Isquemia e Metabolismo de Maonia

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doi:10.1152/ajprenal.00437.2006293:F1342-F1354, 2007. First published 8 August 2007;Am J Physiol Renal Physiol

Jin Kim, Mary E. Handlogten, Christopher A. Adin and I. David WeinerKi-Hwan Han, Hye-Young Kim, Byron P. Croker, Sirirat Reungjui, Su-Youn Lee,ammonia metabolism and the collecting ductEffects of ischemia-reperfusion injury on renal

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Effects of ischemia-reperfusion injury on renal ammonia metabolism

and the collecting duct

Ki-Hwan Han,1 Hye-Young Kim,2 Byron P. Croker,3,4 Sirirat Reungjui,2 Su-Youn Lee,1 Jin Kim,5

Mary E. Handlogten,2 Christopher A. Adin,6 and I. David Weiner2,7

1

Department of Anatomy, Ewha Womans University, Seoul, Korea;

2

Division of Nephrology, Hypertension, andTransplantation and 3 Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of

Medicine, Gainesville, Florida; 4Pathology and Laboratory Medical Service, Gainesville Veterans Affairs Medical Center,

Gainesville, Florida; 5Department of Anatomy and Medical Research Center for Cell Death Disease Research Center, The

Catholic University of Korea, Seoul, Korea; 6 Department of Small Animal Clinical Sciences, Veterinary Medical Teaching

Hospital, University of Florida, Gainesville, Florida; and7 Medical Service, Gainesville Veterans Affairs Medical Center,

Gainesville, Florida

Submitted 3 November 2006; accepted in final form 18 July 2007

Han K-H, Kim H-Y, Croker BP, Reungjui S, Lee S-Y, Kim J,Handlogten ME, Adin CA, Weiner ID. Effects of ischemia-reperfusion injury on renal ammonia metabolism and the collectingduct. Am J Physiol Renal Physiol 293: F1342F1354, 2007. First

published August 8, 2007; doi:10.1152/ajprenal.00437.2006.Acuterenal injury induces metabolic acidosis, but its specific effects on the

collecting duct, the primary site for urinary ammonia secretion, theprimary component of net acid excretion, are incompletely under-

stood. We induced ischemia-reperfusion (I/R) acute renal injury in

Sprague-Dawley rats by clamping the renal pedicles bilaterally for 30min followed by reperfusion for 6 h. Control rats underwent sham

surgery without renal pedicle clamping. I/R injury decreased urinaryammonia excretion significantly but did not persistently alter urine

volume, Na, K, or bicarbonate excretion. Histological examinationdemonstrated cellular damage in the outer and inner medullary col-

lecting duct, as well as in the proximal tubule and the thick ascending

limb of the loop of Henle. A subset of collecting duct cells weredamaged and/or detached from the basement membrane; these cells

were present predominantly in the outer medulla and were less

frequent in the inner medulla. Immunohistochemistry identified thatthe damaged/detached cells were A-type intercalated cells, not prin-cipal cells. Both TdT-mediated dUTP nick-end labeling (TUNEL)

staining and transmission electron microscopic examination demon-strated apoptosis but not necrosis. However, immunoreactivity for

caspase-3 was observed in the proximal tubule, but not in collectingduct intercalated cells, suggesting that mechanism(s) of collecting

duct intercalated cell apoptosis differ from those operative in theproximal tubule. We conclude that I/R injury decreases renal ammo-

nia excretion and is associated with intercalated cell-specific detach-ment and apoptosis in the outer and inner medullary collecting duct.

These effects likely contribute to the metabolic acidosis frequentlyobserved in acute renal injury.

acid-base; apoptosis; intercalated cell; acute tubular necrosis

ACUTE RENAL INJURY is a common clinical occurrence that resultsin abnormalities in multiple renal functions. Glomerular filtra-tion is decreased, impairing the ability to excrete metabolicimpurities (reviewed in 32, 60). Sodium, potassium, and watertransport are impaired, leading to volume overload, hyper-kalemia, and disorders of water balance. Mineral metabolism,including calcium and phosphorus and vitamin D metabolism,

is abnormal, resulting in hypocalcemia, hyperphosphatemia,and hyperparathyroidism. In addition, acid-base disturbancesare common and can contribute to impaired cardiovascularstability due to complications of metabolic acidosis on myocar-

