Chronic renal hypoxia after acute ischemic injury:effects of L-arginine on hypox - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:32 |只看该作者 |倒序浏览 |打印

作者：David P.Basile, Deborah L.Donohoe, KellyRoethe,  David L.Mattson作者单位：Department of Physiology, Medical College of Wisconsin,Milwaukee, Wisconsin 53226
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      【摘要】
   Ischemic acute renal failure (ARF)results in the permanent loss of peritubular capillaries andpredisposes the progression of chronic renal failure. The present studywas undertaken to determine whether renal hypoxia, which may representan important mediator in disease progression, is persistentlyexacerbated after recovery from ARF. Rats were subjected toischemia-reperfusion injury and allowed to recover for 5 or 20 wk. Immunohistochemistry of the hypoxia-sensitive marker 2-pimonidizoleat 5 wk revealed an overall increase in incorporation in the outermedullary region after recovery from ARF compared with sham-operatedcontrols. Unilateral nephrectomy, in combination withischemia-reperfusion injury resulted in greater2-pimonidizole staining than that observed in the bilateral injurymodel. In addition, in the unilateral ischemia-nephrectomymodel, proteinuria, interstitial fibrosis, and renal functional lossdeveloped significantly faster than in the bilateral model of ARF whenanimals were allowed to recover for 20 wk. L -Arginine inthe drinking water (~0.5 g/day) increased total renal blood flow~30%, decreased pimonidizole staining, and attenuated manifestationsof chronic renal disease. These data suggest that a reduction in theperitubular capillary density after ARF results in a persistentreduction in renal P O 2 and that hypoxia mayplay an important role in progression of chronic renal disease after ARF. 7 M2 C$ i/ o8 Z7 r0 G
      【关键词】 fibrosis proteinuria blood flow acute renal failure" B% G. K& I# \% T; T- n2 K
   INTRODUCTION$ c9 u( C# g9 e! V" e

ISCHEMIC INJURY TO THE KIDNEY is a leading cause of acute renal failure (ARF). Thisclinical syndrome is associated with high mortality rates but islargely reversible if patients survive the initiating insult( 17 ). In the setting of renal transplantation, prolongedischemic time contributes to delayed graft function, which ischaracterized by the requirement for dialysis in the immediateposttransplant period ( 29 ). Both of these are associated with tubular cell damage and a reduction in glomerular filtration rate(GFR), which are restored through a recovery and repair response ( 5, 20 ). The occurrence of delayed graft functionimmediately after transplant strongly correlates with development ofhypertension and allograft nephropathy ( 18, 29, 31 ). Todate, there is no strong or established correlation with ARF in nativekidneys and the development of long-term complications; however, renal function is not always fully restored and the development of chronic renal insufficiency often ensues after ARF ( 4, 8, 25, 27 ).These clinical observations argue that reversible renal injurypredisposes the kidney to the development of long-term renal complications.

Rodent models of ischemia-reperfusion (I/R) injury have beenutilized to study some of the features of both ARF and delayed graftfunction. In rats, I/R injury results in tubular necrosis and areduction in GFR; the recovery from this type of injury is well studiedand described ( 5, 20, 21, 35, 39 ). Recently, we reportedthat several elements of renal structure and function are irreversiblyaltered after I/R injury ( 3 ). We showed that there was apermanent reduction in peritubular capillary density in rats afterrecovery from bilateral I/R injury. We also observed that recovery frombilateral I/R injury resulted in a permanent alteration inurinary concentrating ability, increased pressor activity inresponse to a low dose of ANG II, and the development of progressiveproteinuria and interstitial fibrosis ( 3 ).. {7 W% n2 E5 J' n( B
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The mechanisms by which chronic renal dysfunction ensues after recoveryfrom ARF is not well explored. The development of hypoxia in thepostischemic kidney has the potential to activate a number ofprofibrotic pathways, including those involving transforming growthfactor- activity, which have the potential to contribute to renalscarring ( 2, 7, 16, 22, 37 ). It is possible that thealterations in peritubular capillary density may have profound effectson renal function and the development of chronic renal insufficiency.We hypothesize that alterations in peritubular capillary densityexacerbate the extent of renal hypoxia and predispose the developmentof chronic renal disease.
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As an initial step toward addressing this hypothesis, the followingstudy was conducted to investigate whether postischemic recovered kidneys are more hypoxic than those of corresponding sham-operated controls. In addition, we sought to determine whether unilateral nephrectomy (UNX), a treatment that is purported to exacerbate renal hypoxia ( 7 ), hastens the development ofchronic renal insufficiency. Finally, we sought to determine whether L -arginine, which is thought to influence renal blood flow(RBF) ( 6, 14 ), affects renal hypoxia and progression of CRF.0 H# [! w* i* k2 ^$ B. k, ^: O0 m
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METHODS

