Expression of the polymeric immunoglobulin receptor and excretion of secretory IgA in the postischemic kidney

James C. Rice, Jeff S. Spence, Judit Megyesi, Randall M. Goldblum, and Robert L. Safirstein

Departments of Internal Medicine and Pediatrics, University of Texas Medical Branch at Galveston, Galveston, Texas 77555


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The humoral mucosal immune response of the kidney involves the transport of secretory IgA (S-IgA) through renal epithelial cells by the polymeric immunoglobulin receptor (pIgR). The pIgR is cleaved and released as free secretory component (FSC) or attached to IgA (S-IgA). We examined the effects of an ischemic model of acute renal failure (ARF) on the expression of pIgR and the secretion of FSC and S-IgA in the urine. Kidney pIgR mRNA levels decreased in ischemic animals by 55% at 4 h and by 85% at 72 h compared with controls. pIgR protein expression in the medullary thick ascending limb (TAL) decreased within 24 h and was nearly undetectable by 72 h. Urinary S-IgA and FSC concentrations decreased by 60% between days 3 and 6. pIgR mRNA and pIgR protein in the kidney returned to ~90% of control levels and urinary FSC and S-IgA concentrations returned to ~55% of control levels by day 7. We demonstrate that ischemic ARF decreases renal mucosal S-IgA transport in vivo and may contribute to the increased incidence of urinary tract infections.

secretory component; mucosal immunity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE SECRETIONS THAT BATHE mucosal epithelial surfaces contain an array of host defense factors, including polymeric immunoglobulins (pIg), of which IgA is the major class. These secretory immunoglobulins (S-IgA and S-IgM) are transported from the basolateral to the apical surface of mucosal epithelial cells by the polymeric immunoglobulin receptor (pIgR). As in humans, the rat pIgR is a 118- to 120-kDa glycoprotein composed of an amino-terminal immunoglobulin-binding portion, termed secretory component (SC), a membrane-spanning element, and a carboxyl-terminal cytoplasmic domain (20, 34). After transcytosis of the pIgR to the apical region, both pIgR bound to immunoglobulin and unbound pIgR accumulate in endosomal vesicles called "apical recycling endosomes" (3). The pIgR-containing endosomal vesicles fuse with the apical membrane, where most of the ectoplasmic segment of pIgR is proteolytically cleaved and either secreted as free SC (FSC) or bound to polymeric immunoglobulins as secretory Ig (S-Ig).

The pIgR is expressed in renal tubule epithelial cells of humans (1) and rodents (27), both with and without IgA (1, 27). During urinary tract infections (UTI), secretion of SC is increased. This includes SC that is attached to IgA (12, 33), where it may function to prevent degradation of secretory IgA (16), and FSC (12), which may inhibit adhesion of some bacteria to cell membranes (9). Thus the concentration of S-IgA and FSC in the urine may influence susceptibility to UTI. Consistent with this view, bacterial growth is more rapid in urine obtained from animals undergoing water diuresis (13), and bacterial growth rates are higher in the kidneys of diuretic animals, compared with antidiuretic controls, in some models of pyelonephritis (7).

The effects of ischemia on the mucosal immune response in the kidney are of interest because UTI are the most common infectious complications during acute renal failure (ARF) (40) and in the early (postischemic) renal transplant period (29). We have shown recently that renal pIgR levels are influenced by hydration status, urine flow rate, and arginine vasopressin (27); factors that are altered in ischemic renal failure (2, 25).

In the present study, we investigated the effect of renal ischemia and reperfusion on pIgR expression in the rat kidney and both SC and secretory IgA (S-IgA) secretion in the urine. We speculated that ischemic ARF might decrease pIgR expression and impair the mucosal immune response. We found that renal ischemia and reperfusion resulted in a rapid and progressive decline in pIgR mRNA expression in the kidney. Immunohistology demonstrated that staining for pIgR in the thick ascending limb (TAL) was decreased in ischemic animals (compared with sham-operated controls) on day 1 and fell to nearly undetectable levels on day 3. The urine concentration of both FSC and S-IgA fell significantly by day 3, due primarily to the increased urine flow. The rapid decrease in pIgR mRNA, the decline in intracellular pIgR protein levels, and the fall in urine SC and S-IgA concentrations may impair the mucosal immune response of the urinary tract and predispose the postischemic kidney to infection.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Sprague-Dawley rats (Harlan, Houston, TX) weighing between 250-350 g were used for all experiments. We placed rats in individual metabolic cages for an equilibration period of 3-6 days before starting the study. Animals received food and water ad libitum until 3 days before surgery, after which they were pair fed (Formulab diet 5008; PMI Feeds, St. Louis, MO). Animals were randomized to two surgical study groups: 1) bilateral renal artery ischemia, in which rats underwent decapsulation of both kidneys and clamping of the renal vessels for 50 min, and 2) sham-ischemia (control) animals, which underwent decapsulation of both kidneys and manipulation of renal vessels without clamping. Rats were anesthetized with pentobarbital sodium (~65 mg/kg injected intraperitoneally). Renal ischemia was induced by simultaneous clamping of the renal hilum of both kidneys using 50 mm "bulldog-type" serrated vascular clamps (Kent Scientific, Litchfield, CT). We visually documented reperfusion of both kidneys after removing the vascular clamps at the end of the ischemic period and before closing the abdomen with 4-0 silk suture. All animals were placed in metabolic cages, after recovery from the anesthesia, until the completion of the study periods. Before harvest, we perfused the kidneys in situ by clamping the aorta and inferior vena cava above the renal arteries, venting the left renal vein, and injecting 20 ml 0.9% saline at 4°C via the infrarenal aorta using a 21-guage "butterfly" needle (Sureflo winged infusion set, Terumo, Elkton, MD). mRNA was isolated from one kidney; immunohistology and pIgR quantification was performed on the other kidney.

