Kidney transplantation has become the standard treatment for children with end-stage renal failure (1). Although the outcomes of renal transplantation have been improved over the past several decades (2), progressive loss of renal function from either immunologic or nonimmunologic processes remains the main hurdle in pediatric kidney transplantation (3–5).
In addition to repeated immunologic injury and chronic calcineurin inhibitor toxicity, renal graft ischemia associated with the donor-recipient size discrepancy is a significant risk factor for chronic ischemic graft injury in transplantation of size-discrepant young infant recipients, particularly those with a body surface area (BSA) of less than 0.5 m2 (6, 7). Children who received kidneys from size-compatible pediatric donors, without any vascular compromise, demonstrate less chronic histologic injury, with slower decline in graft function in the early years after transplantation, as compared with kidneys from adult donors (7).
Our group and others have shown that posttransplantation renal function is influenced by the size discrepancy between the donor and the recipient. When pediatric donor kidneys are transplanted into the adult recipients, a rapid adaptation to the size of the recipient is seen (8). In contrast, an attenuation of the glomerular filtration rate (GFR) is observed when adult donor kidneys are transplanted into small pediatric recipients, both in humans (9) and in animal models (10). The discrepancy between the vasculature size of pediatric renal allograft recipients and the adult-sized kidney (ASK) grafts leads to renal hypoperfusion, decrease in absolute GFR (aGFR), and finally a shrinkage of about a third in renal volume (9–11); these factors have been referred to as “functional adaptation” of these kidneys to the recipients’ size. We have reported recently that this functional adaptation may also be structural because the changes in the allograft are not completely reversible in the smallest recipients and are associated with irreversible histologic damage (6). The underlying molecular mechanisms for the intragraft changes remain unknown but warrant investigation because they may offer clues for therapeutic intervention.
To evaluate this further, we hypothesized that the adaptation of ASK into the smallest pediatric recipient is not only a passive physiologic consequence of low renal blood flow and GFR but also associated with gene expression alterations that may involve the regulation of renal vascular resistance and autoregulation and with the altered expression of renal growth factors, which could add to the irreversible tissue injury. Our aim of the study was to unveil the molecular mechanisms underlying the functional adaptation of ASKs transplanted into small pediatric recipients.
Functional Adaptation of ASKs Transplanted Into Small Pediatric Recipients
The study cohort consisted of 21 pediatric patients (demographics are presented in Table 1), who were recipients of ASK transplants; have undergone three serial early protocol biopsies each at time 0 (engraftment), 3 months, and 6 months; and have serial graft function measurements for a minimum of 1 year after transplantation (aGFR and GFR by Brandt et al. (12)) (6). To avoid the confounding effect of early dysfunction and immunologic injury from acute rejection, only those patients whose transplant was an ASK, with pristine condition of the kidneys at implantation with the absence of any delayed graft function, and with no interval acute rejection episodes in the first 2 years after transplantation were selected. As expected, there was no evidence of significant inflammation on any of the protocol biopsies that were blindly examined and scored by a single pathologist. Chronic allograft damage index (CADI) score revealed only minimal chronic histologic damage at 3 months after transplantation. However, protocol biopsies from the smallest recipients showed worse chronic histologic damage at 6 months as compared with larger recipients, supporting the allograft’s susceptibility to vascular perfusion injury in the smallest children (6).
Renal allograft function at 3 months after transplantation showed a mean aGFR, which represents real graft function unadjusted for recipient size, of 61.8 (SD, 22.9) mL/min and a mean GFR by Brandt et al. (12) (which represents another measure of aGFR) of 70.2 (SD, 33.0) mL/min and correlated with BSA at the time of transplantation (r=0.93, P<0.0001 and r=0.96, P<0.0001, respectively), supporting our previous findings that, in small children, the functional adaptation of the ASK is correlated to the recipients’ body size (6, 13) (Fig. 1A,B). The chronicity of tissue injury as scored by the CADI score on the protocol biopsy at 6 months also significantly correlated with BSA at the time of transplantation (r=−0.61, P<0.01) (Fig. 1C,D).
Microarray Analysis of Transplant Transcriptome According to the Functional Adaptation
Allograft biopsies on all patients were transcriptionally profiled by HG U133 Plus 2.0 gene expression microarrays (Affymetricx, Santa Clara, CA) and normalized and processed for analysis by standard protocols (14). Probe sets that were significantly associated with graft function are indicated in red and green. For significance, we used the conservative threshold of a false discovery rate (FDR) less than 5%. In total, the expression of 1051 probe sets, representing 724 unique genes, were significantly correlated with graft function. Figure 2 shows the signal intensity of a selection of significantly correlated probe sets with graft function at 3 months after transplantation. The single most differentially expressed gene (FDR=0) that correlated best with aGFR was stanniocalcin 1 (STC1)(Fig. 2A).