Animal and surgical procedures. Care of the rats before and during the experimental procedures wasconducted in accordance with the policies of the Animal ResourceCenter, Medical College of Wisconsin, and the Guide for the Careand Use of Laboratory Animals (Washington, DC: National AcademyPress, 1996). All protocols had received prior approval by the MedicalCollege of Wisconsin Institutional Animal Care and Use Committee.
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Male Sprague-Dawley rats (~250 g; Harlan, Madison, WI) were housed inpairs in standard shoebox cages with 12:12-h light-dark cycle (lightson, 0600-1800) and access to water and standard laboratory ratchow (0.8% NaCl, Purina Lab Diets 5001) available ad libitum. Animalswere anesthetized with ketamine (100 mg/kg ip) for 10 min followed byadministration of pentobarbital sodium (25-50 mg/kg ip). ARF wasinduced in rats according to surgical procedures previously described( 3 ). Briefly, animals were placed on heated surgicaltables and midline incisions were made to expose the kidneys. Bloodsupply to the kidneys was interrupted by applying microaneurysm clampsfor the indicated times. After occlusion, the clamps were removed andreflow was verified visually. In some studies, animals were subjectedto right UNX in which the right renal pedicle was ligated with a2-0 silk suture just before the excision of the right kidney.Immediately after this, the animal was subjected to left I/R injury orsham treatment.! ]1 ^1 a, f! K

The schema for the basic experimental design is shown in Fig. 1. Short-term studies (i.e., 5 wk) wereused to address the effects of I/R injury on hypoxia and RBF(see below). This time point was chosen because it is generallyassociated with a restoration of renal tubular structure and GFR in abilateral I/R injury model ( 20 ). In addition, this timepoint is reported to be associated with the restoration of total RBFafter I/R injury ( 11, 12 ). In our hands, there is noevidence of proteinuria or substantial fibrosis. In short-term studies,a bilateral I/R protocol was used, which is referred to as ourtwo-kidney I/R injury (2-K) model. In addition, we also utilized amodel in which we induced unilateral I/R injury with simultaneous UNX;this is referred to as our one-kidney I/R injury (1-K) model." B; z2 }* `. {  Y' f4 v! S

Fig. 1. Schema of experimental design to determine the effects of L -arginine on renal function postischemic injury.All animals were subjected to sham surgery or 30, 45, or 52 min ofischemia-reperfusion (I/R) injury. The 24-h serum creatininevalues were utilized to determine the extent of renal injury. Animalswere assigned to treatment groups (tap water alone vs. tap watersupplemented with L -arginine) such that the 24-h serumcreatinine values were similar in both groups. In long-term studies,renal function was monitored for up to 20 wk. Renal histology wasevaluated at 5 and 20 wk postsurgery. In all 20-wk studies, all animalswere subjected to right unilateral nephrectomy (UNX) just before I/Rinjury [i.e., 1-kidney I/R injury (1-K) model]. 2-K, 2-kidney I/Rinjury model.6 v8 n) K0 `0 a) s- U, |, a- Q

Studies were also performed allowing animals to recover for 20 wkpostsurgery. This protocol was designed to assess the effect oftreatments on the development of chronic renal insufficiency. Alllong-term studies were performed with the 1-K model.8 \4 k! D8 s8 V8 }
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Because of the possibility that L -arginine might affect theinitiating ischemic insult, treatment of animals with L -arginine did not commence until 72 h of recovery.Animals were placed into groups that were maintained on tap water(untreated) or tap water supplemented with L -arginine (10 g/l) as described by other investigators ( 30 ). Under theseconditions, L -arginine from dietary sources accounted for~200-250 mg/day whereas that from the drinking water comprised~500 mg/day (i.e., ~50 ml of water drunk/day). The total amino acidintake attributable to the diet was ~4,500-5,000 mg/day.

In these studies, values of serum creatinine at 24 hpostreperfusion were utilized to match groups placed into the untreated or L -arginine groups (Fig. 1 ). L -Arginine wasreplaced daily, and animals were maintained on this treatment for theduration of the study.
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Measurement of renal function. Renal function was measured at 24 h and 1, 2, 4, 8, 12, 16, and 20 wk postsurgery. Blood samples were collected as follows. Rats wereplaced into a closed anesthesia tank containing halothane until theywere relaxed but not unconscious. The animals were placed into arestrainer, and the tip of the tail (1-2 mm) was cut with asterile steel blade. Blood was collected into heparinized tubes andplasma obtained after centrifugation. Urine collection was for 24 h in metabolic cages (Nalgene). Serum and urine creatinine weredetermined by using standard assays (creatinine kit 555A, Sigma). Urinevolume was determined gravimetrically. Creatinine clearance over24 h was calculated as(U creatinine · V )/P creatinine, in which U creatinine is urinary creatinine,P creatinine is plasma creatinine, and V is flow rate.
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Urinary protein excretion was determined with a protein assay kit(Bio-Rad) by using the microassay format for enhanced sensitivity. Urine osmolarity was determined with the microosmette osmometer (Precision Systems), which functions on the basis of freezing-point depression.

