Background

The underlying mechanisms of renal transplant dysfunction are poorly understood. There is little information on tubular function in kidney grafts. The cDNAs encoding kidney-specific cell surface proteins required for renal reabsorption of sodium (sodium cotransporter in thick ascending limb of Henle, rBSC1) and water (apical water channel in collecting duct, AQP2) have been recently identified. Since transcripts of these proteins are up-regulated in dehydration in association with maximal concentration of urine, we examined urinary concentrating ability and expression levels of mRNA of these proteins in kidney isografts.

Methods

Male Sprague-Dawley rats underwent syngeneic renal transplantation or unilateral nephrectomy (UNX) and were deprived of water for 24 hours at six weeks after the operation when histological and functional compensation of the intact kidney was complete. Blood and urinary samples were collected before and after dehydration. The amount of rBSC1 or AQP2 mRNA was measured using competitive polymerase chain reaction (PCR) by inducing a point mutation at the middle of PCR product for rBSC1 or by deleting 180 bp from 780 bp PCR product for AQP2, respectively. The protein expression was examined by Western blot analysis.

Results

Both groups of rats demonstrated the same levels of compensatory renal hypertrophy (~60% weight increase) and plasma creatinine values. Histological examination revealed enlarged glomeruli and tubules, but no findings of ischemic damage, such as tubular atrophy or interstitial changes. Urinary concentration was noted in the UNX rats but not in rats with kidney grafts. Competitive PCR demonstrated that dehydration did not increase rBSC1 and AQP2 transcripts in rats with kidney transplantation. Immunoblot analysis confirmed that the marked increase of both rBSC1 and AQP2 proteins was noted only in the remnant kidney of dehydrated rats.

Conclusions

Rats with kidney isografts have a limited capacity to concentrate urine and, at the same time, fail to increase rBSC1 and AQP2 transcripts. This suggests that there is a prolonged damage of renal tubules by ischemia or denervation of the donor kidney, both of which are inevitable in the transplantation procedure.

METHODS

Animals

Male Sprague-Dawley rats weighing approximately 200 g were used both as graft recipients and donors. Forty-eight rats were included in the study: 16 for renal transplantation, 16 for UNX, and 16 for sham operation of left kidney. Rats had free access to standard rat chow and water ad libitum, and were maintained in a humidity- and temperature-controlled room with a 12/12-hour light-dark cycle. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation at the Tohoku University School of Medicine.

Kidney transplantation and UNX

The microsurgical technique used in this study is a modification of the technique described by Lee³⁰. All surgical procedures were performed with the animal under ether anesthesia. The donor kidney was flushed with 1 mL of ice-cooled University of Wisconsin solution and transplanted into the recipient rat, in which the left kidney had been removed. Renal blood flow of the grafted organ was restored by release of the vessel clamps after 35 to 45 minutes. At the end of surgery, each rat received 100,000 U of penicillin intraperitoneally. The right kidney was removed seven days later.

Unilateral nephrectomy was performed on the left kidney, which was followed by sham operation of the right kidney seven days later. In the sham-operated group, the left kidney was sham operated first, and then the right kidney was sham operated seven days later.