Ingenuity canonical pathway (http://ww.ingenuity.com) analysis identified significant overrepresentation of relevant and overlapping pathways involved in regulation of tubular salt reabsorption and enzymatic pathways for organ development and hypertrophy (Fig. 2B). The top-ranked pathway was aldosterone signaling in epithelial cells, which contained three components of epithelial sodium channels (SCNN1A, SCNN1B, and SCNN1G) and sodium/hydrogen exchanger (SLC9A1) in the collecting duct and their related enzymatic pathways. Other top-ranked pathways were mainly involved in the regulation of organ development and tissue hypertrophy. In addition, interesting genes for the functional adaptation were associated with the overrepresentation of calcium signaling and cyclic AMP (cAMP)-mediated signaling (see Table, SDC,http://links.lww.com/TP/A656).
Histologic Localization of STC1 Messenger RNA and Protein
The cellular distribution of STC1 messenger RNA (mRNA) and protein in the human kidney was investigated by in situ hybridization and immunohistochemistry (IHC). Hybridization with the antisense probe labeled tubular epithelial cells, vascular endothelium, and glomerular mesangial cells (Fig. 3A). Hybridization with the sense probe did not specifically label any renal structures (Fig. 3B). IHC revealed that STC1 protein expression was present in the transplanted kidney in all proximal straight tubule cells, all cortical thick ascending limb cells, all distal convoluted tubule cells, and some cells of the collecting duct. Figure 3(C) illustrates the weak staining for STC1 in the biopsy specimen at 6 months of a 1-year-old pediatric recipient of an ASK, whereas Figure 3(D) shows the strong staining for STC1 in the biopsy specimen at 6 months of a 15-year-old recipient.
Confirmation of STC1 by QPCR and Correlates of Renal expression of STC1
To confirm the association between the expression of STC1 and graft function in the gene expression microarrays, we measured renal cortical STC1 expression by quantitative real-time polymerase chain reaction (QPCR). The QPCR data confirmed the dysregulation of STC1 expression with similar directional change by QPCR and microarray. Renal STC1 mRNA expression was highly correlated with the aGFR at 3 months (r=0.79, P<0.0001) (Fig. 4A) and with BSA at the time of transplantation (r=0.69, P=0.0003).
Based on the known functions of STC1 such as decrease of intracellular calcium, promotion of angiogenesis, and decrease in inflammation and apoptosis, we compared the renal expression of STC1 with the early histologic features of the graft such as the CADI score, percentage of tubular atrophy, and percentage of microcalcifications. Grafts with tubular microcalcifications at 3 or 6 months, a finding that has previously been suggested to be related with lower recipient size, showed lower renal STC1 mRNA expression as compared with those without microcalcifications (P<0.05) (Fig. 4B). Although renal expression of STC1 at 3 months showed no significant association with histologic finding at 3 months (Fig. 4C), STC1 mRNA levels at 3 months were significantly associated with the chronic injury CADI scores at 6 months (r=−0.63, P=0.006) (Fig. 4D).
This transcriptional analysis of pristine living donor ASKs—which do not experience any delayed graft function or rejection episodes, receive the same immunosuppression, and differ only in their placement into either size-discrepant small infant recipients or size-compatible young adult recipients—demonstrates alterations in the gene expression of the allograft specific to renal autoregulation and growth factors. The adaptation of ASK grafts to the pediatric recipients is not only a passive physiologic consequence of lower renal blood flow but also associated with gene expression alterations involved in the regulation of tubular salt reabsorption, growth factor signaling pathways, and intracellular calcium and in cAMP signaling pathways, specific molecular pathways that associate with the progression of irreversible tissue injury (6, 11).
Analysis of the ASK transcriptome from the infant recipient shows overexpression of tubular sodium reabsorption pathways and enzymatic pathways for organ development and hypertrophy. These genes correlate with early absolute graft function of the ASK at 3 months after transplantation. Because the number of nephrons does not change after transplantation, reducing renal blood flow would result in a reduction in the single nephron GFR and structural hypotrophy. Differences of renal growth-related genes and calcium and cAMP signaling genes could account for early compensatory shrinkage and the change of renal blood flow in size discrepancy in pediatric transplantation. A decrease of renal blood flow and GFR of the ASK would be important for normal-volume homeostasis in a size-discrepant small recipient. It can be hypothesized that, to maintain volume homeostasis in a small recipient with an ASK, it would be necessary to attenuate sodium reuptake in the collecting duct and renal blood flow of the ASK. Recently, we also reported that early posttransplantation renal resistive indices and renal volume are size dependent in small pediatric recipients of an ASK (15).