Antibodies. The monospecific polyclonal rabbit antibody against the SC portion of the rat pIgR utilized in this study was described previously (27). Aliquots of the IgG fraction of this anti-rat SC antibody were conjugated with horseradish peroxidase (Sigma) (21) for use in ELISA. A monoclonal mouse anti-rat IgA antibody used for S-IgA quantification was obtained from Zymed Laboratories (San Francisco, CA). Horseradish peroxidase-conjugated sheep anti-rat IgA (alpha -chain) was obtained from The Binding Site (San Diego, CA). S-IgA, used as a standard in quantification, was isolated from rat bile on an HPLC Superdex 200HR 10/30 column (SMART System Pharmacia Biotech, Piscataway, NJ). The purity of the S-IgA was confirmed by electrophoresis of the individual fractions on 8% SDS-PAGE. These gels were subsequently either stained with Coomassie blue or used for Western blots, utilizing sheep anti-rat IgA (alpha -chain):horseradish peroxidase-conjugate antibodies (The Binding Site) and 4-chloro-1-napthol (Sigma) and methods previously described (27). The concentration of IgA in the purified S-IgA standard was determined by ELISA using purified rat myeloma IgA (Zymed Laboratories) as the standard (27).

Urine collection. Urine excreted over 24 h was collected into polypropylene tubes (Falcon 2059; Becton-Dickinson, Bedford, MA) under mineral oil using metabolic cages. Aprotinin (200 mM; Sigma) in sodium borate-buffered saline was added to each urine collection tube (200 µl/tube) to prevent proteolysis and microbial growth.

ELISA. We quantified renal and urinary FSC levels in a sandwich ELISA utilizing purified rat myeloma IgA and polyclonal rabbit IgG anti-rat FSC conjugated to horseradish peroxidase, as previously described (27). Rat FSC, used as a standard, was purified from bile and quantified by ultraviolet absorbance based on an extinction coefficient of 12.66 (14). The sensitivity of the FSC assay (4 ng/ml) was well within the range of pIgR protein levels in the kidney and FSC concentrations in the urine of sham-operated (control) and ischemic animals. Our average coefficient of variation (CV) for the serial dilutions of each sample tested on an ELISA plate was 0.108 (10.8%). When quantification of SC levels was repeated on different ELISA plates (intra-assay variation), the mean CV was 0.156 (15.6%). Identical methods were used in an ELISA for urinary rat S-IgA levels, except that mouse anti-rat IgA (Zymed) in borate-buffered saline (3 µg/ml) was utilized as the capture reagent and purified S-IgA was utilized as the standard. Urines were tested at multiple dilutions. The concentrations of FSC and S-IgA in the samples were determined using the mean of at least two different dilution points and compared with the standard curve using linear regression.

mRNA isolation and Northern analysis. We prepared whole kidney mRNA by rapid homogenization in guanidinium thiocyanate, as previously described (6, 27), or by using the Ultraspec RNA Isolation System (Biotecx, Houston, TX) per the manufacturer's recommendations. Northern analysis of poly(A)+ RNA (5 µg/lane) was performed as previously described (27). We utilized the 1.4-kb Sac I digest of the full-length cDNA probe for rat pIgR mRNA, which was kindly provided by George Banting of the University of Bristol, United Kingdom (4). We assessed the variability in loading of mRNA by probing the same blot for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Quantification of mRNA for both pIgR and GAPDH was determined utilizing a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and/or a Lynx 5000 Densitometer, using version 5.1 of the Imagequant software (Applied Imaging, Santa Clara, CA). The quantity of pIgR mRNA was expressed as a ratio to that of GAPDH in the same sample.