dial contractility, arrhythmogenicity, and vascular reactivity (1).The mechanism by which acute renal injury induces meta-

bolic acidosis is not well understood. Metabolic acidosis couldeither reflect urinary bicarbonate losses resulting from inade-quate bicarbonate reabsorption or inadequate net acid excre-tion. The routine clinical observation that urine pH is 6 inacute renal injury makes urinary bicarbonate losses relativelyunlikely. The primary component of net acid excretion isammonia, both under basal conditions and in response tometabolic acidosis (10, 12, 18). If there is inadequate renalammonia metabolism in acute renal injury, then it is unlikely tobe due to changes in glomerular filtration; essentially none ofurinary ammonia excretion derives from glomerular filtration(20). Instead, ammonia metabolism involves intrarenal ammo-nia production combined with specific transport mechanisms inthe proximal tubule, loop of Henle, and the collecting duct (10,31). Thus changes in renal ammonia excretion in acute renalinjury, if present, are likely to involve changes in one or morecomponents of renal ammonia metabolism.

In the present studies, we sought to examine the effects ofacute renal injury on renal ammonia metabolism. We inducedacute renal injury using an ischemia-reperfusion injury model.First, we examined whether ischemia-reperfusion injury altersglomerular filtration rate (GFR), renal ammonia excretion, andother measures of renal tubular ion transport. We then exam-ined whether ischemia-reperfusion injury damages the renalcollecting duct, the site where 70 80% of urinary ammonia is

secreted (20, 52), and, if so, whether ischemia-reperfusioninjury has specific effects on intercalated cells, principal cells,or both. Finally, we examined whether ischemia-reperfusioninjury-induced cell injury in the collecting duct involved apop-tosis or cellular necrosis.

METHODS

Animals. These studies were approved by the University of Floridaor Ewha Womans University Institutional Animal Care and Use

Address for reprint requests and other correspondence: I. D. Weiner, Univ.of Florida College of Medicine, P.O. Box 100224, Gainesville, FL 32610-0224(e-mail: [email protected]).

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

Am J Physiol Renal Physiol 293: F1342F1354, 2007.First published August 8, 2007; doi:10.1152/ajprenal.00437.2006.

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Committee, depending on the site of the animal studies, and wereperformed in accordance with the Institute for Laboratory AnimalResearch Guide for the Care and Use of Laboratory Animals. MaleSprague Dawley rats weighing 250350 g were obtained from HarlanSprague Dawley (Indianapolis, IN) and maintained in a temperature-controlled room with alternating 12:12-h light-dark cycles. Animalswere fed a standard diet and allowed free access to water.

Surgical procedures. Rats were anesthetized using 5% inhalant

isoflurane in 100% oxygen and maintained with 1.52% isoflurane in100% oxygen through a tracheostomy tube. Animals were placed ona heating pad, and body temperature was monitored using a rectalthermometer (Control Company, Friendswood, TX) and maintained at37 1C. The left femoral vein and artery were catheterized usingpolyethylene tubing (PE-50, Intramedic, Clay-Adams, Parsippany,NJ) for fluid administration and blood sampling, respectively. Allanimals received fluid support including p-aminohippuric acid(PAH) -inulin (inulin 2.5 mg/ml; PAH 0.001 g/ml) at a rate of 3 ml/hiv for the duration of surgery. A polyethylene tubing T-port wasconstructed and attached to the venous catheter to administer theinulin solution. Blood pressure was monitored via the arterial catheter(Transonic Systems, Ithaca, NY) and recorded (Iox software, EmkaTechnologies, Falls Church, VA) during the entire duration of theexperiment. Following a ventral midline celiotomy, the urinary blad-

der was catheterized using PE-160 (Intramedic) tubing, and the renalpedicles were isolated. Renal ischemia was induced by clamping bothrenal pedicles for 30 min using vascular microclamps (AccurateSurgical and Scientific Instruments, Westbury, NY). After clampremoval, the abdomen was sutured closed using 4-0 polyglyconate(Maxon, Sherwood, Davis, and Geck, St. Louis, MO) for the durationof reperfusion. After 6 h of reperfusion, the kidneys were harvested,and the rats were euthanized by an overdose of pentobarbital sodium(Euthasol, Diamond Animal Health, Des Moines, IA). A portion ofeach kidney was preserved using 10% neutral buffered formalin forhistological analysis. In some cases, a similar experimental model wasused except intravenous fluids were not administered during thesurgical period. Similar results were obtained.