Analysis of RBF. For RBF measurements, the rats were anesthetized with Inactin (100 mg/kg ip) and placed on a heated surgical table. The trachea wascannulated to facilitate breathing, and catheters were placed in thefemoral artery to monitor blood pressure and in the femoral vein forintravenous infusion of isotonic NaCl (1.0 ml · 100 g bodywt 1 · h 1 ); in thesestudies, mean arterial pressure ranged between 95 and 115 mmHg andthere was no difference among any treatment groups (data not shown).After a midline abdominal incision, the left renal artery and vein wereseparated and a 2.0- or 2.5-mm-diameter flow probe was placed on therenal artery for measurement of RBF with an electromagnetic flowmeter(model 501, Carolina Instruments, King, NC). After surgery, a 30-minequilibration period was allowed before steady-state blood flow wasmeasured in the rats during a 30-min period. Mean data, averaged overthe 30-min collection period, were collected at 2 Hz by computer usingdata acquisition software (WinDaq acquisition software, DATAQInstruments, Akron, OH).* }' {0 I% x" u+ D9 w

Assessment of renal hypoxia. Renal hypoxia was assessed by using the hypoxia-sensitive marker2-pimonidizole similar to the approach described previously ( 40 ). 2-Pimonidizole and a corresponding mouse monoclonalantibody were obtained from Natural Pharmacia International (Belmont,MA). Ninety minutes before termination, rats were given 2-pimonidizole (60 mg/kg ip). Renal tissue was obtained from rats after measurement ofRBF (described above) and also from other rats that were treated identically. Anesthetized animals were opened with a midline incision, and the kidneys were removed quickly, cut longitudinally, and fixed byimmersion in 10% buffered formalin (Fisher Scientific). The tissue wasthen prepared for routine histological examination.+ p9 g1 G, |$ R" W( O# K4 Z2 I# z6 M' R
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The incorporation of 2-pimonidizole was assessed immunohistochemicallyfrom 5-µm paraffin sections by using standard staining procedures.After deparaffinization and rehydration, tissue was prepared asfollows: 1 ) endogenous peroxidase activity was blocked byincubation in 3% H 2 O 2; 2 )endogenous biotin was blocked with sequential incubations with avidinand biotin (Avidin-Biotin blocking kit, Zymed); and 3 )nonspecific sites were blocked by incubation in 0.01 M PBS containing0.3% Triton X-100, 10% goat serum, and 0.3% BSA. The mousemonoclonal antibody (1:100 dilution) was incubated for 2 h at roomtemperature; detection was performed by using a streptavidin-biotinimmunoperoxidase technique with aminoethylcarbazole as a substrate(Histostain SP, Zymed).
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Analysis of microvascular structure. In some studies, renal capillary density was assessed by using Microfilas described previously ( 3 ). Microfil was visualized underlight microscopy in 20-µm unstained sections with a Nikon EclipseE400 microscope equipped with a Spot Insight color video camera(Diagnostic Instruments, Sterling Heights, MI). Images were capturedonline by using Metamorph imaging software (version 4.0, UniversalImaging). At least five random images of the cortex, outer stripe ofthe outer medulla, and inner stripe of the outer medulla were stored byusing a ×20 objective lens and a field dimension of ~0.26mm 2.' g/ S9 I! {/ k8 X8 k5 q5 d
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Analysis was carried out by using Metamorph software (version 4.0) aspreviously described ( 3 ). Vascular density was calculated on the basis of percent area occupied by Microfil or by the number ofstructures intersecting an arbitrary grid ( 3 ).
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Analysis of renal structure. ECM content was evaluated from Jones silver-stained sections asdescribed previously ( 3 ). Images of sections were acquired as described above. At least five random images of cortex, outer stripeof the outer medulla, and inner stripe of the outer medulla were storedby using a ×20 objective lens and a field dimension of ~0.26mm 2. The images were subsequently analyzed with Metamorphimaging software by a study group member who was blinded to theexperimental groups. In the absence of counterstain, the sharp contrastbetween stained structures and the translucent renal parenchymafacilitated image thresholding by the software program and allowed forcomputer-generated determination of percent area stained for ECM.9 _  Y9 S- O9 o
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Similarly, -actin-containing myofibroblasts were identifiedimmunohistochemically by using an antibody from Zymed. Detection ofthis antibody by using diaminobenzidine as a substrate also generatedimages that were easily thresholded in the absence of a counterstain.Similar image analysis techniques were applied; data were expressed asthe percent surface area stained with -actin.
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Statistical analysis. Unless otherwise indicated, data for renal function, blood flow, andmorphometry were analyzed by one-way ANOVA and a Student-Newman-Keuls post hoc test for significance. These analyses were carried out byusing Sigma-Stat software., R, Z# T4 o! |  E( T$ `
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RESULTS