Experimental protocols

After the subsequent six-week recovery period, the rats were anesthetized by ether and catheterized for blood sampling and dehydrated as previously described²⁹. Briefly, polyethylene PE-50 catheters filled with heparinized (100 U/mL) 0.9% NaCl and were each placed in the femoral artery and vein. On the following day, after a six-hour urine collection, a 3 mL blood sample was withdrawn from the femoral artery, which was replaced with an equal volume of rat donor blood as a simultaneous injection via the femoral venous catheter. Then, access to water was restricted for 24 hours in 8 out of 16 rats, while another group of 8 rats continued to have free access to water. Again, urine was collected during the last six hours of the dehydration period in the water-restricted rats. Another 3 mL blood sample was withdrawn from these dehydrated rats. All rats were then placed in a supine position, and the abdominal aorta and inferior vena cava were exposed and clamped above the renal branches. A puncture in the vena cava below the renal veins allowed blood and perfusion fluid to escape. After perfusion of the kidney with 0.9% NaCl through the femoral arterial catheter, the kidney from each of the six rats was removed, weighed, and divided into three parts: the cortex, outer medulla, and inner medulla. Each sample was homogenized in 4 mol/L guanidine with 25 mmol/L sodium citrate and 0.7% mercaptoethanol or in 2 mL of phosphate-buffered saline (PBS), 1% triton, 1% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF) for RNA or protein extraction, respectively. The homologous quality of RNA of each sample was confirmed by ethidium bromide staining in the agarose gel and by the measurement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using polymerase chain reaction (PCR) with 25 cycles in which the intensity of the bands was linearly enhanced. The kidney from each of the resting two rats was sliced, fixed in phosphate-buffered 4% paraformaldehyde (PFA), and used for histological examination [PAS (periodic acid-Schiff's reagent) stain].

Competitive PCR for rBSC1

Competitive PCR analysis for rBSC1 was performed as previously described^21,22. Briefly, a point mutation for the formation of EcoRI site was induced in the middle of a 352 bp of partial fragment for the kidney-specific sodium cotransporter (rBSC1) cDNA. A series of diluted mimic cDNA was mixed with a constant amount of a sample template cDNA of the renal outer medulla and coamplified using rBSC1 primers. Polymerase chain reaction proceeded for 25 cycles, and the products were coincubated with EcoRI for three hours at 37°C. The sample and mimic cDNA were designed to produce 352 and 180 bp fragments, respectively. After agarose gel electrophoresis, the intensity of the band in each sample and mimic cDNA was measured using a densitometer. The amount of rBSC1 mRNA in the sample was calculated from the equivalent point (40 mimic for rBSC1 at the equivalent point/1 g RNA).

Competitive PCR for AQP2

We constructed a mimic cDNA as described previously^22,29. Briefly, a pair of PCR primers was designed to frame the major part of AQP2 cDNA (760 bp), which contained SphI and SacI restriction enzyme sites in the middle of the product. By deleting 180 bp between these sites, the final PCR product was 580 bp, which was obtained and used to mimic cDNA for competitive PCR. A series of diluted mimic cDNA was mixed with the same amount of sample template cDNA (from 0.05 g RNA) from each part of the kidney and coamplified using the AQP2 primer set. Polymerase chain reaction was performed using 25 cycles. Following agarose gel electrophoresis, the intensity of the bands in each sample and those of mimic cDNA was measured using a densitometer. The amount of AQP2 mRNA in the sample was calculated from the equivalent point (20 amount of mimic cDNA at the equivalent point/1 g RNA).

Western blots

Protein was extracted from renal outer medulla or inner medulla of the rats, and Western blotting analysis was performed as previously reported^21,22. Antibody against rBSC1 and AQP2 was a kind gift from Dr. Steven C. Hebert (Division of Nephrology, University of Vanderbilt, Nashville, TN, USA) and against AQP2 from Dr. Sei Sasaki (Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan). Tissues were homogenized and centrifuged, and the supernatants were resolved by Laemmli sodium dodecyl sulfate-polyacrylamide (8% or 12%) gel electrophoresis (SDS-PAGE) and transferred in 25 mmol/L Tris-HCl, 192 mmol/L glycine, 25% methanol to a polyvinylidene difluoride membrane. After being blocked with 5% powdered milk, the membrane was exposed to anti-rBSC1 (1:500 dilution) overnight at 4°C or to anti-AQP2 (1:500 dilution) for one hour at room temperature. The membrane for rBSC1 was incubated with peroxidase-linked anti-rabbit Ig, and another membrane for AQP2 was incubated with biotin-labeled anti-rabbit IgG antibody (Vector, Burlingame, CA, USA) for one hour at room temperature. Then antigen–antibody complexes were visualized with a chemiluminescence system (ECL Plus; Amersham International, Arlington Heights, IL, USA).