Sham-operated control rats were treated identically, except that therenal pedicles were not clamped.

Reagents. PAH sodium salt (Sigma, St. Louis, MO) was dissolvedin distilled water to form a 0.22 g/ml solution. Seventy-five milli-grams of fluorescein isothiocyanate-inulin (inulin-FITC, Sigma) wasthen dissolved in 29.85 ml sterile saline and added to 150 l of the0.22 g/ml PAH solution. These solutions were prepared before eachexperiment and were protected from light at all times by covering withaluminum foil.

Serum and urine analysis. Blood was collected at the time ofcatheter placement (baseline, 1 ml), 15 minutes before ischemia (200l), at clamp removal (0 h, 300 l), and 3 h (1 ml) and 6 h (2 ml) afterclamp removal. Serum blood urea nitrogen (BUN) and creatinine weremeasured at baseline, 0, 3, and 6 h, and serum sodium and potassiumwere measured at baseline, 3, and 6 h. Serum inulin concentrationswere measured 15 min before ischemia and 3 and 6 h postischemia.Urine was collected for 30 min before ischemia and collectively for

03 h and 36 h post-clamp removal. Urine volumes were recordedand used to determine urine flow rate. Urine sodium and potassiumwere measured from the baseline (preischemia), 0- to 3-h, and 3- to6-h samples. Urine and serum samples were stored at 20C until thecompletion of the experiment. Samples for inulin determination weretransferred to 80C until analyses were performed.

Sodium, potassium, creatinine, urea nitrogen, and HCO3

in urinewere determined using the VetACE clinical chemistry system (AlfaWassermann, West Caldwell, NJ). Urinary ammonia concentrationwas measured using a commercially available assay (AmmoniaReagent Set, Pointe Scientific, Canton, MI).

Antibodies. Affinity-purified antibodies to Rh B-glycoprotein(Rhbg) and Rh C-glyprotein (Rhcg) generated in this laboratory havebeen characterized previously (24, 55, 56, 65, 69). Antibodies to the T

able1.

Effectofischemia-reperfusioninjuryonurinecomposition

GFR,ml/min

UrineVolume,l/min

UrineNa,mol/min

UrineK,mol/min

Urine

HCO3,mol/min

Pre

0

3h

0

6h

Pre

0

3h

3

6h

Pre

0

3h

3

6h

P

re

0

3h

3

6h

Pre

0

3h

3

6h

Ischemia-

reperfusion

1.9

90.5

1

0.4

60.1

7

0.6

40.1

6

13.63.2

66.57.8

84.811.

3

1.5

20.7

1

7.3

91.4

3*

7.8

71.6

6

4.2

10.6

9

1.7

70.3

2*

2.6

50.4

2

0.0

10.0

1

0.1

90.0

6*

0.2

30.0

9

Sham

operated

1.2

10.1

4

1.7

00.2

0

1.8

70.1

4

11.41.8

44.711.8

69.411.

9

0.5

60.3

3

3.1

30.8

9

5.4

40.8

7

4.7

70.6

5

4.0

20.3

8

2.2

00.1

2

0.0

00.0

1

0.0

10.0

1

0.0

40.0

1

Valuesaremeans

SE.

GFR,glomeru

larfiltrationrate;Pre,

30mintimeperiodbeforeischemia-reperfusion.

*P

0.0

5,

P0

.001vs.sham

operated.

F1343EFFECTS OF I/R INJURY ON RENAL AMMONIA METABOLISM AND CD

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B1/B2 subunit of H-ATPase, raised against amino acids 334513 ofthe human V-ATPase B1 subunit, were obtained from Santa CruzBiotechnology (SC-20943). Antibodies to AE1 were obtained fromAlpha Diagnostic (AE11-A), and antibodies to aquaporin-2 (AQP-2)were obtained from Chemicon (AB3274). Antibodies and blockingpeptides to cleaved caspase-3 were obtained from Cell SignalingTechnology (no. 9661 and no. 1050, respectively, Cell Signaling Tech-nology, Beverly, MA).