To determine the potential long-term effects of ARF on thedevelopment of chronic renal hypoxia, we subjected 2-K rats to bilateral I/R injury for 30, 45, or 52 min and allowed them to recoverfor 4-5 wk postinjury. These injuries resulted in 24 h postreperfusion values of serum creatinine of 1.0 ± 0.2, 2.2 ± 0.2, and 2.9 ± 0.5 mg/dl, respectively ( n = 5/group). These injuries were less severe than the one we utilizedpreviously to demonstrate the chronic deleterious effect of I/R injury.Nevertheless, these injuries substantially reduce peritubular capillarydensity ( 3 ). Within 1 wk, serum creatinine values returnedto those observed in sham-operated controls (not shown). At 4-5 wkpostinjury, urinary concentrating ability was the only obviousfunctional alteration observed in these animals; urinary output was11.7 ± 1.4 ml/day in sham, 19.0 ± 1.2 ml/day in 30-minpostischemic animals, 26.9 ± 4.0 ml/min in 45-minpostischemic animals, and 21.45 ± 3.2 ml/day in 52-minpostischemic animals.9 h# G* m& [1 U( G( e
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Figure 2 illustrates the extent of therecovery of tubular morphology in the renal outer medulla after 5 wk ofrecovery from 1-K or 2-K injury. After a 30-min injury, tubularmorphology is essentially restored in the postischemicrecovered animals; tubules appear fully hypertrophied andredifferentiated and express a periodic acid-Schiff-positive brushborder (Fig. 2 B ). In these kidneys, increased numbers oftubulointerstitial cells are occasionally observed (Fig. 2 B,black arrow). Similarly, 5 wk after a 52-min injury, tubular structurein the outer medulla looks essentially normal (Fig. 2 C ).However, there are several focal areas of these postischemickidneys in which structure is clearly abnormal; dilated tubules areobserved occasionally in the outer medulla (Fig. 2, C-E, *) while atrophic-appearing nephrons (Fig. 2 D, white arrow) and dilated tubules are also apparent inthe renal cortex (Fig. 2 D ). Similar structures areoften seen after 45 min of I/R injury (data not shown) but are almostnever observed after the more mild, 30-min I/R injury. When 5 wk ofrecovery is allowed after I/R injury in the 1-K model, normal-appearingproximal tubules are observed, but dilated tubules and interstitialcellularity are commonly present (Fig. 2 E, * and blackarrow, respectively).. d. l& f; P) B4 i0 f! B% q; F
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Fig. 2. Renal histology after recovery from sham surgery or I/Rinjury. Periodic acid-Schiff-stained cross sections of rat kidneys 5 wkafter surgery are depicted. Shown are outer medulla after sham surgery( A ), outer medulla after 30-min 2-K I/R injury( B ), outer medulla ( C ) and cortex ( D )after 52-min 2-K I/R injury, and outer medulla after 45-min 1-K I/Rinjury ( E ). Black arrow, interstitial cellularity; whitearrow, atrophic-appearing glomerulus; *, incompletely regenerateddilated tubules. Bar = 100 µm.

The reactive hydroxylamine intermediate that results from the reductionof 2-pimonidizole binds to cellular thiols in the presence oflow-tissue P O 2 levels ( 1, 34 ). Theparent compound was administered 90 min before death, andimmunohistochemistry was utilized to evaluate incorporation. Stainingwas absent or mild in kidneys of sham-operated rats; when detectable,2-pimonidizole immunoreactivity was observed in the outer medulla (Fig. 3 A ). In contrast, robust2-pimonidizole staining was observed in the outer medulla ofrats after recovery from 30- or 52-min 2-K I/R injury (Fig. 3, C-E ). No signal was detectable in postischemic tissue by using the anti-pimonidizole antibody in tissues from ratsthat did not receive the parent compound (Fig. 3 B ) or when the primary antibody was replaced with nonimmune IgG (data not shown).

Fig. 3. Immunohistochemical detection of pimonidizole uptake after recoveryfrom I/R injury. Shown are low-magnification micrographs of rat renalcortex (c), outer medulla (om), and inner medulla (im) from rats inwhich immunohistochemistry was performed to reveal 2-pimonidizoleincorporation. Representative staining in a kidney is shown from a 2-Ksham-operated rat ( A ), 2-K 30-min postischemic rat( C ), 2-K 52-min postischemic rat ( E ), and1-K 45-min postischemic rat ( F ). G and H : kidneys corresponding to 2-K and 1-K postischemicrats, respectively, treated with L -arginine. B :representation of staining with the 2-pimonidizole antibody of a kidneyfrom a postischemic rat not treated with 2-pimonidizole.Staining patterns are representative of a minimum of 6 samples/group. D : higher magnification of C, demonstratingtubular staining (arrow).
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The degree of 2-pimonidizole staining was substantially andconsistently more intense and had a wider distribution when the samestudies were performed in a 1-K model of 45-min I/R injury (Fig. 3 F ). Additional studies were performed to determine whether oral L -arginine could affect the extent of renal hypoxia.In both 1-K and 2-K models, the degree of 2-pimonidizole staining was consistently less intense and more diffuse in postischemicanimals maintained on L -arginine (Fig. 3, G and H ). Indeed, less staining of 2-pimonidizole was observed inthe most severely damaged regions of L -arginine-treated ratkidneys (Fig. 3 G ). These data suggest that kidneys of ratsat 5 wk postinjury are more hypoxic than kidneys from sham-operatedcontrols and further suggest that the degree of hypoxia can bemanipulated by UNX or administration of oral L -arginine.$ a0 e& D$ d$ l