Immunohistology. Frozen tissue sections were prepared from whole kidney and stained with monospecific rabbit anti-SC antibody, as previously described (27). Biotinylated goat anti-rabbit antibody (Vector Labs, Burlingame, CA) and avidin-biotin peroxidase complex with 0.01% diaminobenzidine (VectaStain Elite ABC; Vector Labs) were utilized to detect the primary antibody. Nonspecific staining was blocked with 5% normal goat serum (Sigma) in 0.1% Triton X-100 (Sigma), and endogenous peroxidase was inactivated by 1% H2O2 in PBS:methanol (1:1). Additional tissue sections were stained with the horseradish peroxidase-conjugated sheep anti-rat IgA (The Binding Site) to assess the distribution and relative amounts of IgA in the rat kidney. In addition, we incubated whole kidney sections with preimmune rabbit sera to assure that the staining was specific.

Statistical analysis. Differences between and within the ischemic and control groups were assessed by two-way ANOVA (32). When a significant difference was present between groups, additional analyses were performed using either the Tukey's method for all comparisons or the unpaired t-test assuming equal variances for planned comparisons (Microsoft Excel, Redmond, WA). P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

pIgR mRNA expression is decreased in the kidney postischemia. ARF induced by 50 min of renal artery clamping, followed by reperfusion, caused the pIgR mRNA level to decrease rapidly to 53% of control (sham-ischemia) by 4 h postischemia (P < 0.02; Fig. 1). The pIgR mRNA level fell to a nadir of 23% of control levels at 72 h (P < 0.001), then returned to near control levels at 7 days postischemia (Fig. 1).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 1.   Ischemia and reperfusion decreases kidney pIgR mRNA. A: representative Northern blot demonstrates polymeric immunoglobulin receptor (pIgR) mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in kidney at various intervals after renal ischemia and reperfusion. pIgR mRNA levels decrease within 6 h after ischemia and reperfusion, and remain at depressed levels until returning to control values by day 7. B: densitometric analysis of Northern blots showing time course of pIgR mRNA levels in postischemic animals. Each circle represents mean pIgR mRNA level normalized to GAPDH in same sample (pIgR/GAPDH mRNA) and expressed as percentage of control pIgR/GAPDH levels. Data at indicated time points were obtained from 4 separate Northern blots. Error bars represent SE of means; n = 3-5 animals per time point; * P < 0.05, compared with controls.

pIgR staining of the TAL is decreased postischemia. In sham-ischemia (control) animals, pIgR protein was expressed in the epithelium of the TAL and appeared darker in the apical region (Fig. 2A). A smaller amount of pIgR protein was also observed in the distal convoluted tubule and on the basolateral surface of proximal tubule cells (Fig. 2B). We have reported similar results for untreated animals (27). In the ischemic animals, the staining for pIgR was decreased throughout the medullary TAL at 24 h postreperfusion (data not shown) and declined to nearly undetectable levels by 72 h (Fig. 2C). The morphology of the cells lining the medullary TAL, where pIgR protein is predominantly located (27), was essentially unchanged after ischemia, suggesting that the decline in pIgR staining was not due to loss of tubular epithelial cells of the TAL (Fig. 2C). pIgR staining of the cortical TAL and distal convoluted tubule was also decreased by 72 h in the ischemic animals (Fig. 2D), but the decrease in staining was less striking than in the medullary TAL at the same time point after ischemia (Fig. 2C). Staining for pIgR recovered to near control levels by day 7 in the inner stripe of the outer medulla (Fig. 2E) and the cortex (Fig. 2F). There was no pIgR staining detected in glomeruli, interstitial cells, vasculature, or inner medulla at any time point in the sham-operated or ischemic animals (Fig. 2, A-F).


View larger version (163K):
[in this window]
[in a new window]
 
Fig. 2.   Ischemia decreases pIgR staining in kidney. Representative tissue sections from inner stripe of outer medulla (A, C, and E) and cortex (B, D, and F) of kidneys isolated from ischemic and controls animals. pIgR staining at apical surface of thick ascending limb (TAL; *) is decreased at 72 h after ischemia-reperfusion (C), compared with controls (A). pIgR staining of proximal (arrows) and distal tubules (arrowheads) is also decreased at 72 h after ischemia-reperfusion (D), compared with controls (B). However, decreases in pIgR staining of the cortical segments are less than in TAL at same time postischemia (C). By day 7, staining for pIgR returned to control levels in both TAL (E) and proximal and distal tubules of cortex (F). There is no staining of glomeruli (g), vessels, or inner medulla for pIgR at any time point. Magnification, ×160.

IgA staining in TAL and distal convoluted tubules decreased after ischemia. Weak IgA staining was observed in the distal convoluted tubule (Fig. 3) and subapical region of the TAL in control animals (Fig. 3, inset). IgA staining was not detected in the cortical TAL or distal convoluted tubules in ischemic animals at 72 h after ischemia-reperfusion, although IgA returned to near control levels by day 7 (data not shown). IgA staining was not detected in lymphoid cells, interstitial cells, proximal tubule cells, or glomeruli at any time point in control or ischemic animals, suggesting that the majority of IgA was delivered to the renal tubules via the renal blood flow.