General histological examination. Routine hematoxylin and eosinstaining of tissue sections and periodic acid-Schiff staining wereperformed in the clinical laboratory of the Gainesville VeteransAffairs Medical Center. Tissue sections were examined by an expertpathologist (B. P. Croker), who was blinded to the treatment conditionfrom which the tissue was obtained. The proximal convoluted tubule,

proximal straight tubule in the outer stripe of the outer medulla,medullary thick ascending limbs of the loop of Henle in the innerstripe of the outer medulla, and medullary collecting ducts wereidentified using standard criteria and were evaluated. In each segment,tubules were evaluated for evidence of normal histology, cell swellingand vacuolization, brush-border loss, nuclear condensation, and evi-dence of karyolysis, karyorrhexis, and cell sloughing. A semiquanti-tative scoring system was used: tubules were graded 0 if nonewere involved, 1 if110%, 2 if1125%, 3 if26 50%, 4 if5175%, and 5 if76100% were involved.

Immunohistochemistry. Immunolocalization was accomplished us-ing immunoperoxidase procedures and a commercially available kit(Dako, DakoCytomation, Carpinteria, CA) as we have reported indetail previously (24, 55, 65, 69). Sections were photographed usinga Nikon E600 microscope equipped with DIC optics, a DXM1200Fdigital camera, and ACT-1 software (Nikon). Control experimentsomitting the primary antibodies revealed no nonspecific immunore-activity.

Double-labeling procedure. Colocalization of two different anti-gens was performed using sequential immunoperoxidase proceduresand a commercially available kit (Dako, DakoCytomation, Denmark).The tissue sections were dewaxed, had endogenous peroxidasequenched using DAKO peroxidase solution for 30 min, and then werewashed, incubated with DAKO serum-free blocking solution, washed,and incubated with Rhbg or AE1 antibody overnight at 4C in ahumidified chamber. The sections were then washed, incubated withbiotinylated anti-mouse and anti-rabbit secondary antibody (DAKOLSAB2 kit) for 30 min, washed, incubated with streptavidin for 30min, washed, exposed to 3,3-diaminobenzidine (DAB) for 5 min, andthen washed. The above procedure was then repeated with the sub-stitution of a second primary antibody (H-ATPase, 1:200; AQP-2,1:200) and the substitution of Vector SG for DAB. Sections werephotographed using a Nikon E600 microscope equipped with DICoptics, a DXM1200F digital camera, and ACT-1 software (Nikon).Control experiments omitted the primary antibodies and revealed nononspecific immunoreactivity.

TdT-mediated dUTP nick end-labeling staining. Cells undergoingapoptosis were identified by use of the FragEL DNA FragmentationDetection Kit from Calbiochem (cat. no. QIA33). Kidney sectionswere deparaffinized with xylene and ethanol, and after rinsing inTris-buffered saline (TBS), the sections were treated with 20 g/mlproteinase K in Tris buffer, pH 8.0, for 20 min at room temperature,followed by rinsing with TBS. Endogenous peroxidase activity wasinactivated by incubation with 3% H2O2 for 5 min at room temper-ature. The sections were then incubated with equilibration buffer for10 min at room temperature, followed by incubation with workingTdT labeling reaction mixture at 37C for 1h. The reaction was

terminated by incubation in stop buffer for 10 min at 37C. Afterbeing treated with blocking buffer, the sections were incubated withthe peroxidase-substrate solution, a mixture of 0.05% DAB and 0.01%H2O2, for 15 min at room temperature. After being rinsed with PBS,double immunolabeling was performed using the H-ATPase anti-body and Vector SG as described above. Control experiments omittedthe primary antibodies and revealed no nonspecific immunoreactivity.

Quantification of TdT-mediated dUTP nick-end labeling staining.

High-resolution, 36 megapixel digital micrographs of tissue samplesdouble-labeled for H-ATPase and TdT-mediated dUTP nick-end

Fig. 1. Effect of ischemia-reperfusion injury on urinary ammonia excretion.The rate of urinary ammonia excretion was calculated as urinary ammoniaconcentration multiplied by urine production rate. Ischemia-reperfusion injuryresults in significant inhibition of ammonia excretion rate compared withsham-operated control rats at both the 0- to 3-h and the 3 to 6-h time points(*P 0.05).

