Background

A loss of proximal tubule cell polarity is thought to activate tubuloglomerular feedback, thereby contributing to glomerular filtration rate depression in postischemic acute renal failure (ARF).

Methods

We used immunomicroscopy to evaluate the segmental distribution of Na⁺/K⁺-ATPase in tubules of recipients of cadaveric renal allografts. Fractional excretion (FE) of sodium and lithium was determined simultaneously. Observations were made on two occasions: one to three hours after graft reperfusion (day 0) and again on post-transplant day 7. An inulin clearance below or above 25 ml/min on day 7 was used to divide subjects into groups with sustained (N = 15) or recovering (N = 16) ARF, respectively.

Results

In sustained ARF, the fractional excretion of sodium (FE_Na) was 40 6% and 11 5%, and the fractional excretion of lithium (FE_Li) was 76 5% and 70 2% on days 0 and 7, respectively. Corresponding findings in recovering ARF were 28 2% and 6 2% for the FE_Na and 77 4% and 55 3% (P < 0.05 vs. sustained) for FE_Li. Na⁺/K⁺-ATPase distribution in both groups was mainly basolateral in distal straight and convoluted tubule segments and collecting ducts. However, Na⁺/K⁺-ATPase was poorly retained in the basolateral membrane of proximal convoluted and straight tubule segments in sustained and recovering ARF on both days 0 and 7.

Conclusions

We conclude that loss of proximal tubule cell polarity for Na⁺/K⁺-ATPase distribution is associated with enhanced delivery of filtered Na⁺ to the macula densa for seven days after allograft reperfusion. Whether an ensuing activation of tubuloglomerular feedback is an important cause of glomerular filtration rate depression in this form of ARF remains to be determined.

METHODS

Patient population

Our experimental population consisted of 31 consecutive patients undergoing cadaveric kidney transplantation. Each gave informed consent to a protocol that had been approved previously by the Committee for Research in Human Subjects at Stanford University. They ranged in age from 30 to 71 years. The corresponding age of the kidney donors was 10 to 62 years. A determination of the GFR, as measured by an inulin clearance at the end of the first post-transplant week, was used to arbitrarily divide the subjects into two groups. Those in which GFR was depressed by two thirds or more below optimal GFR for a renal allograft were classified as exhibiting "sustained ARF" (N = 15). In the remainder, GFR depression was by less than two thirds, and they were classified as "recovering ARF" (N = 16). The optimal GFR for a single transplanted kidney was defined as 77 ml/min/1.73 m². The latter value is based on the average level of inulin clearance that we recorded in long-standing recipients of renal allografts that were donated by a closely related living donor (sibling or parent); these recipients had never undergone an episode of rejection¹³.

A control group of living donors (N = 9) was used to establish the intensity and distribution of Na⁺/K⁺-ATPase staining in healthy renal tubular cells. Each living donor had routinely undergone an extensive evaluation prior to donation in order to exclude the presence of any detectable kidney disease¹⁴. Kidney tissue for histochemical analysis was obtained in each instance by an intraoperative needle biopsy, which was performed immediately before the renal artery was clamped in preparation for donor nephrectomy.

Protocol

Subjects underwent a study that included serial clearances and, in selected cases, serial biopsy. The initial study was performed between one and three hours after graft reperfusion on the day of transplantation (day 0). The repeat study was at the end of the first post-transplant week (day 7). Prior to each examination, the subject was given an oral dose of 600 mg of lithium carbonate. On the day of transplantation, the lithium carbonate was given just before surgery. On day 7, it was given one to two hours before beginning the clearance study. Eight of the 31 subjects (4 from each group) were given intravenous lasix (40 to 80 mg) intraoperatively after completion of the anastomosis. The remainder were infused with 12.5 g of 20% mannitol at this time. Diuretic agents were withheld for at least 24 hours before the day 7 study. Between the two studies, each recipient was given a diet containing 2.3 to 3.0 g of sodium per 24 hours and was immunosuppressed according to a standardized regimen that has been described in detail elsewhere^5,14.

The study on the day of transplantation (day 0) began intraoperatively approximately one hour after completion of the vascular anastomosis and reperfusion of the transplanted kidney. An allograft biopsy was performed using a gun biopsy device with a 16-gauge needle (Monopty; CR Bard Inc., Covington, CA, USA). Use of a needle rather than a surgical wedge biopsy permitted examination of medullary as well as cortical tubule segments. Immediately after completion of the transplantation procedure, the recipient was moved to a recovery room. A bladder catheter was used to make two consecutive, timed collections of urine, each 30 minutes in duration. A sample of blood was drawn at the midpoint of each collection. Urine and plasma samples were analyzed for creatinine, sodium, and lithium. The creatinine clearance was used as a surrogate measure of GFR. The clearance of sodium and lithium divided by the creatinine clearance and multiplied by 100 yielded fractional excretions (FE) of each cation.