View larger version (150K):
[in this window]
[in a new window]
 
Fig. 3.   IgA distributed in distal tubule and TAL. Representative tissue section of renal cortex from control animals reveals low levels of IgA staining in apical aspect of distal convoluted tubules (arrowheads). High-power view of outer stripe of outer medulla (inset) demonstrates localization of IgA in subapical surface of TAL (*). There was no staining of IgA-containing lymphocytes in interstitium or tubular basement membrane. Magnification, ×140; inset, ×350.

Urinary flow rates increased and urine FSC concentration fell in postischemic rats. The daily urine volume in ischemic animals was increased by fourfold on day 2 (from 5.3 ± 0.7 to 23.2 ± 2.3 ml/24 h) compared with controls, and remained at this level through day 6 (Fig. 4). Urine FSC concentration was significantly less in ischemic animals than in controls from day 1 through day 6 (Fig. 4; P < 0.002). Urine FSC concentration fell to <40% of preischemic levels from day 2 through day 6 in the ischemic group (P < 0.001) and remained below preischemic values at day 7 (P < 0.01; Fig. 4). Within the control group, urine FSC concentration was increased from pre-sham ischemia only on day 1 (the first 24 h postsurgery; P < 0.001) and day 5 (P < 0.05). Although the concentration of SC was significantly different between the ischemic and sham-ischemic (control) groups on day 1 through day 6, the total (daily) urinary SC (µg SC/24 h) did not differ. The ability to maintain the daily SC urine excretion in the ischemic group appeared to result in the depletion of stored intracellular pIgR/SC, as noted by immunohistology (Fig. 2).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   A: ischemia increases urine flow rate. There was a significant increase in urine volume per 24 h in ischemic () vs. control () animals; n = 9-11 animals per group; * P < 0.001, ischemic vs. control. B: urine free secretory component (FSC) concentration is decreased postischemia. Urine FSC decreased from day 1 through day 6 in ischemic group () vs. controls (); n = 9-11 animals per group; * P < 0.002, ischemic vs. control.

Urinary S-IgA concentration was decreased postischemia. The S-IgA concentration in the urine of ischemic animals was significantly different from control (sham-ischemia) animals on day 4 through day 6 (Fig. 5; P < 0.05). In the ischemic group, urinary S-IgA concentration fell to 40% of control levels at 72 h and remained at that reduced level through day 6 after ischemia and reperfusion (Fig. 5). Urine S-IgA concentration in the ischemic animals returned to 63% of urine S-IgA concentration of controls on day 7. Daily S-IgA excretion (total µg/24 h) in the ischemic animals did not differ significantly from controls, except when S-IgA excretion increased transiently during the onset of the diuresis, between 24 h and 48 h (P < 0.01, day 2, data not shown).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   A: ischemia results in decreased secretory (S)-IgA concentration in urine. Urine S-IgA levels are decreased from day 4 to day 6 in postischemic group, compared with controls; n = 9-11 animals per group; * P < 0.05, ischemic vs. controls. B: S-IgA-to-FSC concentration ratio is increased in urine of ischemic animals. S-IgA-to-FSC concentration ratio is increased on day 2 in ischemic animals vs. controls, suggesting that ischemia has an unequal effect on SC and S-IgA secretion early after ischemia and reperfusion. Ratios were determined from paired values (S-IgA and FSC) for each urine sample; n = 8-10 animals per group; * P < 0.05, ischemic vs. control.

Increased urine S-IgA-to-SC ratio after ischemia-reperfusion. The ratio of S-IgA concentration to FSC concentration (µg S-IgA/µg FSC), measured on the same sample of urine, increased early after ischemia-reperfusion (Fig. 5). The S-IgA-to-FSC ratio in the urine of ischemic animals increased threefold from preischemic values on day 2 (P < 0.05; ischemic vs. control). These results suggest that ischemia affected urinary FSC excretion to a greater extent than urinary S-IgA excretion. There was no significant change in the S-IgA/FSC ratio in the urine of control animals.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To our knowledge, this is the first study to evaluate the effects of ischemic stress on pIgR expression in the kidney and FSC and S-IgA excretion in the urine. We demonstrated that renal ischemia, followed by reperfusion, decreased pIgR mRNA levels in the kidney, pIgR staining in the TAL, and FSC and S-IgA concentrations in the urine.