Although our analyses suggest intuitive pathways for the functional adaptation of ASKs to small recipient size, the mechanism underlying the development of later histologic consequences such as interstitial fibrosis, tubular atrophy, and tubular microcalcifications, which prevail in smaller children, required additional analysis. To this end, we further investigated STC1, the gene most significantly associated with early decline in graft function at 3 months. We confirmed the differential expression of renal STC1 mRNA expression as seen on the microarrays by replicating these data by QPCR. STC1 expression correlated best with renal allograft function in the early posttransplantation period, and its expression correlated with the presence of subsequent histologic changes such as intragraft tubular atrophy and microcalcifications. Because STC1 is known to reduce intracellular calcium, promote angiogenesis, decrease inflammation, and reduce apoptosis (16, 17), we hypothesized that renal STC1 could be an interesting candidate gene that could be driving the functional, structural, and molecular adaptation of size-discrepant ASK transplants in children.
STC1 is a hypocalcemic glycoprotein hormone originally discovered in teleost fishes (18). However, the mammalian STC1 gene is widely expressed, and mRNA levels are very high in the ovary, kidney, adrenal, prostate, and heart (18). The expression of STC1 is found to be involved in numerous developmental and pathophysiologic processes, including plasma calcium-phosphorus homeostasis (19), pregnancy, lactation (20), and organogenesis (21). In the kidney, STC1 is involved in early nephrogenesis (22) and renal phosphate reabsorption (19) and potentially in renal salt-and-water balance (23). The regulatory mechanisms of renal expression of STC1 remain obscure, but vitamin D (24) and hypertonicity (25) are involved in renal STC1 regulation. In vitro experiments suggest that angiogenic stimulation (26), dibutyryl cAMP (27), and hypoxia-inducible factor 1 (28) also affect the renal expression of STC1.
Although STC1 mRNA has been previously shown to be exclusively expressed in the collecting duct of a rat kidney (29) and in the renal tubules of a mouse kidney (30), we detected human STC1 mRNA expression in renal proximal and distal tubules, vascular endothelium, and glomerular mesangial cells. ASKs transplanted into infant recipients showed greater tubular atrophy, interstitial fibrosis, and tubular microcalcification on the protocol biopsies at 3 and 6 months (6) and demonstrated lower renal STC1 mRNA expression than ASK biopsies from size-compatible recipients. Because STC1 can control calcium signaling and the rate of apoptosis in chronic hypoxia (31) and most importantly could play a role in renal salt-and-water balance (23), we hypothesize that dysregulation of renal STC1 in size-discrepant ASK transplants is the consequence of hypoperfusion and could play a role in the worse histologic outcome of the size-discrepant ASK transplants in children.
A limitation of the current study was that we used an estimation of the aGFR instead of direct measurement. However, there was a highly significant correlation between the values of aGFR and GFR by Brandt et al. (12) (r=0.95, P<0.0001), and the GFR by Brandt et al. (12) is known to be comparable to measured GFR with iothalamate-measured GFR in children (12). An additional limitation is that, in this human study, we could not manipulate the increased expression of STC1 in the ASK to evaluate if this maneuver could alter the pathophysiologic mechanisms that drive the progression of histologic injury in size-discrepant ASK transplantation. Additional studies with validation of these data in an independent pediatric patient cohort, evaluation of the time-dependent changes in the transcriptome, and longer clinical and histologic follow-up will be necessary to verify the pathophysiologic role of STC1 in worsening renal allograft function and progression of chronic histologic damage.
Further elucidation of the molecular mechanisms of the adaptation of ASKs to the small recipients’ size and of the pathophysiologic role of STC1 will likely lead to a better understanding of the specifics of renal autoregulation and the grafts’ adaption to chronic renal hypoperfusion.