The second examination was performed on post-transplant day 7. A priming dose of inulin (50 mg/kg) was followed by a sustaining infusion calculated to maintain plasma inulin concentration constant at 20 mg/dl. After a 60-minute equilibration period, four timed urine collections were made. A blood sample was drawn to bracket each urine collection. "Effective" GFR was calculated as the average of the four individual inulin clearances. The same urine and blood samples were used to calculate the simultaneous clearances of endogenous creatinine, sodium, and lithium. Because inulin and creatinine clearances did not differ in either sustained or recovering ARF (vide infra), the creatinine clearance was again used as the denominator in the calculation of FE_Li and FE_Na, as was the case on day 0. To evaluate the distribution of Na⁺/K⁺-ATPase during the maintenance and recovery stages of ARF, the transplant recipients were requested to undergo a repeat biopsy after completing the clearance determination, and nine agreed to do so. On this occasion, it was performed as a closed, transcutaneous procedure but employed the same Bard Monopty device with a 16-gauge needle that was used for the intraoperative biopsy an day 0.

Laboratory determinations.

Concentrations of inulin in urine and plasma were determined by a colorimetric method using an autoanalyzer. After acid hydrolysis to break inulin down into fructose, the concentration of the latter was determined with resorcinol¹⁵. The concentration of creatinine in urine and plasma was determined by an automated picrate method using a creatinine autoanalyzer (Analyzer II; Beckman Instruments, Palo Alto, CA, USA). This method is rate dependent and minimizes the influence of slowly reacting noncreatinine chromogens, thereby providing an estimate of true creatinine concentration. Measurements of lithium and sodium were performed by ion selective electrode (NOVA Biomedical, Newton, MA, USA)¹⁶. After measurement of urinary sodium and potassium concentrations, a second aliquot of urine was diluted with 200 mM sodium chloride to achieve sodium and potassium concentrations in the range observed in plasma. This step was necessary because the electrode was programmed to measure lithium in plasma. Recovery of lithium from the diluted samples was estimated by adding lithium chloride over a concentration range of 0 to 20 mmol/liter to standard solutions or blank samples of urine or plasma. The recovery averaged 104 6% (mean 1 SD).

Tubule morphology

Portions of biopsy tissue were prepared for light and electron microscopy. Tubule cell morphology was evaluated using semiquantitative techniques that have been described in detail elsewhere¹⁴. In brief, the percentage of tubule cells in seven grid fields that had exfoliated into the tubule lumen was examined by light microscopy (900). The apical and basolateral membranes of proximal tubule cells were evaluated by electron microscopy. Cross-sections of 60 to 90 tubules were examined at a magnification of 4800. The percentage of cells exhibiting either grossly diminished or absent apical brush border was determined. The frequency of interdigitations along the basal surface of the same cells was computed.

Immunohistochemistry

Antibodies.

Rabbit polyclonal antibodies against the subunit of Na⁺/K⁺-ATPase were used at a dilution of 1:100, as described previously⁵. Simultaneous double or triple immunofluorescence staining with antibodies against aquaporin-1, cytokeratin K8, and Tamm-Horsfall protein was used to identify the following discrete nephron segments: convoluted and straight proximal tubules, distal straight tubules (or thick ascending limbs of the loops of Henle) and distal convoluted tubules and collecting ducts. Rabbit polyclonal antibody against aquaporin-1 was raised against KI-stripped human red blood cell ghosts (CHIP 28; a gift from Dr. Alan S. Verkman, University of California, San Francisco, CA, USA), and was used at a dilution of 1:500. Aquaporin-1 was used as a marker for cells of both the convoluted and straight segments of the proximal tubule. Troma-1 rat monoclonal supernatant was used to detect cytokeratin K8. It was obtained from the Developmental Studies Hybridoma Bank (Department of Biology, University of Iowa, IA, USA, under contract N01-HD-2-3144 from the National Institute of Child Health and Human Development) and was used at a dilution of 1:1. The pattern of cytokeratin K8 distribution permitted proximal segments to be distinguished from distal convoluted segments, collecting ducts, and distal straight tubules. Antihuman Tamm-Horsfall protein monoclonal antibody was used to identify distal straight tubules and was purchased from Accurate Chemical & Scientific Corporation (Westbury, NY, USA); the antibody was used at a dilution of 1:200. By distinguishing medulla from cortex, the Tamm-Horsfall protein staining also facilitated differentiation between the straight and convoluted segments of the aquaporin-1–positive proximal tubule.

Tissue preparation for immunohistochemistry.