pIgR mRNA declines rapidly after ischemia. The rapid (4 h) and sustained (72 h) decrease in pIgR mRNA levels postischemia (Fig. 1) cannot be explained by the simple loss of cells that produce pIgR. The predominant sites of pIgR expression in the kidney are the TAL and distal convoluted tubule, where, unlike the proximal tubule, the morphological changes induced by ischemia are moderate and reversible (8). The decline in pIgR mRNA levels after ischemia follows a pattern similar to that observed for prepro-epidermal growth factor (prepro-EGF) mRNA and Tamm-Horsfall mRNA in the TAL, as reported by our group (30). The decrease in pIgR mRNA is not due to a nonspecific decline in TAL mRNA, as the expression of other genes that localize to the TAL and distal tubule are upregulated after ischemia (19). The changes in the pIgR/GAPDH ratio in the kidney after ischemia-reperfusion are not due to changes in GAPDH mRNA levels, as we have previously noted that GAPDH mRNA levels in the kidney are not affected by ischemia (31).

We have previously suggested that pIgR mRNA levels in the kidney in vivo are regulated, in part, by cAMP (27). Specifically, we found that arginine vasopressin administration increased pIgR mRNA levels fourfold by 24 h, compared with controls. The vasopressin-V2 receptor is localized to the collecting duct and TAL in the rat (23) and is coupled, through adenylyl cyclase, to the production of cAMP (11, 17). Despite higher vasopressin levels in rats during the polyuric phase of acute tubular necrosis (25), the outer medulla of the postischemic kidney has a decreased adenylyl cyclase response to vasopressin (2). The administration of dibutyryl cAMP ameliorates the increase in serum creatinine and the fall in urine volume after acute ischemic renal failure in rats in vivo (15). It is possible, therefore, that decreased cAMP levels resulting from the decreased adenylyl cyclase response to vasopressin in the postischemic kidney may contribute to the decrease in pIgR mRNA expression. Local cytokine (10, 36) and chemokine expression (30) are also altered in the postischemic kidney. For example, gamma interferon (IFN)-gamma , which increases pIgR mRNA in colon adenocarcinoma cells in vitro (24), is increased in the kidney by day 3 postischemia (10, 36). Hence, the extent to which the pIgR mRNA level falls in the postischemic kidney may depend on the effects of multiple factors, including decreased cAMP levels and changes in local cytokine expression.

pIgR protein levels decrease in the kidney after ischemia. The pIgR protein, either free or bound to IgA, is stored in subapical vesicles before its transport across the apical membrane and excretion into the lumen of mucosal tissues (3, 35). We have previously shown that intact pIgR is preferentially expressed near the luminal surface of the TAL in normal rat kidney (27), presumably in "apical-recycling endosomes" (3). In this report, we demonstrate that renal ischemia, followed by reperfusion, caused a significant decrease in immunohistochemical staining for pIgR in the TAL by 24 h, which continued to decrease up to 72 h after reperfusion (Fig. 2).

The loss of staining that occurred primarily in the TAL of ischemic animals suggests depletion, or "washout", of pIgR-stored protein. Although the mechanisms that result in loss of stored pIgR are unclear, we have demonstrated a similar decline in apical staining in normal animals during the diuresis induced by water loading (27). The increase in urinary FSC excretion during the polyuric phase may reflect the ischemia-induced decline in ATP (38) and cAMP levels (37). cAMP has been suggested, by some authors, to inhibit FSC secretion in pIgR-transfected Madin-Darby canine kidney (MDCK) cells in vitro (5). Hence, the decreased cAMP levels in the kidney during ischemia may facilitate the depletion of stored pIgR. We are unable to determine, at the present time, whether the decrease in pIgR staining in the TAL, specifically the loss of darker staining on the apical surface of these cells, is due to loss of pIgR stored in apical vesicles.

We have estimated that, in normal animals, >60% of the total intracellular pIgR in the kidney is secreted into the urine per day (unpublished observations). The depletion of intracellular pIgR in ischemic animals noted by 72 h (Fig. 2), therefore, is likely due to the combined effects of continued SC excretion and a decrease in pIgR mRNA levels (Fig. 1). Although we did not directly measure the production of pIgR/SC protein after ischemia, the unchanged daily SC excretion, coupled with the immunological evidence of pIgR depletion and the decreased pIgR mRNA levels, suggests that pIgR production is decreased by ischemia. These effects of renal ischemia would result in a decrease in the amount of pIgR available for IgA transport into the tubule lumen of the kidney.

Urine SC concentration decreases early postischemia. Urine FSC concentrations are constant throughout a wide range of urine flows in untreated animals (27). In contrast, we found that urine FSC concentration fell significantly with increased urine flow in ischemic animals (Fig. 4). Hence, SC excretion did not increase in proportion to the increase in urine flow after ischemia. The lack of a proportional increase in urine SC excretion during the diuresis after ischemia is different than the proportional increase in urine SC excretion that occurs during the diuresis induced by water loading in normal animals (27).