Measurements of AVP

Arginine vasopressin was measured by radioimmunoassay (RIA) as described previously³¹. Briefly, AVP was extracted using octacasyl-silane packed in a cartridge (Sep-Pak C18 cartridge; Waters Associates, Milford, MA, USA) and assayed using specific antibodies to AVP (Mitsubishi Petrochemical, Tokyo, USA). The recovery rate was 72.4 6.8% (N = 30).

Other measurements and statistical analysis

The osmolality of plasma and urine samples was determined using an osmometer (3D2; Advanced Instruments, Needham Heights, MA, USA) and plasma Na was determined by flame photometry (Hitachi flame photometer, 205D). Plasma creatinine concentration was measured by an autoanalyzer. Differences in laboratory data were examined for statistical significance using unpaired t tests followed by the Student t test. The results are expressed as mean SEM. A P value <0.05 was considered significant.

RESULTS

Renal hypertrophy and absence of ischemic damages

The kidney weight to body weight ratio is shown in Table 1. Significantly high ratios were noted in both the UNX and transplanted rats compared with that of the left kidney to body weight ratio in the sham-operated rats. However, there was no difference in this ratio between UNX and grafted rats, indicating the same level of renal hypertrophy in these two groups. The microscopic findings of renal cortex confirmed the enlargement of glomeruli and tubules in kidney-grafted rats Figure 1 and UNX (data not shown). However, histological findings such as focal atrophy of renal tubules, and interstitial cell infiltration or fibrosis, all suggest that the ischemic renal damage was not observed Figure 1.

Figure 1.

Histological sections of the renal cortex. (A) Sham-operated rat. (B) Graft kidney (periodic acid-Schiff's reagent stain, magnification 100).

Full figure and legend (229K)

Table 1 - Changes in various renal function parameters induced before and after water restriction.

Full table

Effects of dehydration on urinalysis, body fluid, and renal function

Plasma creatinine concentrations and urinary osmolality before water restriction were similar in rats with UNX and kidney isograft Table 1. Water restriction increased urinary osmolality in UNX rats to levels similar to those in sham-operated and normal control rats described in our previous study²⁹. However, urinary concentration by water deprivation was limited in rats with a kidney isograft Table 1. Consequently, more urinary volume during the water-restriction period and further increases in the weight reduction rate, plasma osmolality, sodium, urea, and AVP concentrations were observed in rats with the kidney isograft compared with the respective values in UNX rats, although all of these changes were not significant. Urinary sodium excretion before the water restriction was quite similar in all three groups. During the water deprivation period, however, the marked reduction of urinary sodium excretion observed in the sham-operated or UNX rats was limited in rats with the kidney isograft.

rBSC1 transcripts

Figure 2 shows the results of the competitive PCR analysis of rBSC1 mRNA in the renal outer medulla. The mean of six determinations demonstrated that rBSC1 transcripts were significantly higher in uninephrectomized and grafted rats compared with those of the sham-operated controls Figure 2a. Figure 2b demonstrates the effects of dehydration (24-hour water restriction) on the expression level of the rBSC1 transcript. Dehydration caused a significant increase in rBSC1 signals in uninephrectomized rats, but no such increase was noted in rats with the kidney isograft. The dehydration-induced increase in rBSC1 mRNA in UNX rats was almost the same as that observed in sham-operated controls²¹.

Figure 2.

Competitive polymerase chain reaction (PCR) of rat kidney-specific sodium cotransporter (rBSC1) transcripts. (A) Mean data under euhydration. Six determinations (SEM) of renal outer medulla from sham-operated (Sham), unilateral nephrectomy (UNX), and transplanted rats (Graft). *P < 0.05 compared with sham-operated rats. (B) Effects of 24-hour water restriction on rBSC1 transcripts from UNX and transplanted rats (Graft). Six determinations (SEM). *P < 0.05 between euhydration and dehydration conditions.