The remaining portion of the biopsy core was immediately dropped into 10 ml of paraformaldehyde-lysine-periodate fixative on ice for 30 minutes. After fixation, the tissue was washed three times in ice-cold phosphate-buffered saline (PBS). Each wash was carried out for 10 minutes on ice. After this step, the tissue was cryoprotected by transferring it to a 50 ml conical tube containing 40 ml of 2.5 M sucrose in PBS for at least 48 hours at 4°C. Tissue was then immersed in OCT cryoembedding compound (Miles), frozen in liquid N₂, and stored at -70°C.

Immunofluorescence staining

The frozen tissue blocks were mounted onto chucks and were sectioned using a 2800 Frigocut N cryostat (Reichert-Jung, NuBloch, Germany). Six-micrometer thick sections were transferred onto glass slides coated with 0.1% gelatin and 0.01% chromium potassium sulfate, were allowed to warm to room temperature, and were then extracted for 10 minutes in cytoskeleton buffer [50 mM NaCl, 300 mM sucrose, 10 mM piperazine-N, N'-bis 2-ethane-sulfonic acid (pH 6.8), 3 mM MgCl, 0.5% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride]. Each of the following steps was performed at room temperature unless otherwise stated, and each PBS wash involved two rinses, which were each 10 minutes in duration. After extraction, slides were washed in PBS, incubated in blocking solution (PBS containing 20% normal goat serum, 0.2% bovine serum albumin, 50 mM NH₄Cl, 25 mM glycine, and 25 mM lysine) for two hours in a humidified chamber, and washed again in PBS containing 0.2% bovine serum albumin. They were then incubated overnight at 4°C in a humidified chamber with the appropriate primary antibody diluted in PBS containing 20% normal goat serum and 0.2% bovine serum albumin. For the double-/triple-labeling experiments, sections were incubated with the relevant primary antibodies concurrently. The following day, slides were washed with PBS containing 0.2% bovine serum albumin and were then incubated with the appropriate secondary antibody for two hours in a humidified chamber; for double-/triple-labeling experiments, sections were simultaneously incubated with multiple secondary antibodies. Rhodamine-fluorescein-conjugated and AMCA-conjugated secondary antibodies were diluted 1:200 in PBS containing 20% normal goat serum and 0.2% bovine serum albumin. At the end of this incubation, slides were washed in PBS containing 0.2% bovine serum albumin and were then mounted with glass coverslips in PBS containing 16.7% Mowiol (CalBiochem Corp., La Jolla, CA, USA), 33% glycerol, and 0.1% paraphenylene diamine. Slides were viewed and photographed using a Zeiss Axioplan epifluorescence microscope equipped with differential interference contrast optics. Photographic slides (Kodak Ektachrome ASA 400; Eastman Kodak, Rochester, NY, USA) were used to assess the distribution of each protein. Photographs were taken in at least three different fields for each protein with 40, 63, or 100 objectives.

An arbitrary scale of 1 to 4 was used to assess Na⁺/K⁺-ATPase distribution and is illustrated in Figure 1. A normal basolateral distribution was classified as grade 1 Figure 1a. Complete redistribution of Na⁺/K⁺-ATPase from the cell membrane to cytoplasm was classified as grade 4 Figure 1d. Grades 2 and 3 were intermediate, with predominantly basolateral but some cytosolic staining in grade 2 Figure 1b and predominant cytosolic with some residual basolateral staining in grade 3 Figure 1c.

Figure 1.

Distribution of Na⁺/K⁺-ATPase in proximal convoluted tubule cells (PCT), grading of the severity of injury. The injury is heterogeneous, and the PCT in each panel, which exemplifies the degree of injury being described, is designated PCT¹. Abbreviations are: *, distal straight tubule; DCT, distal convoluted tubule; PST, proximal straight tubule. Grades are defined as: Grade 1 (A), normal, basolateral staining; Grade 2 (B), mild injury, diminished basolateral staining with some cytoplasmic redistribution; Grade 3 (C), moderate injury, further diminution of basolateral staining with increased cytoplasmic redistribution; Grade 4 (D), severe injury, virtually complete cytoplasmic redistribution.

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Statistical analysis

The significance of differences in physiological or clinical findings between the two groups was tested using either an unpaired Student's t-test or Wilcoxon's rank sum test, depending on the distribution of the findings. Intragroup differences between days 0 and 7 were evaluated with paired versions of the aforementioned tests. Results are expressed as the mean 1 standard error.

RESULTS

Clinical features

Clinical characteristics of the allografts and patient population are summarized in Table 1. The number of patients and their gender and age distribution were similar in the groups with sustained versus recovering ARF Table 1. The total time of allograft ischemia (cold storage and rewarming times) tended to be shorter in the recovering than the sustained ARF group. However, the difference reached statistical significance only for the warm ischemic time, where the latter is defined as the rewarming time between removal of the graft from cold storage and completion of the vascular anastomosis between recipient and donor vessels.

