital camera interfaced to a PC with commercially available newCAST software (Visiopharm, Hørsholm, Denmark). Patient characteristics were unknown to the investigator analyzing the biopsies.

The total number of glomerular profiles in the biopsy material varied from 10 to 26. Half glomerular profiles were not included, but glomerular profiles >75% were included if it was possible to visualize the lacking outline. Glomerular sclerosis was defined as global sclerosis while segmental sclerosis was not included in this term. Because the number of glomeruli in the biopsy was known, we obtained an exact percentage of sclerotic glomerular profiles.

The mean area of all glomeruli in a biopsy was determined by measuring the area of all glomerular profiles in all sections using the 2-dimensional nucleator together with a ×4 lens (NA 0.13) and the outline of the glomerular tuft defined as the boundary. Afterward, it was known exactly how many glomerular profiles occurred in the biopsy and how many sections they appeared on. Assuming a spherical shape of the glomerulus, and based on the Weibel-Gomez method, glomerular volume (Vglom) was then estimated.21,23 Vglom was further corrected for loss of glomerular perfusion pressure24 and tissue shrinkage during paraffin embedding.25

Systematically scattered test points (≈100) across the Masson trichrome–stained sections estimated the amount of kidney fibrosis using a ×4 lens (NA 0.13). Likewise, the proportion of test points landing on cortical tissue was used to estimate total biopsy cortex area. The glomerular number density was estimated by dividing the number of glomerular profiles per area by the average glomerular diameter taking section thickness into account.21 Finally, the total number of glomeruli per kidney could then be calculated by multiplying glomerular number density with the CT- or MRI-based kidney cortex volume.

All arteriolar profiles on the periodic acid-Schiff–stained sections were identified and measured using a ×60 oil lens (NA 1.25). Lumen and tunica media areas were estimated using the 2-dimensional nucleator. The media:lumen area ratio, based on ≥1 arteriolar profiles, was calculated by dividing tunica media area by the combined luminal and media areas.21

Statistical Evaluation

Data are presented as means with SD or median with 95% confidence intervals. Simple linear regression is used to visualize the associations between skGFR and cortex volume or histological parameters at baseline and between GFR at 1 y and histological parameters in LKDs and recipients. GFR was not corrected for body surface, as kidney cortex volume and histological parameters were also unadjusted. The influence of the histological parameters on GFR at follow-up was also tested using multiple linear regression or logistic regression with adjustment for age, sex, BMI, 24-h mean BP (for donors, donor data were used, and for recipients, recipient data were used), baseline skGFR, and the exact number of days between transplantation and follow-up. P values <0.05 were considered statistically significant.

RESULTS

The clinical characteristics of the included LKDs and living donor transplant recipients are shown in Table 1. The recipients were slightly younger, more often men, and had significantly higher 24-h mean ambulatory BP as compared to the donors. At follow-up, mean arterial pressure remained unchanged in donors but decreased in the recipients. The 15 donors not included due to lack of mGFR at follow-up did not differ from those included regarding gender distribution and average age (52 y). eGFR was accessible in 13 of these with a mean value of 66 ± 19 mL/min/1.73 m2 using the CKD-EPI equation.26

TABLE 1. - Clinical characteristics of living kidney donors and renal transplant recipients with available measurements of GFR at 1-y follow-up
Donors Recipients
n 49 51
Women, % 53 24
Age, y 50.7 ± 11.8 44.2 ± 13.1
Diabetes, % 0 12
Active smokers, % 24 20
Body mass index, kg/m2
 Baseline (pre-transplantation) 25.7 ± 3.3 24.4 ± 3.1
 Follow-up 26.0 ± 3.7 25.0 ± 3.7
24-h mean BP, mm Hg
 Baseline (pre-transplantation) 90 ± 9 102 ± 15
 Follow-up 90 ± 9 98 ± 8 a
Data are mean ± SD.
aP < 0.05 as compared to baseline.
BP, blood pressure; GFR, glomerular filtration rate.

For LKDs, the linear association between single-kidney cortex volume and skGFR is shown in Figure 2A, whereas the association between the number of nonsclerosed glomeruli and skGFR is depicted in Figure 2B demonstrating skGFR to be dependent on both factors.

F2
Figure 2.:
Linear associations between cortex volume and pretransplantation (baseline) skGFR (A) and glomerular number and skGFR (B) in living kidney donors. Data are based on the 53 donors investigated pre-transplantation (106 kidneys). skGFR, single-kidney glomerular filtration rate.

skGFR at baseline and GFR at follow-up are presented in Table 2 and Figure 3A and B for the nondonated and donated kidneys, respectively. skGFR was similar in nondonated and donated kidneys, and significant correlations between skGFR and follow-up GFR were observed for both nondonated and donated kidneys. Hyperfiltration at follow-up was highly variable among donors and recipients, and the data are presented in Table 2 and Figure 3C–F. The percentage of hyperfiltration was higher for nondonated as compared to donated kidneys (P < 0.05). The degree of hyperfiltration correlated negatively to baseline skGFR for nondonated and donated kidneys. Especially in recipients‚ a low skGFR seems to be associated with more hyperfiltration‚ whereas this was less obvious in donors. As expected, the degree of hyperfiltration correlated positively to GFR at follow-up‚ and this was significant for both donors and recipients.