The possibility that oral L -arginine manifested its effectson renal hypoxia by influencing RBF was addressed by usingelectromagnetic flowmetry. RBF was measured ~5 wk after 2-K I/Rinjury. When expressed on a per kidney basis, total RBF was notdifferent between postischemic and sham-operated controls(sham, 6.5 ± 0.5, vs. I/R, 6.1 ± 0.3 ml/min). However, dueto the mild hypertrophy present in postischemic kidneys in thisexperiment (~10%), there was a modest but significant reduction inRBF when data were expressed on a per gram-tissue basis (Fig. 4 ). Total RBF was significantly enhancedin both sham-operated and postischemic animals that weremaintained on L -arginine compared with the untreatedpostischemic group (Fig. 4 ).
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Fig. 4. Effects of I/R injury and oral L -arginine(ARG) on renal blood flow (RBF). Animals were subjected to the protocoldepicted in Fig. 1 and allowed to recover for 5 wk. Blood flow throughthe left renal artery was measured by using electromagnetic flowmetry.Values are means ± SE. * P P L -arginine-treated animals vs. their respectivetap water-treated controls by ANOVA and Student-Newman-Keuls test., Steady-state blood flow measurements from individualanimals.6 ]8 ^! R9 N3 i2 U  {+ a% \

We performed an additional study in which vessel density was measuredby using Microfil in 2-K animals 5 wk after 45-min I/R injury(Fig. 5, Table 1 ). Untreated postischemicanimals showed a significant decrease in vessel density through thecortex, outer stripe of the outer medulla, and inner stripe of theinner medulla relative to sham-operated controls. L -Arginine had no effect on vessel density at 5 wkpostinjury.1 l$ r" O5 H% T3 y$ J2 D
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Fig. 5. Effect of ischemic injury and L -arginine on blood vessel density. Representative sectionsof microfilled kidneys from a sham-operated animal ( A ),postischemic animal ( B ), and postischemicanimals maintained on L -arginine ( C ). See Table 1 for analysis. CX, cortex; OS, outer stripe of the outer medulla; IS,inner stripe of the inner medulla.9 i* P% Q# r9 ]7 U3 ?

Table 1. Effect of L -arginine on capillary density after 45-min I/Rinjury
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The effects of renal mass reduction and oral L -argininesupplementation on the long-term effects of I/R injury were addressed in the 1-K model (45-min ischemia). Serum creatinine valueswere determined 24 h postinjury, and the animals were stratifiedon the basis of these values; L -arginine treatmentcommenced on day 3 (see the schema in Fig. 1 ). Unilateralischemic injury in combination with contralateral UNX resultedin an immediate decrease in renal function. The increase in serumcreatinine and decrease in creatinine clearance was evident at 24 h postsurgery (Fig. 6, A and B ). Serum creatinine values24 h postischemia were similar in the untreated groupcompared with the group supplemented with L -arginine (Fig. 6 A ). The recovery of GFR-related measurements was apparentin the first week. In our previous report that used a 2-K model of I/Rinjury, serum creatinine and creatinine clearance were unchanged afterthe initial recovery for up to 40 wk postinjury ( 3 ). However, in the 1-K model, there was an increase in serum creatinine vs. corresponding sham-operated controls at week 20 (Fig. 6 A ), whereas creatinine clearance values were significantlydecreased at 16 and 20 wk postsurgery (Fig. 6 B; week20 values: sham, 3,904 ± 411, vs. postischemic,2,901 ± 441 ml/day). Conversely, creatinine clearance values ofpostischemic animals maintained on L -arginine werenot different from those of sham-operated controls (Figs. 6, A and B ).& d; B0 J* r. I0 ]5 a! d

Fig. 6. Renal function postischemia. A : serumcreatinine values are shown at 24 h and 1 and 20 wk postinjury. B : creatinine clearance values were determined on the basisof 24-h urine collections. The number of animals decreased after the5-wk time point during the course of the study. Thus n = 20 and 12 in the postischemic water group before and afterthe 5-wk time point, respectively. In addition, 2 animals in this groupwere euthanized before 20 wk (1 each after weeks 8 and 12 ). Thus n = 10 at the completion of thestudy. Values are means ± SE, expressed relative to the valuesobtained in the sham-operated/water-treated group. * P6 S4 m1 u% c% ^: r

Urine protein excretion increased dramatically after ischemicinjury in the untreated compared with the corresponding sham-operated UNX controls (Fig. 7 ). The development ofproteinuria in postischemic animals maintained on L -arginine was significantly repressed vs. levels observedin postischemic animals maintained on tap water (Fig. 7 ). Inaddition, protein excretion developed more severely in 1-Kpostischemic animals than what was observed previously by usingour 2-K model (Fig. 7 ) ( 3 ).