Table 1 - Characteristics of patient population.

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Renal function

The subjects were classified as belonging to the sustained or recovering ARF groups according to the finding on post-transplant day 7 of an inulin clearance below or above 25 ml/min, respectively. Of note, however, the inulin and simultaneous creatinine clearances on day 7 did not differ significantly. In the sustained ARF group, the respective values were 9 2 ml/min for inulin and 10 3 ml/min for creatinine (P = NS). The corresponding values in the recovering ARF group were 55 5 and 56 9 ml/min, respectively (P = NS). Because we did not perform an inulin clearance on day 0, we have chosen to make a direct comparison between the day 0 and day 7 findings by using the creatinine clearance on each day as a measure of GFR, as well as the denominator in calculating fractional excretions of sodium and lithium.

Our classification criterion of GFR below or above 25 ml/min at the end of the first week post-transplant also separated the subjects on the day of transplantation Figure 2. Those destined to exhibit sustained ARF had a day 0 GFR of only 5 2 versus 23 2 ml/min in those destined to recover rapidly (P < 0.001). Also noteworthy is that even in the recovering group, the GFR on postoperative day 7 did not achieve the "normal" range observed in long-standing, stable renal allografts transplanted from living related donors (shaded area in Figure 2). Sustained ARF was also distinguished from recovering ARF in that the rate of urine flow was lower both on the day of transplantation (1.7 0.4 vs. 9.2 1.5 ml/min, P < 0.001) and on post-transplant day 7 (1.8 0.5 vs. 4.5 0.8 ml/min, P < 0.01). Of the 15 subjects with sustained ARF, 11 required dialysis during the first post-transplant week. In the remaining four subjects, the serum creatinine level at the time of the day 7 evaluation was higher than in recovering ARF, 3.5 1.2 vs. 1.7 0.2 mg/dl, respectively.

Figure 2.

Creatinine clearance (C_Cr) in the first post-transplant week. Symbols are: () sustained ARF; () recovering ARF. Values are expressed as mean SEM. *P < 0.05 on postoperative day 7 vs. postoperative day 0 within each group; **P < 0.05 sustained vs. recovering ARF. The shaded area indicates the corresponding range for GFR in an optimally functioning transplanted kidney¹⁵.

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Judged by an FE of sodium (FE_Na) of approximately 1%, depending on dietary sodium intake, the fraction of filtered sodium that is excreted by healthy individuals is very small. In the freshly transplanted kidney, however, FE_Na was massive Figure 3. On day 0, it averaged 40 6% and 28 2% in the sustained and recovering ARF groups, respectively (P = NS). On day 7, the corresponding values fell significantly in each group to 11.1 4.6% and 5.7 1.6%, respectively Figure 3. Worthy of note is that despite a trend to a lower value approaching the physiological range in recovering ARF, the difference from sustained ARF on day 7 did not reach statistical significance.

Figure 3.

Fractional excretion of sodium (A) and lithium (B) in subjects with sustained () and recovering () acute renal failure (ARF) in the first post-transplant week. Values are expressed as mean SEM. *P < 0.05 day 7 vs. day 0 within each group; **P < 0.05 sustained vs. recovering ARF.

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As stated previously, the fraction of filtered sodium delivered out of the proximal tubule and the distal straight tubule (Henle's thick ascending limb) was estimated using the FE_Li. Unlike the physiological value of 31 2% reported for healthy individuals⁹, the FE_Li was markedly elevated in both the sustained and recovering ARF groups. The respective values were 76 5% and 77 4% on day 0 and 70 2% and 55 3% on day 7. The latter value in recovering ARF, albeit markedly elevated, was significantly lower than the corresponding day 7 value in sustained ARF Figure 3. Subtracting FE_Li from 100 in each individual yields an approximate value for fractional proximal sodium reabsorption on day 0 of 25 5% and 23 4% in sustained and recovering ARF, respectively (P = NS). Corresponding values on day 7 were 30 2% versus 45 3%, respectively (P < 0.05). Of note, however, the latter value in recovering ARF remains well below the physiological range of approximately 70% in healthy individuals^89101112.

Tubule morphology

Some proximal tubule cells appeared collapsed and necrotic by light microscopy. In keeping with our earlier observations¹⁴, however, an average of fewer than 3% of proximal tubule cells in seven grid fields (900) had exfoliated into the tubule lumen in both the sustained and recovering ARF groups on day 0. On day 7, the corresponding percentage of exfoliated proximal tubule cells was less than 1% in each group. Electron microscopy of proximal tubule cells, by contrast, revealed widespread abnormality (Figure 4 and Table 2). The percentage of cells exhibiting either complete absence or severe reduction of apical brush border was substantial Table 2. The finding was more frequent in sustained than recovering ARF, both on days 0 and 7, but the trend failed to achieve statistical significance. Basolateral interdigitations were sparse in both sustained and recovering groups on day 0 Figure 4, the number of basal infoldings per mm basal cell surface length averaging only 634 86 and 700 75, respectively Table 2. The basal infolding number remained depressed in the sustained group on day 7 (731 147) but increased significantly to 1311 838 in the recovering group (P < 0.05 vs. day 0).