Although the SC concentration in the urine of ischemic animals was significantly below control levels on day 1 through day 6, there was no difference in total SC excretion (µg/24 h) between the ischemic and control groups. The similar urine SC excretion between groups, during a time of decreased pIgR mRNA in the ischemic animals, may account for the loss, or washout, of stored pIgR/SC in the kidneys after ischemia-reperfusion (Fig. 2). The decrease in SC concentration in the urine may be biologically significant, as recent evidence suggests that SC can inhibit bacterial adhesion to cell membranes (9). Hence, the decline in SC concentration after ischemia may, in part, account for the increased susceptibility of the postischemic kidney to infection.

Urine S-IgA concentration is decreased postischemia. Total S-IgA excretion was similar between groups, except for an increase in S-IgA excretion that occurred in the ischemic group during the onset of diuresis (between day 1 and day 2). This may have contributed to the depletion of stored S-IgA in the kidney, as evidenced by immunohistology on day 3 (data not shown). The more striking and perhaps biologically relevant result was the significant decline in S-IgA concentration in the urine on day 4 through day 6. The concentration of S-IgA decreased in the urine by 35-70% between day 3 and day 7 in the ischemic group (Fig. 5), primarily due to the increase in urine flow rate (Fig. 4). Previous reports emphasize that S-IgA is important in the mucosal defense of the urinary tract (18, 28) and may be the primary factor in the urine that inhibits the binding of bacteria to urinary tract epithelial cells (33). The lowering of S-IgA concentration in the urine of ischemic animals from day 4 through day 6 correlates with a time period that UTI frequently complicate ARF (40). The significance of urine SC and S-IgA concentrations in preventing UTI is suggested by the observation that bacterial growth is more rapid in urine obtained from animals undergoing water diuresis (13). In addition, bacterial growth rates are higher in the kidneys of diuretic animals, compared with antidiuretic controls in some models of pyelonephritis (7).

The major immunological function of pIgR/SC is thought to be transport of S-IgA. Because IgA-containing B cells were not seen in the tissue sections of any of the rats, the predominant source of IgA for transport by pIgR into the urine appears to be via the renal blood flow, which is decreased early after ischemia (39). Thus the impairment in local mucosal immunity in ARF may be due to either decreased availability of pIgR for immunoglobulin transport, decreased IgA delivery, or both.

Ischemia affects the ratio of S-IgA to FSC in the urine. Although ischemia decreased both S-IgA and FSC concentrations in the urine, the ratio of S-IgA to FSC increased threefold by day 2 (Fig. 5). These results, measured on the same urine specimens, indicate that ischemia decreases SC concentration to a greater extent than it decreases S-IgA concentration. This disproportionate effect of ischemia on urine S-IgA and FSC concentrations indicates that the decrease in FSC and S-IgA concentrations are not purely due to increases in urine volume, as this would affect the concentration of both of these excreted proteins equally. The mechanisms in ischemia that underlie this disproportionate effect on S-IgA and FSC excretion by day 2 are unknown. However, it is likely that reduced pIgR synthesis results in diminished tubular expression of pIgR. If IgA delivery to these sites were not proportionally decreased, the proportion of pIgR occupied by IgA would be expected to increase. This would be reflected in an increase in the S-IgA-to-FSC ratio in the urine, as demonstrated in these experiments.

In summary, we demonstrated that ischemia decreases the level of pIgR expression in the kidney and decreases the concentration of S-IgA and FSC in the urine. Although it is known that immune function is impaired in ARF (22, 26), the interactions of ischemia on the mucosal immunity of the kidney have heretofore been unexplored. Our results suggest that acute ischemic injury compromises the local mucosal immune response of the kidney, in part, by decreasing the expression of the polymeric immunoglobulin receptor and SC levels in the urine. These findings may help to explain the frequent association of UTI with ARF (40).


    ACKNOWLEDGEMENTS

We acknowledge the technical support of Esther Tamayo, the research guidance and manuscript review of Dr. David Good, and the secretarial assistance of Kristi Raynaud and Barbara Knotts.


    FOOTNOTES

This work was supported, in part, by John Sealy Memorial Endowment Fund for Biomedical Research Grant 2585-95, the American Heart Association Beginning Grant-in-Aid 98-BG615, Texas Affiliate (to J. C. Rice), and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-49340 (to R. M. Goldblum) and DK-54471-01 (to J. Megyesi and R. L. Safirstein).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. C. Rice, Univ. of Texas Medical Branch, 4.200 John Sealy Annex, 301 Univ. Boulevard, Galveston, TX 77555-0562 (E-mail: JRICE{at}UTMB.EDU).

Received 22 September 1998; accepted in final form 26 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abramowsky, C. R., and G. L. Swinehart. Secretory immune responses in human kidneys. Am. J. Pathol. 125: 571-577, 1986[Abstract].