Fig. 7. Development of proteinuria postischemic injury.Daily urinary protein excretion was determined at the indicated timesfrom 24-h urine collections. Development of proteinuria in animalsafter bilateral renal ischemia ( ) wasoriginally presented in a previous publication ( 3 ). Valuesare means ± SE for all groups in this study. bw, Body wt. *, a, b: P L -arginine, and UNX postischemic group on L -arginine, respectively (by ANOVA and Student-Newman-Keulstest); c, P t -test)." E9 t: q% v- D- T( n( N
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Two of twelve animals in the untreated postischemic groupwere euthanized during the course of the recovery period before the20-wk time point. These animals had elevated serum creatinine valuesand a high urine protein content (data not shown); the loss of thesemost severely affected animals reduced the number of individuals inthis group and resulted in diminution of the apparent reduction inrenal function after the initial recovery period.- C2 Y! K" L8 G: ~: t# @: c

Renal ischemia resulted in a large increase in urine flowthat resolved partially during the first 4 wk of recovery; however, urine flow remained significantly elevated at all time points duringthe recovery period (Fig. 8 A ).The effects of L -arginine on urine flow are shown in Fig. 8, B and C. Animals maintained on L -arginine manifested significantly larger urine outputcompared with either the untreated sham-operated or untreatedpostischemic groups. Measurement of urine osmolaritydemonstrated the reciprocal relationship; i.e., postischemicanimals and animals maintained on L -arginine had moredilute urine compared with their appropriate control groups (data notshown).
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Fig. 8. Urinary output postischemic injury. Urine flowrates are on the basis of 24-h urine collections at the indicated timepoints. A : urine flow rate after UNX and either sham rats orpostischemic rats maintained on tap water. B : urineflow rate of rats after UNX and sham surgery maintained on either tapwater or L -arginine. C : urine flow rate of ratsafter UNX and I/R injury maintained on either water or L -arginine. Values are means ± SE. * P P L -arginine group vs. tap watergroup in sham and postischemic groups, respectively.
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Renal structural data are shown in Figs. 9 and 10.Representative cortical sections stained with silver to highlight ECMare shown for rats 20 wk after surgery (Fig. 9, A-C ). Basement membrane thickening and tubulointerstitial ECM material can be observed in thepostischemic group compared with the sham-operated group (compare Fig. 9 B with 9 A ). There was asignificant increase in cortical and outer medullary ECM contentcompared with the sham-operated group; the increase in ECM staining wasattenuated in the postischemic group maintained on L -arginine (Fig. 9 D ).
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Fig. 9. ECM staining in kidneys of rats after I/R. Sections of rat kidneys(5 µm) were stained with Jones silver stain. Shown are sectionsthrough renal cortex of rats 20 wk after sham surgery ( A ),I/R injury maintained on water ( B ), and I/R injurymaintained on L -arginine ( C ). Note basementmembrane thickening (arrow, C ) in the postischemicgroups and the prominent interstitial cellularity (arrow, B )in the postischemic group maintained on water. D :ECM staining was scored by using computer-aided image analysis. Valuesare means ± SE, normalized in each zone to values obtained in thesham-operated/water groups (indicated by line) and are expressed asrelative percent surface area with positive staining.* P t -test). # P L -arginine group (byStudent's t -test). Bar = 100 µm.

Fig. 10. Imunohistochemical characterization and scoring of -actin inkidneys of rats after I/R. Sections of rat kidneys (5 µm) werestained immunohistochemically for -actin. Shown are sections throughrenal outer medulla of rats 20 wk after sham surgery ( A ),I/R injury maintained on water ( B ), and I/R injurymaintained on L -arginine ( C ). D : -actin staining was scored by using computer-aided image analysis.Values are means ± SE, expressed as percent surface area withpositive staining in the outer medulla. * P t -test).# P L -arginine group (by Student's t -test). Bar = 100 µm.

The presence of tubulointerstitial scarring characterized by thepresence of myofibroblasts was revealed by -actinimmunohistochemistry (Fig. 10 ).Staining of -actin in kidneys of sham-operated control rats waspresent in renal blood vessels and only moderately in the interstitialregion (Fig. 10 A ). In kidneys from untreatedpostischemic animals, there was a substantial and significantincrease in -actin-containing interstitial cells (Fig. 10 B ); the presence of these cells was largely attenuated inkidneys from L -arginine-treated animals (Fig. 10, C and D ). Taken together, the data suggest that L -arginine attenuates or delays the progression of chronicrenal insufficiency after recovery from acute ischemic injury.7 P# K. m& n/ M5 r% B; R
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DISCUSSION
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Both experimental and clinical studies demonstrate that therecovered postischemic kidney is not normal. The observationthat recovery from I/R injury in rats predisposes the development of features indicative of chronic renal failure has now been demonstrated by several different groups of investigators ( 3, 9, 13, 19, 33 ). These observations may have clinical implications withregard to the recovery of patients after ARF or delayed graft function.: v7 J9 U0 J+ X8 G- Z

In an effort to study the potential factors that predispose thedevelopment of chronic renal failure after I/R injury, we have examinedrenal tissue at time points in which renal function, and to a largedegree tubular structure, is restored after injury (i.e., 5 wkpostinjury). Pagtalunan et al. ( 33 ) have performed morphometric analyses of kidneys after recovery from I/R injury. Theydemonstrated the incomplete recovery of a large population of nephronsand that some tubules lost continuity with their parent glomeruli. Wedid not perform the same type of elegant nephron reconstructionutilized by these investigators, but results of gross morphology at 5 wk postinjury are consistent with abnormal tubular structure indicativeof incomplete recovery. However, on gross examination, it is apparentthat there is heterogeneity with regard to the degree of incompleteregeneration at 5 wk postinjury. The degree and number of persistentlydilated tubules appear to be dependent on the severity of the injury aswell as renal mass, such that renal structure is more disrupted in 1-K animals.