Figure 4.

Electron photomicrograph of proximal tubule in subject with sustained acute renal failure (ARF). Notice the reduction of apical brush border and basal infoldings.

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Table 2 - Morphometry.

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Identification of nephron segments

CHIP28 (Aquaporin 1).

Strong staining was detected along the entire proximal tubule and descending thin limb of Henle Figure 5 and 6. Staining was densely localized to the apical membrane and apical cytoplasm of proximal tubule cells, but was confined to the apical membrane of the flattened cells in the descending thin limb of Henle.

Figure 5.

Nephron segment localization by staining with CHIP28 (left) and cytokeratin K8 (middle) and Tamm Horsfall protein (right) in cortex (upper panels) and medulla (lower panels) in biopsy tissue from a healthy living kidney donor (control). Abbreviations are: PCT, proximal convoluted tubule; PST, proximal straight tubule; CD, collecting duct; *, distal straight tubules (thick ascending limb) are identified by positive staining for Tamm-Horsfall protein.

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Figure 6.

Distribution along the nephron of Na⁺/K⁺-ATPase, CHIP28 (Aquaporin-1), cytokeratin K8, and Tamm-Horsfall protein (THP). The presence of a line indicates staining, and its breadth indicates the intensity of staining at each site. Abbreviations are: CD, collecting duct; DCT, distal convoluted tubule; DST, distal straight tubule (thick ascending limb); DTL and ATL, descending and ascending thin limbs of Henle's loop; Glom, glomerulus; IMCD, inner medulla collecting duct; PCT, proximal convoluted tubule; PST, proximal straight tubule.

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Cytokeratin K8.

All tubule segments, except the ascending thin limb of Henle, were stained, but the various segments tended to have distinctly different staining patterns Figure 5 and 6. Proximal convoluted tubule staining exhibited a "shaggy or irregular rooty" pattern in the apical-basal axis of cells. Staining of proximal straight tubule and the thin limb of Henle, by contrast, was associated with the basolateral membrane. Distal straight tubules showed only faint staining that was present mostly at the basolateral membrane. Distal tubule segments beyond the straight tubule displayed two different characteristic staining patterns. The segments immediately beyond distal straight tubules exhibited weak basolateral staining similar to that in distal straight tubule cells. Distal tubule cells adjacent to collecting ducts and cells of collecting ducts, by contrast, stained more strongly than any other tubule segments, and the pattern of the staining was characteristically rectangular with staining at both apical and basolateral plasma membranes.

Tamm-Horsfall protein.

Cells of distal straight tubules were stained Figure 5 and 6. The staining was localized to the cytoplasm and/or basolateral and apical membranes. The distribution of staining along these plasma membrane domains was more pronounced in cells of distal straight tubule from the inner stripe of the outer medulla. Cytoplasmic aggregates of Tamm-Horsfall protein staining were also observed in distal convoluted tubule cells immediately adjacent to the distal straight tubule. All other nephron segments exhibited weak staining for Tamm-Horsfall protein that was not above the background level.

Histochemical analysis of Na⁺/K⁺-ATPase

In control tissue sections, Na⁺/K⁺-ATPase was expressed in glomeruli, proximal convoluted tubules, proximal straight tubules, distal straight tubules, and distal convoluted tubule segments, and collecting ducts Figure 6. The intensity of staining along the nephron was variable. Distal straight and convoluted tubules exhibited the highest intensity, and staining was at the basolateral membrane. However, whereas basal and lateral membrane staining were similarly intense in cells of the distal convoluted tubule and collecting duct, there was more abundance of basal than lateral membrane staining in cells of proximal convoluted and distal straight tubules.