TABLE 2. - Renal function, cortex volume, and histological characteristics of nondonated and donated kidneys
Nondonated kidney Donated kidney
Single-kidney GFR, mL/min
 Baseline (pre-transplantation) 55.1 ± 11.4 55.9 ± 14.5
 Follow-up 71.2 ± 15.9 62.8 ± 18.4
Hyperfiltration, % 30.4 ± 22.9 16.5 ± 35.3 a
Kidney volume, cm3
 Total 161 ± 30 163 ± 35
 Cortex 95 ± 20 96 ± 23
Glomerular number, b 103 910 ± 460 900 ± 410
Glomerular volume, 106 μm3 4.6 ± 1.6 4.7 ± 1.6
Globally sclerotic glomeruli, % 0 (0-1.9) 0 (0-1.8)
Kidney fibrosis, % 6.3 (5.6-7.0) 5.8 (5.2-6.5)
Arteriolar media-lumen ratio 0.22 ± 0.1 0.20 ± 0.1
Data are mean ± SD or median with 95% confidence intervals.
aP < 0.05.
bNonsclerotic.
GFR, glomerular filtration rate.

F3
Figure 3.:
The upper panel figures show linear associations between pretransplantation skGFR of the nondonated kidney and the corresponding GFR at 1-y follow-up in donors (A) and skGFR of the donated kidney and GFR at 1-y follow-up in recipients (B). The middle panel figures demonstrate the relation between skGFR of the nondonated kidney and percentage hyperfiltration at 1 y in donors (C) and between skGFR of the donated kidney and percentage hyperfiltration at 1 y in recipients (D). The lower panel figures show the relation between percentage hyperfiltration and GFR at 1-y follow-up in donors (E) and recipients (F). GFR, glomerular filtration rate; skGFR, single-kidney GFR.

Total and cortex volumes of the nondonated and donated kidneys were similar (Table 2). The linear associations between cortex volume and GFR at 1-y follow-up are shown in Figure 4A and B for the nondonated and donated kidneys, respectively. The relation between cortex volume and percentage hyperfiltration was not significant for nondonated kidneys (Figure 4C). However, for the donated kidneys, there was a negative association between cortex volume and the degree of hyperfiltration (Figure 4D).

F4
Figure 4.:
The upper panel figures show linear associations between cortex volume of the nondonated kidney and the corresponding GFR at 1-y follow-up in donors (A) and cortex volume of the donated kidney and GFR at 1-y follow-up in recipients (B). The lower panel figures demonstrate the relation between cortex volume of the nondonated kidney and percentage hyperfiltration at 1 y in donors (C) and between cortex volume of the donated kidney and percentage hyperfiltration at 1 y in recipients (D). GFR, glomerular filtration rate.

Histological parameters based on the perioperative biopsies from the donated kidneys are shown in Table 2. The linear associations between the number of nonsclerosed glomeruli and Vglom and GFR at follow-up are shown in Figure 5A–D. Besides a weak correlation between glomerular number and GFR in the nondonated kidneys, these glomerular measures were not associated with GFR at follow-up. The relations between kidney interstitial fibrosis and GFR are depicted in Figure 5E for nondonated kidneys and Figure 5F for donated kidneys. More interstitial fibrosis was associated with lower GFR at follow-up in donors but not recipients. It was not possible to establish the relations between the percentage of sclerosed glomeruli and GFR at 1-y follow-up in either donors or recipients because of the large number of kidneys without any globally sclerosed glomeruli (Figure S1, SDC, https://links.lww.com/TP/C496). Linear associations between arteriolar media-lumen ratio and GFR were insignificant for both nondonated (P = 0.31) and donated kidneys (P = 0.49; Figure S2, SDC, https://links.lww.com/TP/C496).

F5
Figure 5.:
The upper panel figures show linear associations between glomerular number of the nondonated kidney and the corresponding GFR at 1-y follow-up in donors (A) and glomerular number in the donated kidney and GFR at 1-y follow-up in recipients (B). The middle panel figures demonstrate the relation between mean glomerular volume in the nondonated kidney and the corresponding GFR at 1-y follow-up in donors (C) and the relation between mean glomerular volume in the donated kidney and the corresponding GFR at 1-y follow-up in recipients (D). The lower panel figures demonstrate the linear associations between percentage interstitial fibrosis in the nondonated kidney and the corresponding GFR at 1-y follow-up in donors (E) and percentage fibrosis in the donated kidney and the corresponding GFR at 1-y follow-up in recipients (F). GFR, glomerular filtration rate.