2.   Anderson, R. J., J. A. Gordon, J. Kim, L. M. Peterson, and P. A. Gross. Renal concentration defect following nonoliguric acute renal failure in the rat. Kidney Int. 21: 583-591, 1982[Medline].

3.   Apodaca, G., L. A. Katz, and K. E. Mostov. Receptor-mediated trancytosis of IgA in MDCK cells is via apical recycling endosomes. J. Cell Biol. 125: 67-86, 1994[Abstract].

4.   Banting, G., B. Brake, P. Braghetta, J. P. Luzio, and K. K. Stanley. Intracellular targetting signals of polymeric immunoglobulin receptors are highly conserved between species. FEBS Lett. 254: 177-183, 1989[Medline].

5.   Barroso, M., and E. S. Sztul. Basolateral to apical trancytosis in polarized cells is indirect and involves BFA and trimeric G protein-sensitive passage through the apical endosome. J. Cell Biol. 124: 83-100, 1994[Abstract].

6.   Chirgwin, J. M., A. E. Przybylz, R. J. MacDonald, and W. J. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299, 1979[Medline].

7.   Danzig, M. D., and L. R. Freedman. Experimental pyelonephritis XVII. Enhancement of pyelonephritis by water diuresis following direct inoculation of E. coli in the renal medulla of the rat. Yale J. Biol. Med. 45: 1-11, 1972[Medline].

8.   Donohoe, J. F., M. A. Venkatachalam, D. B. Bernard, and N. G. Levinsky. Tubular leakage and obstruction after renal ischemia: structural-functional correlations. Kidney Int. 13: 208-222, 1978[Medline].

9.   Giugliano, L. G., S. T. G. Ribeiro, M. H. Vainstein, and C. J. Ulhoa. Free secretory component and lactoferrin of human milk inhibit the adhesion of enterotoxigenic Escherichia coli. J. Med. Microbiol. 42: 3-9, 1995[Abstract].

10.   Goes, N., J. Urmson, V. Ramassar, and P. F. Halloran. Ischemic acute tubular necrosis induces an extensive local cytokine response. Transplantation 59: 565-572, 1995[Medline].

11.   Grantham, J. J., and M. B. Burg. Effect of vasopressin and cyclic AMP on permeability of isolated collecting tubules. Am. J. Physiol. 211: 255-259, 1966[Medline].

12.   Greenwell, D., J. Petersen, A. Kulvicki, J. Harder, R. Goldblum, and D. E. Neal, Jr. Urinary secretory IgA and free secretory component in pyelonephritis. Am. J. Kidney Dis. 26: 590-594, 1995[Medline].

13.   Guze, L. B., J. Z. Montgomerie, C. S. Potter, and G. M. Kalmanson. Pyelonephritis XVI. Correlates of parasite virulence in acute ascending Escherichia coli pyelonephritis in mice undergoing diuresis. Yale J. Biol. Med. 46: 203-211, 1973[Medline].

14.   Kobayashi, K. Studies on human secretory IgA comparative studies of the IgA-bound secretory piece and the free secretory piece protein. Immunochemistry 8: 785-800, 1971[Medline].

15.   Koiwai, T., H. Oguchi, M. Terashima, Y. Yanagisawa, M. Higuchi, and S. Furuta. Beneficial effect of dibutyryl cyclic AMP (DBcAMP) on ischemic acute renal failure in the rat. Ren. Fail. 14: 461-465, 1992[Medline].

16.   Lindh, E. Increased resistance of immunoglobulin A dimers to proteolytic degradation after binding of secretory component. J. Immunol. 114: 284-286, 1975[Abstract].

17.   Liu, J., and J. Wess. Different single receptor domains determine the distinct G protein coupling profiles of members of the vasopressin receptor family. J. Biol. Chem. 271: 8772-8778, 1996[Abstract/Free Full Text].

18.   Marx, M., M. Weber, D. Schafranek, E. Wandel, K.-H. M. zum Büschenfelde, and H. Köhler. Secretory immunoglobulin A in urinary tract infection, chronic glomerulonephritis, and renal transplantation. Clin. Immunol. Immunopathol. 53: 181-191, 1989[Medline].

19.   Megyesi, J., J. Di Mari, N. Udvarhelyi, P. M. Price, and R. Safirstein. DNA synthesis is dissociated from the immediate-early gene response in the post-ischemic kidney. Kidney Int. 48: 1451-1458, 1995[Medline].

20.   Mostov, K. E., M. Friedlander, and G. Blobel. The receptor for transepithelial transport of IgA and IgM contains multiple immunoglobulin-like domains. Nature 308: 37-43, 1984[Medline].

21.   Nakane, P. K., and A. Kawaoi. Peroxidase-labeled antibody. A new method of conjugation. J. Histochem. Cytochem. 22: 1084-1091, 1974[Medline].