In allograft tissue sections, Na⁺/K⁺-ATPase was expressed in the same tubule segments as in controls, but the distribution of staining was heterogeneous, varying both among members of each group and among cells in a given section and segment. Figure 7 shows that adequate tissue was available to assess proximal convoluted or proximal straight tables in 11 to 12 members of both the sustained and recovering ARF groups. On day 0, the staining pattern for Na⁺/K⁺-ATPase was rarely restricted to the basolateral membrane (corresponding to grade 1) in proximal tubule cells of the allograft recipients. This was true regardless of whether the ARF was subsequently categorized as sustained or recovering. As shown, more than half of the biopsy tissues from the sustained ARF group were interpreted as showing grade 4 maldistribution in cells of both the convoluted and straight segments of the proximal tubule. The maldistribution was more variable in the recovering group. Worthy of note, however, is that the most abnormal pattern of redistribution (grade 4) was seen in only proximal straight but not proximal convoluted tubules among those patients who recovered rapidly. The distal tubule distribution of Na⁺/K⁺-ATPase is illustrated in Figure 8. Adequate tissue and localization of Na⁺/K⁺-ATPase permitted the distal straight tubule in all but 1 of the 31 recipients to be evaluated. The corresponding number with distal convoluted tubules or collecting ducts that were evaluable was smaller, however, varying between 7 and 10 members of each group. Maldistribution of Na⁺/K⁺-ATPase was far less prevalent in cells of the distal nephron on day 0 Figure 8. Except for occasional severe grades 3 or 4 injury to the distal straight tubule Figure 8a, the pattern of Na⁺/K⁺-ATPase distribution was predominantly normal (grade 1) or mildly deranged (grade 2) throughout the distal nephron. A trend to greater maldistribution of proximal Na⁺/K⁺-ATPase in the sustained than recovering group Figure 7 was not evident in the distal nephron.

Figure 7.

Grading of the distribution of Na⁺/K⁺-ATPase in proximal convoluted (A) and proximal straight (B) tubule cells on the day of transplantation. Symbols are () sustained ARF; () recovering ARF.

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Figure 8.

Grading of the distribution of Na⁺/K⁺-ATPase in distal straight (A), distal convoluted (B), and collecting duct (C) tubule cells on the day of transplantation. Symbols are: () sustained ARF; () recovering ARF.

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The pattern of Na⁺/K⁺-ATPase distribution remained highly variable in day 7 biopsies. In addition to variability, another feature shared with day 0 findings was prominent derangement of proximal but minor derangement of distal distribution. Given the variability and small sample size, a clear trend toward greater abnormality in sustained than recovering ARF was not discernible at this time. Among the five subjects with sustained ARF who underwent biopsy on day 7, proximal convoluted tubules exhibited a normal appearance in two (grade 1) and moderately severe maldistribution of Na⁺/K⁺-ATPase (grade 3) in three Table 3. Corresponding findings for proximal convoluted tubules in the four subjects with evaluable tissue in recovering ARF were three with grade 3 and one with grade 4 maldistribution Table 3. The percutaneous day 7 biopsies were less successful than the open biopsies on day 0 in sampling medullary tissue. Thus, the former contained adequate proximal straight tubules in only one individual with sustained ARF; these exhibited grade 3 maldistribution. Similarly, only two members of the recovering ARF group had evaluable proximal straight tubules in the day 7 biopsy, and these were scored as showing a normal appearance (grade 1) in one individual and severe maldistribution (grade 4) in the other Table 3. All day 7 biopsies, whether from the sustained or recovering groups, exhibited either a normal (grade 1) or mildly abnormal (grade 2) distribution of Na⁺/K⁺-ATPase in distal straight tubules. In the case of distal convoluted tubules and collecting ducts, the distribution of Na⁺/K⁺-ATPase on day 7 was always normal (grade 1). Of note, redistribution of Na⁺/K⁺-ATPase from the basolateral to the apical membrane was not observed in either the proximal or distal tubule cells of either ARF group.

Table 3 - Grading of Na⁺/K⁺-ATPase distribution in proximal tubule segments on postoperative day 7.

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DISCUSSION

Transient interruption of blood flow to the kidney for 30 to 60 minutes is followed by ARF¹⁷. The brunt of the postischemic injury is borne by proximal tubular cells, which exhibit disruption of the actin-based cytoskeleton^18,19. An ensuing loss of cell polarity is manifest by a redistribution of Na⁺/K⁺-ATPase from its normal location on the basolateral plasma membrane^4,20. The normal localization of Na⁺/K⁺-ATPase to the basolateral membrane is regulated by direct interactions with membrane-associated cytoskeletal proteins^21,22 and generates an electrochemical gradient across the cell that is required for vectorial Na⁺ transport from the tubule lumen to the surrounding interstitium and peritubular circulation²³. Redistribution of Na⁺/K⁺-ATPase has thus been proposed as an explanation for the high fraction of filtered sodium that is typically excreted by patients with postischemic ARF¹.

Because patients with postischemic ARF of native kidneys are too ill to undergo biopsy, we have used the reperfused, cadaveric kidney transplant to test this hypothesis. Between the time of procurement from the donor and subsequent implantation into the recipient, a kidney transplanted from a cadaveric source undergoes a prolonged interval of nonperfusion, one that typically exceeds 18 hours Table 1. Despite measures to protect the renal cells, including storage at 4°C so as to bring metabolism to a halt, the cadaveric kidney transplant is often the site of a postischemic injury²⁴. In fact, based on the assessment of function and structure one to three hours after reperfusion in this study, it seems reasonable to suggest that postischemic injury is invariable. Judged by corresponding findings on day 7, we infer that approximately one half of cadaveric kidneys exhibit a sustained form of ARF that often requires dialysis therapy. The remainder exhibits varying but largely incomplete degrees of recovery that can be identified by a trend for the GFR to increase toward the normal range.