Full figure and legend (71K)

AQP2 transcripts

Aquaporin 2 mRNA signals are shown in Figure 3. A slight increase in AQP2 transcript signals was observed in the inner medulla of both uninephrectomized and kidney-grafted rats Figure 3a. Figure 3b demonstrates that AQP2 transcripts were markedly increased in the water-restricted rats with uninephrectomy, whereas no increase was noted in the kidney grafted rats.

Figure 3.

Competitive PCR of aquaporin 2 (AQP2) transcripts. (A) The mean data of six determinations (SEM) of the inner medulla under euhydration. *P < 0.05 compared with sham-operated rats. (B) Effects of 24-hour dehydration on AQP2 transcripts. Six determinations (SEM). *P < 0.05 between euhydration and dehydration conditions.

Full figure and legend (61K)

rBSC1 proteins

After confirming the similar protein expression of rBSC1 between remnant kidney and kidney isograft in the euhydrated condition and dehydration-induced increase of signal intensity in UNX rats as previously reported (data not shown)²¹, we compared the rBSC1 signals between remnant kidney and kidney isograft in a dehydrated condition Figure 4. The results clearly demonstrated that the kidney isograft signals were less intense in the dehydrated condition.

Figure 4.

Western blots for rBSC1. Total protein (150 g) from outer medulla of dehydrated rats is loaded in each lane. In dehydrated conditions, the signals are much intensified in UNX rats.

Full figure and legend (10K)

AQP2 proteins

Since the association between AQP2 up-regulation and maximal urinary concentration has been well established^{25,26,27,28,29}, signals of remnant kidney and kidney isograft of dehydrated rats were compared. The results also clearly demonstrated that signals of kidney isograft were much less intense in the dehydrated condition Figure 5.

Figure 5.

Western blots for AQP2. Total protein (40 g) from inner medulla of dehydrated rats is loaded in each lane, again showing that signals are much more intensified in UNX rats in dehydrated condition. M, size marker.

Full figure and legend (13K)

DISCUSSION

The present study analyzed the physiological responses of the kidney graft to dehydration and investigated the transcripts of kidney-specific cell surface proteins that are known to be closely involved in urinary concentration. The major findings of our study are that the kidney isograft has a limited capacity to concentrate urine and—at the same time—to increase the transcripts of Na transporter in TAL and of apical water channel in the collecting duct during water restriction.

Previous studies have demonstrated that UNX results in a process of compensation for the lost kidney manifested by enlargement of individual nephrons in the remaining kidney^{9,10,11,12,13,14}. These morphological and functional compensation processes are completed four to six weeks after UNX^9,10,11. Accordingly, our study was designed to examine the urinary concentration in response to dehydration at six weeks after the operation of UNX and kidney transplantation, when proteinuria caused by late graft dysfunction, probably in association with progression in glomerular sclerosis, is still absent⁵. Interestingly, rats with the graft kidney demonstrated the same level of renal hypertrophy and the same values for plasma creatinine as UNX rats, indicating that morphological and functional compensatory alterations also occur in the kidney graft. However, maximal urinary concentration, where a large amount of energy is required to reabsorb sodium and water in the kidney, was not noted in rats with the transplanted kidney. These findings suggest that only limited tubular function exists in the kidney graft despite the morphological compensation.

The salt-absorptive form of the apical Na-K-Cl cotransporter in TAL supplies a critical source of energy for the countercurrent multiplier^19,20, which cotransports luminal NaCl at the relatively water-impermeable TAL. Gamba et al succeeded in cloning a cDNA encoding the rat Na-K-Cl cotransporter, rBSC1, which has a high affinity for loop diuretics such as bumetanide and is expressed in the apical side of TAL^17,18. We reported that dehydration alters the amount of rBSC1 transcript²¹. Our results demonstrated that the mRNA of this transporter is increased in TAL in the outer medulla of dehydrated rats, together with an increased apical expression of protein, suggesting increased sodium transport for the formation of hypertonic medulla under dehydration conditions. In the present study, a significant increase in rBSC1 transcripts in the outer medulla was noted in rats with a UNX and kidney isograft in the euhydration condition. Since enhanced transcription in hypertrophied kidney has been reported³², this increase in basal rBSC1 transcripts is likely to be due to a transcriptional enhancement associated with renal hypertrophy. The dehydration-induced increase, however, was observed only in rats with UNX, while the transcript signals in rats with a kidney isograft were not affected by dehydration. Western blot analysis also demonstrated that marked increase of protein signals was noted only in dehydrated UNX rats. Thus, the responsive up-regulation of rBSC1 to dehydration is impaired in the transplanted kidney.