The influence of cortex volume and each of the histological parameters on 1-y GFR is shown in Table 3 with the nondonated kidney representing the donor and the donated kidney representing the recipient. The predictive value of these parameters was further evaluated using multiple linear regression analysis after adjustment for age, sex, BMI, 24-h mean arterial pressure, and baseline skGFR and the exact number of days between transplantation and follow-up. For the nondonated kidneys, none of the histological parameters predicted GFR, but for the donated kidneys, the degree of interstitial fibrosis was significantly associated to GFR at follow-up. After multiple adjustments, cortex volume remained predictive for GFR in donors but not in recipients.

TABLE 3. - Predictors of measured GFR in living kidney donors and renal transplant recipients 1 y following donation or renal transplantation
Unadjusted Adjusted a
β P R2 β P
Nondonated Kidney Cortex volume, cm3 0.56 <0.001 0.5 0.42 <0.001
Glomerular number, 103 0.01 0.03 0.1 −0.0027 0.54
Glomerular volume, 103 μm3 1.76 0.22 0.03 1.6 0.13
Sclerotic glomeruli, % −0.59 0.085 0.06 0.013 0.96
Kidney fibrosis, % −0.99 0.29 0.02 −0.73 0.31
Arteriolar M:L ratio −16.39 0.31 0.02 1.93 0.87
Donated Kidney Cortex volume, cm3 0.22 0.047 0.08 −0.22 0.17
Glomerular number, 103 0.004 0.54 0.008 −0.0077 0.23
Glomerular volume, 103 μm3 2.00 0.22 0.03 1.83 0.22
Sclerotic glomeruli, % −0.10 0.08 0.001 0.29 0.44
Kidney fibrosis, % −2.72 0.013 0.12 −2.37 0.017
Arteriolar M:L ratio −13.32 0.49 0.01 4.9 0.81
The nondonated kidney represents the donor at follow-up, and the donated kidney represents the recipient at follow-up. The β-value refers to the change in GFR (mL/min) for each unit increase in the parameters listed in column 2.
aFor age, gender, body mass index, smoking status, 24-h mean blood pressure at follow-up, baseline single-kidney glomerular filtration rate, and the exact number of days between transplantation and follow-up.
GFR, glomerular filtration rate; M:L, media to lumen.

A logistic regression was also performed to establish the value of cortex volume and histology to predict a GFR below 60 mL/min. In the fully adjusted analysis, cortex volume predicted a GFR <60 mL/min in donors with borderline significance (P = 0.06). However, for recipients, interstitial fibrosis could not significantly predict a GFR <60 mL/min (P = 0.13).

DISCUSSION

Based on the pretransplantation donor angiography and stereological investigation of the kidney biopsies obtained during the transplantation procedure, we aimed to establish parameters able to predict renal function after 1 y. This is the first study examining the possible use of these parameters at the same time and using the same methods in donors and recipients.

Renal Outcome in LKDs

A high predonation GFR of the nondonated kidney seems to ensure a high postdonation GFR in nearly all donors. The degree of hyperfiltration is highly variable, but we noticed a weak inverse relation between predonation skGFR and hyperfiltration. The adaption of GFR postdonation has in some studies been related to age, hypertension, and vascular stiffness,27-29 whereas others report less influence of these factors.30,31 The discrepancies may‚ to a large extent‚ depend on the methods used to assess skGFR and especially that formula-based eGFR can be an imprecise measure of GFR in uninephric donors.16 Our baseline data suggest a stronger association between cortex volume and skGFR than between estimated glomerular number and skGFR as also previously observed in healthy kidneys obtained at autopsy.32 This could reflect cortex volume as a better indicator of true glomerular number and the uncertainty related to determining total glomerular number based on 1 or 2 biopsies.33 However, it may also be explained by the complex physiological regulation of GFR being influenced by functional parameters as glomerular pressure and hydraulic permeability not measurable by quantitative histology.

The influence of cortex volume on postdonation renal function has been addressed previously, demonstrating correlations between volume and GFR after 1 y.30,34-36 Our data further reveal that cortex volume is independently associated with renal outcome even after adjustment of multiple factors including GFR of the nondonated kidney. Recent reports suggest that contrast-enhanced CT scans with determination of cortex volume could substitute measurement of skGFR.35,37,38 However, our observations emphasize the importance of determining both split renal function and cortex volume as part of the LKD examination program.