In an earlier study of the marked depression of GFR that attends sustained allograft ARF, we evaluated each of the determinants of GFR¹⁴. Using a mathematical model of glomerular ultrafiltration, we calculated that abolition of the net pressure for ultrafiltration accounted for the observed hypofiltration. In experimental animals with postischemic ARF, a corresponding loss of filtration pressure has been attributed in large part to obstruction of tubule lumina by exfoliated tubule cells^25262728. In the reperfused renal allograft, by contrast, there is little, if any, evidence in support of tubular obstruction. As was the case in this study, we have found the prevalence of exfoliated tubule cells to be very low. Moreover, as judged by fractional volumes of Bowman's space and proximal tubule lumina, neither of these structures appears to be distended as a result of obstruction at some downstream site¹⁴. It follows that loss of filtration pressure in postischemic allograft ARF is more satisfactorily explained by a reduced perfusion pressure in glomerular capillaries than by an elevated pressure in Bowman's space¹⁴. The fall in glomerular capillary pressure, in turn, is most likely to be a consequence of afferent arteriolar constriction.

A number of potential mediators of afferent arteriolar vasoconstriction have been implicated in lowering glomerular capillary perfusion pressure in postischemic ARF in the rat. These include enhanced production and activity of angiotensin II and endothelin, along with increased sympathetic nervous traffic²⁹³⁰³¹. Conversely, afferent arterioles in ARF have been shown to be resistant to nitric oxide-dependent vasodilators, such as acetyl choline and bradykinin^31,32. The transplanted kidney is denervated, thereby precluding a role for enhanced sympathetic nervous traffic under these conditions. However, we have shown that the protracted injury associated with delayed allograft function is accompanied by sustained elevation in plasma of renin activity and endothelin-1 levels¹⁴. In addition, there appears to be resistance of afferent arterioles to the vasodilator actions of endogenous atrial natriuretic peptide, which is present in excess³³. Thus, postischemic allograft injury appears to be accompanied by an imbalance that favors constrictor hormones over those that dilate the afferent arteriole. This, in turn, could contribute to GFR depression by lowering glomerular perfusion pressure.

Another phenomenon that could mediate afferent arteriolar constriction and could lead to a fall in glomerular perfusion pressure is tubuloglomerular feedback³⁴. The findings in this study are consistent with the conditions needed for activation of tubuloglomerular feedback. We have confirmed our earlier observation¹⁴ that a remarkably large fraction of filtered sodium is excreted during the first few hours following reperfusion of a cadaveric renal allograft Figure 3. Administration of diuretic agents has never been reported to achieve fractional Na⁺ excretion in excess of 10% in the human kidney. We thus infer that neither the intraoperative administration of lasix to occasional subjects nor of mannitol to others can possibly explain the fractional sodium excretions in the 30% to 40% range observed on day 0 in this study Figure 3. That the underlying impairment of sodium reabsorption is upstream from the macula densa is suggested by our determinations of FE_Li. Lithium has been shown to undergo cotransport with Na⁺ in the proximal tubule and in the distal straight tubule. Each site is upstream from the macula densa and accounts for 90% and 10% of lithium reabsorption, respectively^9,10. Our finding that FE_Li is elevated by a factor of two in both groups on day 0 and by a similar amount in the sustained ARF group on day 7 is thus consistent with massive enhancement of the fraction of filtered sodium that is delivered to the macula densa. Conceivably, subsequent activation of tubuloglomerular feedback could lead to constriction of afferent arterioles.