In an earlier study, we examined vessel density by using Microfil afterI/R injury to 2-K animals at 4-8 wk. In contrast to theheterogeneity of dilated, atrophic, or incompletely regenerated nephrons whose presence in the recovered kidney is sporadic and focal,the pattern of vessel reduction revealed by Microfil after I/R injuryis homogeneous and was observed after even mild, i.e., 30 min, of 2-KI/R injury ( 3 ). In addition, we have not observed anyevidence of recovery of vessel density.+ q/ T% a* H* b$ @# x$ h6 X

Coinciding with a reduction in peritubular capillaries, we alsodemonstrated a persistent urinary concentrating defect after theapparent recovery from ARF ( 3 ). The persistent urinary concentrating defect is consistent with medullary dysfunction and couldpotentially be attributed to hypoxia ( 7 ). Although reduction in peritubular capillary density is the greatest in the innerstripe of the inner medulla, all zones of the kidney manifested asignificant decrease in vessel density and the potential forexacerbated hypoxia ( 3 ). Our present hypothesis is that exacerbated hypoxia occurs secondary to ischemic injury andpredisposes the kidney to develop chronic renal disease. The purpose ofthe present study was to test one particular component of thishypothesis; i.e., whether postischemic recovered kidneys aremore hypoxic than kidneys of sham-operated control animals. Inaddition, we hoped to determine whether manipulation of tissueP O 2 might influence the development of chronicrenal dysfunction.
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Clearly, other studies are required before the hypothesis as a wholecan gain wide acceptance. For example, it would be useful to devise atreatment that blocks the reduction in vessel density and measureshypoxia; however, we do not yet know the means by which to accomplishthis. Of interest to this issue are studies by Kang et al. ( 23, 24 ) in which VEGF reversed the reduction of peritubularcapillary density after either cyclosporin A treatment or reduced renalmass; VEGF in these settings ameliorated the progression of chronicrenal disease. However, in those studies, hypoxia was not directly addressed.

A number of investigators have suggested that exacerbated medullaryhypoxia might trigger the development of chronic disease ( 2, 7, 16, 22, 37 ). Hypoxia can trigger a number of profibroticpathways; e.g., transforming growth factor-, collagen, andfibronectin ( 2, 7, 16, 22, 37 ). It is also possible thatthese pathways can become activated in the cortical region as a resultof focal hypoxia too small or subtle to be detected with our presentmethodology. Exacerbated medullary hypoxia might affect corticalstructure and function either directly or indirectly. For example, itis thought that scarring in the outer medulla exacerbates localischemia and the spread of the fibrotic area to encompass eventhe most superficial cortex ( 7 ). In addition, it is alsopossible that hypoxia in the renal medulla can reduce Na reabsorptionin the thick ascending limbs of Henle's loop and contribute toafferent vasoconstriction and increased ANG II generation at the maculadensa. This locally produced ANG II could affect glomerular scarring,structure, and development of proteinuria ( 7 ). It isimportant to note that Pagtalunan et al. ( 32 ) demonstratedthe beneficial effects of blocking the renin-angiotensin system on thesecondary development of proteinuria after recovery from I/R injury toa solitary kidney.. Q9 m& ?( h4 Q9 K* R$ N# ~
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Although evidence in favor of hypoxia as a mitigating factor inprogression of renal disease after ARF is compelling, other suggestionshave been raised. The activation of costimulatory pathways in responseto ischemic injury, such as the B7 pathway, have been shown toplay a contributory role in the development of renal disease afterischemic injury ( 9 ). Moreover, the observation that there is incomplete regeneration of the tubule has led to thesuggestion that remnant nephrons can cause the development of scarringwhile hyperfiltration of surviving nephrons can result in progressivenephron damage ( 33 ). Whether hypoxia plays a primary rolein the genesis of secondary renal dysfunction after ARF or whether itmodifies the rate of disease progression set in motion by some othertrigger is presently unclear. In our opinion, costimulatory pathways,hyperfiltration of remnant nephrons, and development of renal hypoxiamay all be considered potentially interrelated phenomena.
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The results of the present study are consistent with exacerbated renalhypoxia, at least within the medullary zone, after recovery froman I/R insult. These results are on the basis of studies showingenhanced 2-pimonidizole uptake. The pimonidizole technique isbecoming utilized with increasing frequency because this compound andcorresponding monoclonal antibodies are now available commercially.Reports have demonstrated that the binding of the reactivehydroxylamine intermediate to cellular thiols occurs atP O 2 below ~10 mmHg ( 1 ). Studiesin tumor models have verified the reliability of this technique byperforming simultaneous measurements with oxygen electrodes( 34 ). To date, at least one other study has utilized thistechnique in kidney in which acute cyclosporin A administration wasshown to exacerbate renal hypoxia ( 40 ).