22.   Newberry, W. M., and J. P. Sanford. Defective cellular immunity in renal failure: depression of reactivity of lymphocytes to phytohemagglutinin by renal failure serum. J. Clin. Invest. 50: 1262-1271, 1971[Medline].

23.   Nonoguchi, H., A. Owada, N. Kobayashi, M. Takayama, Y. Terada, J. Koike, K. Ujiie, F. Marumo, T. Sakai, and K. Tomita. Immunohistochemical localization of V2 vasopressin receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts. J. Clin. Invest. 96: 1768-1778, 1995[Medline].

24.   Piskurich, J. F., J. A. France, C. M. Tamer, C. A. Willmer, C. S. Kaetzel, and D. M. Kaetzel. Interferon-gamma induces polymeric immunoglobulin receptor mRNA in human intestinal epithelial cells by a protein synthesis dependent mechanism. Mol. Immunol. 30: 413-421, 1993[Medline].

25.   Pruszczynski, W., H. Caillens, L. Drieu, L. Moulonguet-Doleris, and R. Ardaillou. Renal excretion of antidiuretic hormone in healthy subjects and patients with renal failure. Clin. Sci. 67: 307-312, 1984[Medline].

26.   Rice, J. C., and T. P. Haverty. Vitamin D and immune function in uremia. Semin. Dialysis 3: 237-239, 1990.

27.   Rice, J. C., J. S. Spence, J. Megyesi, R. L. Safirstein, and R. M. Goldblum. Regulation of the polymeric immunoglobulin receptor by water intake and vasopressin in the rat kidney. Am. J. Physiol. 274 (Renal Physiol. 43): F966-F977, 1998[Abstract/Free Full Text].

28.   Riedasch, G., P. Heck, E. Rauterberg, and E. Ritz. Does low urinary sIgA predispose to urinary tract infection? Kidney Int. 23: 759-763, 1983[Medline].

29.   Rubin, R. H. Infectious disease complications of renal transplantation. Kidney Int. 44: 221-236, 1993[Medline].

30.   Safirstein, R., J. Megyesi, S. J. Saggi, P. M. Price, M. Poon, B. J. Rollins, and M. B. Taubman. Expression of cytokine-like genes JE and KC is increased during renal ischemia. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F1095-F1101, 1991[Abstract/Free Full Text].

31.   Safirstein, R., P. M. Price, S. J. Saggi, and R. C. Harris. Changes in gene expression after temporary renal ischemia. Kidney Int. 37: 1515-1521, 1990[Medline].

32.   Sokal, R. R., and F. J. Rohlf. Biometry: The Principles and Practice of Statistics in Biological Research. New York: Freeman, 1995, p. 1-887.

33.   Svanborg-Edén, C., and A.-M. Svennerholm. Secretory immunoglobulin A and G antibodies prevent adhesion of Escherichia coli to human urinary tract epithelial cells. Infect. Immun. 22: 790-827, 1978[Medline].

34.   Sztul, E. S., K. E. Howell, and G. E. Palade. Biogenesis of the polymeric IgA receptor in rat hepatocytes. II. Localization of its intracellular forms by cell fractionation studies. J. Cell Biol. 100: 1248-1254, 1985[Abstract].

35.   Sztul, E., A. Kaplin, L. Saucan, and G. Palade. Protein traffic between distinct plasma membrane domains: isolation and characterization of vesicular carriers involved in transcytosis. Cell 64: 81-89, 1991[Medline].

36.   Takada, M., K. C. Nadeau, G. D. Shaw, K. A. Marquette, and N. L. Tilney. The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney: inhibition by a soluble P-selectin ligand. J. Clin. Invest. 99: 2682-2690, 1997[Abstract/Free Full Text].

37.   Tomita, I., M. Sawa, T. Munakata, K. Tanaka, and S. Kasai. The beneficial effect of dibutyryl cyclic adenosine nonophosphate on warm ischemic injury of the rat liver induced by cardiac arrest. Transplantation 62: 167-173, 1996[Medline].

38.   Van Why, S. K., A. S. Mann, G. Thulin, X. Zhu, M. Kashgarian, and N. J. Siegel. Activation of heat-shock transcription factor by graded reductions in renal ATP, in vivo, in the rat. J. Clin. Invest. 94: 1518-1523, 1994[Medline].

39.   Zager, R. A., T. P. Timmerman, and A. J. Merola. Effects of immediate blood flow enhancement on the post-ischemic kidney: functional, morphologic and biochemical assessments. J. Lab. Clin. Med. 106: 360-368, 1985[Medline].

40.   Zech, P., R. Bouletreau, J. F. Moskovtchenko, M. Beruard, S. Favre-Bulle, N. Blanc-Brunat, and J. Traeger. Infection in acute renal failure. Adv. Nephrol. Necker Hosp. 1: 231-258, 1971[Medline].


Am J Physiol Renal Physiol 276(5):F666-F673
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society