Our examination of the localization and distribution of Na⁺/K⁺-ATPase provides a possible structural basis for enhanced sodium delivery to the macula densa. The observations in healthy transplant donors confirm that Na⁺/K⁺-ATPase is normally distributed along the basolateral membrane of tubular epithelial cells. As is the case in rodents, increased intensity of staining points to greater abundance of Na⁺/K⁺-ATPase in the distal nephron^35,36. Whereas staining in the mouse nephron is the most intense in distal convoluted tubule cells, the cells of the distal straight tubule exhibit the most intense staining in the human nephron Figure 5. In addition to exhibiting weaker staining than in distal cells, the cells of the proximal tubule show less distinct lateral than basal staining, a finding that is attributable to numerous interdigitations of the lateral cell membrane is this region of the nephron. Despite its lower intensity of staining, however, it is the proximal tubule that exhibits the greatest maldistribution of Na⁺/K⁺-ATPase Figure 7. Because of its low oxygen supply, the medullary portion of the distal straight tubule is considered the nephron segment most susceptible to postischemic damage^38,39. However, it is the convoluted and straight segments of the proximal tubule, rather than the distal straight tubule, that exhibit the most severe histopathological change in humans with postischemic ARF⁴⁰, and it is these former segments that demonstrate the greatest inability to retain Na⁺/K⁺-ATPase in the basolateral membrane Figure 7. Moreover, both elevated fractional excretions of lithium and sodium and failure to retain Na⁺/K⁺-ATPase in basolateral membranes of proximal tubule cells persisted on day 7, suggesting that each abnormality is prolonged and possibly linked over at least the maintenance stage of ARF. Persistent linkage of impaired polarity and sodium reabsorption in the proximal tubule also appears to attend the recovery stage of ARF on day 7, however. This raises the possibility that alterations in mediators of afferent vasoconstriction other than tubuloglomerular feedback account for the improvement in GFR observed on day 7 in those with recovering ARF.

We wish to point out that most of the evaluable medullary sections that we successfully stained were from the outer stripe of the outer medulla, as judged by the presence of adjacent cortical structures Figure 1. We cannot exclude the possibility that we have failed to examine adequately the distal straight tubules in the more precariously oxygenated inner stripe of the outer medulla. It is conceivable that failure to retain Na⁺/K⁺-ATPase in the basolateral cell membrane at this site was more marked than in the outer stripe, contributing substantially to impaired Na⁺ reabsorption. Similarly, restoration to normal by day 7 of an undetected earlier maldistribution of Na⁺/K⁺-ATPase in the inner stripe segments of the distal straight tubule could have contributed to improved vectorial Na⁺ and Li⁺ transport and hence to the increasing GFR on that day.

The precise mechanism by which dislocation of Na⁺/K⁺-ATPase leads to impaired reabsorption of sodium remains to be fully elucidated. Transient interruption of renal blood flow to the rat kidney in vivo or temporary anoxia of cultured tubule cells in vitro have been associated in some studies with redistribution of Na⁺/K⁺-ATPase to the apical membrane¹⁸¹⁹²⁰. The latter finding has been attributed to diffusion of the enzyme past an incompetent intercellular tight junction^3,20,41,42. In theory, apical Na⁺/K⁺-ATPase could then pump intracellular sodium into the tubular lumen. This interpretation is not supported by a recent analysis of cultured Madin-Darby canine kidney (MDCK) cells subjected to anoxia and ATP depletion, however,⁴². In this analogue of ischemia/reperfusion injury, the "fence" function of tight junctions in the MDCK monolayer was preserved; flow of protein and lipid between apical and basolateral membranes remained inhibited. Consistent with similar preservation of "fence" function in the postischemic injury of this study, we have never observed staining for Na⁺/K⁺-ATPase in the apical membrane of tubule cells in the reperfused renal allograft. We submit that an apical redistribution of Na⁺/K⁺-ATPase is not required to explain impaired tubular reabsorption of sodium. Our observation that Na⁺/K⁺-ATPase is dislocated from the basolateral membrane to the cytoplasm would effectively result in depolarization of the tubule cell and could well serve to inhibit sodium reabsorption.

To summarize, this study has provided incremental evidence that implicates a loss of proximal tubule cell polarity in the genesis of postischemic ARF. First, we have shown that FE_Li, a surrogate measure for the fraction of filtered sodium that is delivered to the macula densa, is massively enhanced. Second, we have demonstrated that failure to retain Na⁺/K⁺-ATPase in the basolateral cell membrane is confined to or is proportionately more severe in cells of the proximal tubule. Finally, we have shown that the aforementioned abnormalities persist for seven days, a period that is representative of the maintenance stage of this form of postischemic ARF. We submit that the foregoing evidence attests to the existence of the conditions that are necessary to invoke a role for tubuloglomerular feedback in the filtration failure that accompanies ARF. On the other hand, we have not been able to demonstrate convincingly repolarization of tubule cells in the recovering ARF group on day 7 when GFR, FE_Na, and FE_Li are all returning to the normal range. We suspect that this disparity reflects both the semiquantitative nature of our histochemical analysis and the small number of biopsy samples that were available for analysis on day 7. Unfortunately, no techniques are currently available to evaluate the contribution of tubuloglomerular feedback to glomerular hypofiltration in the intact, human kidney. Thus, the precise role of loss of proximal tubule cell polarity in lowering the GFR in postischemic allograft ARF must remain a matter for speculation.

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Acknowledgments

This study was supported by grants R01 DK50712 and M01-RR00070 from the National Institutes of Health. Dr. Kwon's postdoctoral fellowship was supported by the Satellite Dialysis Centers Fund in Nephrology.