Semiquantitative and more advanced histological assessments of donor kidney biopsies have been undertaken in several studies as an attempt to further predict postdonation renal outcome. We estimated nondonated kidneys to contain 9.4 × 105 glomeruli, which is similar to that reported by Issa et al39 and Denic et al10 in large donor cohorts but slightly higher than described by others.40,41 We found glomerular number to be the only histological parameter associated with 1-y GFR in agreement with previous reports,39,41 but after adjustment for relevant clinical parameters, this association weakens.39 The presence of completely sclerosed glomeruli has been associated with worse donor renal outcome,42,43 but although influenced by age, a large proportion of donors have no sclerotic glomeruli in the biopsy material rendering glomerular sclerosis difficult to use as a single parameter. Hori et al44 recently reported that the degree of interstitial fibrosis/tubular atrophy predicts donor eGFR after 1 y; however, using measured GFR, this was not the case in our material. In the present study, we also evaluated arteriolar changes in terms of media-lumen ratio, as increased luminal stenosis has previously been shown to predict development of hypertension in LKDs.39 However, in our cohort, increasing luminal narrowing had no effect on GFR. Taken together, cortex volume contributes with information regarding donor renal outcome, whereas quantitative kidney histological examination adds little value when predicting GFR 1 y after kidney donation when adjusting for relevant clinical and more easily accessible parameters.

Renal Outcome in Living Kidney Transplant Recipients

As compared to nondonated kidneys, transplanted kidneys showed on average less hyperfiltration with a considerable proportion having lower GFR at follow-up as compared to their corresponding skGFR before transplantation. The loss of graft function is multifactorial being influenced by surgical complications, immunosuppressive medication, episodes of rejection, infections, and various recipient characteristics. Still, the histological findings from the perioperative biopsy may potentially predict future graft function of the recipient. Issa et al15 analyzed 2293 living donor biopsies and concluded that nephrosclerosis, interstitial fibrosis, and large nephron size were modestly associated with death-censored graft failure. This is in accordance with a recent Danish study analyzing biopsies from living or deceased donors using light and electron microscopy and with a follow-up time of 14 y concluding that less hyaline arteriolar thickening and less interstitial fibrosis is associated with long-term graft survival.13 In the present investigation, focusing on 1-y GFR of the transplanted kidney, the degree of interstitial fibrosis remained a significant marker rendering the use of quantitative glomerular histology of limited use as regards short-term graft function. Fibrosis development is especially worsened by calcineurin inhibitor (CNI) treatment, which probably explains the predictive value of this parameter in recipients and not in LKDs. Longer follow-up time may have revealed a role of media-lumen ratio as arterial hyalinosis is also a feature of calcineurin inhibition.

Strengths and Limitations

A significant strength of our study is the use of a gold standard plasma clearance method to determine skGFR before donation and GFR at follow-up rendering calculation of hyperfiltration as precise as possible. An important assumption is that histology of the nondonated kidney resamples the donated kidney, which is likely in healthy persons with kidneys of equal size and GFR distribution as in our material. In general, long-lasting hyperfiltration is supposed to be deleterious and lead to loss of renal function. However, nephrectomy is associated with an immediate increase in blood flow and hyperfiltration of the remaining kidney.45 We did not evaluate postdonation cortex hypertrophy in either donors or recipients. However, in donors, this may be substantial,30,46 reflecting glomerular growth and increased single-nephron ultrafiltration coefficient,45 possibly protecting the kidney from glomerular hypertension.

An important limitation is the relative limited sample size that refrained us from demonstrating weak associations or to include more variables in the regression analysis. Estimation of glomerular number based on limited tissue material, and in addition algorithms that differ between labs, is associated with uncertainty,33 possibly rendering glomerular quantitative histology more uncertain than determination of interstitial fibrosis.

Clinical Implications

When focusing on the clinical usefulness of donor renal imaging and perioperative graft histology, our data suggest (1) the CT- (or MRI) based renal imaging of the donor should result in a precise determination of cortex volume (not only a qualitative description), and if cortex volume is low, donation should be reconsidered; (2) the histological assessment of the graft biopsy should mainly focus on quantifying interstitial fibrosis. Potential clinical consequences of this could be early considerations concerning immunosuppressive treatment regimens toward lower CNI doses or changing to a non–CNI-based treatment.

Conclusions

Our study demonstrates differential prognostic information of pretransplantation cortex volume and perioperative biopsy-based quantitative histology regarding 1-y renal outcome in LKDs and their recipients. Although cortex volume is the strongest independent predictor of GFR in donors, the degree of interstitial fibrosis is the best predictor in recipients. In contrast‚ neither estimated glomerular number nor histology predicted renal outcome when adjusting for relevant clinical parameters including pretransplantation skGFR. Taken together, the data support pretransplantation determination of absolute cortex volume and quantitative assessment of interstitial fibrosis in the perioperative graft biopsies.

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