MATERIALS AND METHODS
The study population consisted of consecutive pediatric ASK (donors >18 years of age) recipients (1–20 years of age) enrolled in the steroid-free immunosuppression protocol at Stanford University Medical Center from November 1999 to June 2006 (6). Patients who experienced delayed graft function and a biopsy-proven acute rejection in the first 6 months after transplantation were excluded, to avoid interference of immunologic or ischemic injury per se with functional adaptation to donor-recipient size discrepancy. Among 81 acute rejection-free pediatric ASK (donors >18 years of age) recipients, 21 (age [SD], 8.4 [6.7] years) patients presented with sufficient RNA from all of the intended samples for microarray analyses and were enrolled into this study. All patients or their parents gave written informed consent, and the study was approved by the institutional review board of Stanford University. All adequate protocol biopsies (n=39; 21 at 3 months and 18 at 6 months) were reviewed blindly by one experienced pathologist. Histologic lesions were semiquantitatively scored according to the revised Banff criteria (32). The CADI score (33) and a chronic calcineurin inhibitor toxicity score (6, 34) were calculated in all biopsies. BSA-adjusted GFR (“relative GFR” in mL/min per 1.73 m2) was estimated by the Schwartz formula. The aGFR (mL/min), which represents real graft function, was calculated as Schwartz GFR×BSA/1.73 at these time points; BSA was calculated with the Mosteller formula (35). aGFR by Brandt et al. (12) was also calculated as GFR (mL/min) = k×
, where k=0.95 for females and k=1.05 for males (12).
Microarray Analysis and Real-time PCR
Total RNA was extracted from biopsy specimens at 3 months using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA was reverse transcribed into complementary DNA using the SuperScript Choice System (Invitrogen, Carlsbad, CA). Complementary DNA was in vitro transcribed to complementary RNA (cRNA) and biotin labeled (Affymetrix, Santa Clara, CA). Biotinylated cRNA was purified and fragmented. Fragmented cRNA (15 μg) was hybridized overnight to the Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA), which comprised 54,675 probe sets. After washing and staining, the gene chips were scanned with the GeneArray scanner controlled by Affymetrix GCOS 1.4 software. For processing and normalization of the scanned images, dChip 2006 software was used, with perfect match/mismatch difference modeling and invariant set normalization. Unsupervised and supervised average-linkage hierarchical clustering and visualization were performed using Cluster and TreeView. The multiclass response utilities in Significance Analysis of Microarrays (SAM) (version 3.0) were used to assess gene expression fold differences and significance of genes correlating with aGFR. Probe sets with more than twofold change and an FDR less than 5% were selected as differentially expressed. Ingenuity pathway program (Ingenuity Systems, Redwood City, CA) was used to assess their biologic functions and examine canonical pathways based on the Ingenuity Pathways Knowledge Base. Real-time PCR was performed in an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA) by applying gene expression assays for TaqMan primer sets that were predesigned and optimized by Applied Biosystems for STC1 (Hs00174970_m1). Quantification of gene expression was performed using the ΔCt method. 18S was used as the endogenous reference gene.
In Situ Hybridization
In situ hybridization of the normal portion of a nephrectomized human kidney was performed based on a protocol published previously (36). Briefly, sections were deparaffinized in xylene for 5 min, followed by hydration in graded ethanols for 5 min each. Next, sections were digested in a 10-μg/mL dilution of Proteinase K (Qiagen, Valencia, CA) at 37°C for 30 min, followed by hybridization overnight at 55°C with a 200-ng/mL dilution of antisense or sense riboprobes in mRNA hybridization buffer. On the following day, sections were serially washed and incubated with RNAase A(Qiagen, Valencia, CA) Next, slides were washed twice in 2× standard saline citrate/50% formamide at 55°C, followed by one wash at 0.08× standard saline citrate at 55°C. The signal amplification was achieved by incubation of sections with biotinyl-tyramide, followed by secondary streptavidin complex (GenPoint kit; DAKO, Glostrup, Denmark). The final signal was developed with diaminobenzidine chromogen (GenPoint kit; DAKO).
IHC staining for STC1 was performed using 2-μm sections of paraffin-embedded tissue of graft biopsies using standard protocols based on antigen retrieval. Antihuman STC1 antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA) were used, at a dilution of 1:50. Endogenous peroxidase was blocked, and the DAKO Envision system was used for detection. Staining was optimized using appropriate positive and negative controls.
Data are expressed as mean (SD). The correlation between the variables was assessed using Spearman test. For variance analysis of continuous variables in different groups, nonparametric Mann-Whitney test was used. Dichotomous variables were compared using the chi-square test. All P values were two sided, and those less than 5% were considered to indicate statistical significance. Data analysis was performed using SAS software (version 9.1; SAS institute, Cary, NC).
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