Water transport across the epithelium of the collecting duct is mediated by at least two regulatory mechanisms, that is, a short-term regulation and long-term regulation¹. The short-term regulation involves a rapid increase in water permeability in the collecting ducts through cAMP-dependent AQP2 redistribution^24,25,26, which is completed within 30 to 40 minutes after exposure to AVP. The long-term regulation of water reabsorption by the renal collecting ducts is manifested by a stable increase in water permeability after water restriction for 24 hours³³. In the long-term regulation, an increase in the expression of AQP2 occurs at both the mRNA²⁹ and protein²⁸ levels in the whole collecting duct, probably resulting in a persistent increase in water reabsorption. Therefore, in the present study, we investigated the expression of AQP2 mRNA in a rat model of renal transplantation. Again, competitive PCR demonstrated a slight increase in basal expression of AQP2 mRNA in rats with a single kidney as well as those with kidney isograft. On the other hand, a significant increase in water channel transcripts was noted only in the UNX rats in response to dehydration, but not in rats with kidney isograft. In addition, a clear enhancement of protein expression was observed only in these dehydrated UNX. These results are likely to be due to the failure of increasing the number of apical water channels expressed in the kidney isograft during dehydration.

Although the linkage between enhanced sodium and water reabsorption and de novo synthesis of rBSC1 and AQP2 has not been fully determined, dehydration induced by water deprivation for 24 to 48 hours has been reported to be associated with up-regulation of rBSC1²¹ and AQP2^28,29, as mentioned previously in this article. In addition, endogenous AVP stimulation has clearly demonstrated an increased protein synthesis of AQP2²⁷ and rBSC1³⁴. Therefore, our findings of an impaired up-regulation of rBSC1 and AQP2 transcripts in rats with a kidney graft are thought to be closely linked to the limited urinary concentration in these rats.

In kidney transplantation, the underlying mechanisms of chronic transplant dysfunction are still to be elucidated^2,3. Recently, Kouwenhoven et al demonstrated that prolongation of cold ischemia enhances late graft failure, even when antigen-dependent graft rejections are minimized by syngeneic transplantation⁵. However, even an optimized transplant procedure of rat kidney transplantation still requires a short-term ischemia of the donor kidney (30 to 45 min), which may damage renal tubules in spite of the absence of morphological alterations in the tubules or interstitium. Since a large amount of energy is required to achieve the maximum concentration of urine, such a small degradation of renal tubules may manifest as limited urinary concentration. In addition to ischemic damage, denervation is another inevitable factor of renal transplantation that influences urinary sodium excretion⁶ and renal blood flow⁸. Thus, the influence of these factors on urinary concentration and expression of rBSC1 and AQP2 need to be elucidated individually in future studies.

In summary, our study demonstrates a defective up-regulation of rBSC1 and AQP2 transcripts in the kidney isograft of rats in response to dehydration. This defective up-regulation may be involved in the limited urinary concentrating capacity of the transplant kidney.

References

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Acknowledgments

This study was supported by Research Grants for Scientific Research (12877163 and 13671095) from the Japanese Ministry of Science and Education. We thank Miss Mika Ishikawa, Ms. Noriko Tsuchiya, and Ms. Yoshiko Iwami for excellent technical assistance. We also thank Dr. Steven C. Hebert (Division of Nephrology, University of Vanderbilt, Nashville, TN, USA) and Dr. Sei Sasaki (Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan) for providing the antibodies used in this study.