Although our results are encouraging, the immunohistochemical method isnonquantitative. Furthermore, reductions in P O 2 that 10 mmHg are undetectable. Moreover, the generation of thethiol-binding intermediate is dependent on reduction via NADH orreduced NADP, which may affect the intensity of the signal ( 1 ). Thus the method has several limitations. It is ofinterest that pimonidizole uptake in the kidney was consistently lower in the inner medulla vs. the outer medulla. The reason the inner medulla demonstrates consistently less staining is unknown at this timebut may be related to delivery, transport, or conversion of the parentcompound in the deeper region of the medulla.8 k8 [% k0 _# w

Nevertheless, our ability to consistently observe differences in theintensity and the area of staining in kidneys from postischemic animals vs. those from sham-operated controls suggests that recovery from I/R injury is associated with exacerbated renal hypoxia, which islikely secondary to the reduction in peritubular capillary density.With this in mind, it is important to mention the early models ofoxygen transport proposed by Krogh ( 26 ) that suggest thatreduced capillary density increases the effective distance of oxygentransport to the tissues and promotes tissue hypoxia ( 36 ).Although more detailed models now exist, the basic tenets of Krogh'smodel are widely held. In addition, the model also suggests thatincreased flow through the same number of vessels will decrease hypoxiaby increasing convective delivery of oxygen ( 26, 36 ). Thusit is possible to reduce hypoxia by increasing flow through a reducednumber of vessels.

Two important observations in this study are that 2-pimonidizole uptakeis increased and progressive renal disease occurs more rapidly in the1-K model than in the 2-K model. In this study, we utilized serumcreatinine and creatinine-clearance determinations to measure loss ofrenal function postischemia. Whether changes in creatinineclearance truly reflect alterations in GFR are unclear because of theknown contribution of tubular secretion of creatinine and how this maybe affected by loss of peritubular capillaries. Nevertheless, there isa clear enhancement of proteinuria and renal fibrosis, suggesting thatprogressive renal disease is present. These results are similar tothose reported by Cruzado et al. ( 13 ), who have alsoreported an effect of renal mass on the development of secondarycomplications after I/R injury. Thus it is clear that in a rat model ofI/R injury, renal mass affects the rate of progression of secondaryrenal dysfunction after injury. Because UNX is associated withincreased metabolic demand and exacerbated hypoxia ( 7 ),our observations are also consistent with a role for chronic hypoxia asa mediator of renal disease progression.; ]. Y6 H% G1 O; Z

However, it should be emphasized that other potentially deleteriouscompensatory adaptations of reducing renal mass may predispose thedevelopment of chronic renal disease and that there are clear differences in the number of incompletely regenerated nephrons in the1-K model of I/R injury. It is simplistic but reasonable to suggestthat renal dysfunction in patients after delayed graft function is moreprevalent than in patients after recovery from ARF in their nativekidneys, in part, because of mitigating factors related to compensationfor reduced renal mass.

L -Arginine has been utilized by many investigators toattenuate the progression of chronic renal disease induced by a variety of stimuli including cyclosporine, ureteral obstruction, hyperglycemia, and various forms of hypertension ( 10, 14, 15, 30, 38 ). However, the mechanism of the protection afforded by L -arginine in these models has not been directly addressed.Infusion of L -arginine either systemically or directly intothe renal medullary interstitial space increases RBF in a nitricoxide-dependent fashion ( 14, 28 ). Thus it is possible that L -arginine might attenuate deleterious effects of renalpathological stimuli by affecting total and/or medullary RBF and renalhypoxia. In this study, oral L -arginine increased RBF andpartially attenuated the uptake of 2-pimonidizole. This effect of L -arginine on RBF and hypoxia is likely the result ofvasodilation and appears to be independent of any effect on vasculardensity (Fig. 5 ). In addition, oral L -arginine profoundly enhanced urinary output; this observation is consistent with washout ofthe medullary tonicity that would be expected with increased medullaryblood flow.1 ^* n- h7 T. \( V2 c

The effects of L -arginine on the long-term function ofpostischemic kidneys was consistent with the protectionafforded in other models characterized by interstitial fibrosis( 10, 14, 15, 30, 38 ). L -Arginine-treatedanimals had less scarring and a reduced rate of proteinuria comparedwith animals maintained on tap water. Although other potentialprotective mechanisms might be postulated for L -arginine inthis setting, it is tempting to speculate that renal hypoxia may be acentral mechanism that contributes to the progression of severaldisease models that are characterized by interstitial fibrosis and havebeen shown, experimentally, to be amenable to L -arginine intervention.
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In summary, there are a number of alterations in thepostischemic kidney that might affect long-term outcome andfunction. We suggest that chronic renal hypoxia present after recoveryfrom I/R injury is one important parameter that may influenceprogressive disease. We suggest hypoxia is secondary to the alterationin peritubular capillary density after I/R injury, but evidence for adirect link between these two observations has not yet been made. Thepossibility that enhanced renal hypoxia postrecovery represents animportant element of progressive disease is suggested by studies inwhich exacerbated hypoxia (via UNX) hastens development of long-termcomplications while L -arginine attenuates the development of long-term complications.8 N, q6 q. c: ?3 R! B$ _8 P7 L3 r
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ACKNOWLEDGEMENTS

The authors thank Katherine Fredrich and Carla Meister fortechnical assistance. The authors appreciate discussions with Dr. GavinArteel (University of North Carolina).0 j2 u4 X8 e$ f; V+ R- V% ]" p
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