Table 2. Effect of ischemia-reperfusion injury on renal cell histology

Segment/Condition Normal Cell Swelling, Vacuolization BB Loss Nuclear Condensation Karyolysis, Karyorrhexis, Cell Sloughing

Proximal convoluted tubuleIschemia-reperfusion 0.670.42 1.670.42 2.670.33 1.500.50 0.500.22Control 5.000.00* 0.000.00 0.000.00* 0.000.00 0.000.00*

Proximal straight tubuleIschemia-reperfusion 0.000.00 2.500.34 3.000.00 3.000.00 2.170.31Control 5.000.00* 0.000.00* 0.000.00* 0.000.00* 0.000.00*

MTALIschemia-reperfusion 1.170.54 1.330.42 N/A 1.670.33 1.330.21Control 5.000.00* 0.000.00 N/A 0.000.00* 0.000.00*

Collecting ductIschemia-reperfusion 0.000.00 2.330.33 N/A 2.670.33 2.670.33Control 5.000.00* 0.000.00* N/A 0.000.00* 0.000.00*

Values are means SE. Scoring system: 0 if none were involved, 1 if approximately 110%, 2 if approximately 1125%, 3 if approximately2650%, 4 if approximately 5175%, and 5 if approximately 76100% were involved. MTAL, medullary thick ascending limb of the loop of Henle;BB loss, brush-border loss; N/A, not applicable. *P 0.001, P 0.01, P 0.05 vs. ischemia-reperfusion.

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labeling (TUNEL) from control and ischemia-reperfusion kidneyswere obtained in the cortex, outer stripe of the outer medulla, innerstripe of the outer medulla, and the initial inner medulla by aninvestigator blinded to the treatment status of the kidneys using aNikon E600 microscope equipped with DIC optics, a DXM1200Fdigital camera, and ACT-1 software (Nikon). The number of TUNEL-positive and TUNEL-negative intercalated cells were quantified in theleast three micrographs per kidney. The number of TUNEL-positive

and TUNEL-negative intercalated cells in each animal was averaged,and the average was used for statistical analysis.

Transmission electron microscopy. Glutaraldehyde-fixed kidneyswere removed, and tissue from various regions of the renal medullawas cut into 1-mm3-sized blocks and postfixed with 1% osmiumtetroxide in phosphate buffer for 2 h, dehydrated in a graded series ofethanols, and embedded in poly/Bed-812 resin (Polysciences, War-rington, CA). One-micrometer semithin sections were stained withtoluidine blue and examined by light microscopy. Ultrathin sectionswere stained with uranyl acetate and lead citrate and photographedwith a transmission electron microscope (HITACHI H-7650).

Caspase-3 and H-ATPase double labeling. To detect caspase-3,deparaffinized tissue sections were treated in 10 mM sodium citratebuffer (pH 6.0) and placed in a microwave oven for antigen retrieval.After endogenous peroxidase quenching and serum blocking, tissue

sections were incubated with antibodies to cleaved caspase-3(Asp175) at 4C overnight. The sections were then washed andincubated with peroxidase-conjugated donkey anti-rabbit IgG Fabfragment (Jackson ImmunoResearch laboratories, West Grove, PA)for 1 h. Sections were then incubated with peroxidase substratesolution, a mixture of 0.05% DAB and 0.01% H2O2, for 5 min at roomtemperature. The above procedure was then repeated with the substi-tution of a second primary antibody (H-ATPase) and the substitutionof Vector SG for DAB. A blocking peptide to activated caspase-3 wasused to confirm specificity of activated caspase-3 immunoreactivity.

Statistical analysis. Data were analyzed using unpaired Studentst-test, and P 0.05 was taken as evidence of statistical significance.When comparing the number of TUNEL-positive and -negative in-tercalated cells in control and ischemia-reperfusion kidneys, we usedchi-squared analysis to determine statistical significance; n refers to

the number of animals studied. In all cases, tissue from six control andsix ischemia-reperfusion injury animals was studied, unless otherwisenoted.

RESULTS

Physiological parameters. Acute ischemia-reperfusion injury significantly decreased GFR compared with sham-oper-ated control animals (Table 1). There were no differences inurine production rates between ischemia-reperfusion andsham-operated control rats at 03 h and 36 h; urinary Na

rates were increased and K rates were decreased significantlyat the 0- to 3-h time point but not at the 3- to 6-h time pointwhen kidney samples were obtained. Since GFR was de-

creased, whereas Na

and K

excretion rates at 6 h wereunchanged, fractional excretion of both Na and K wasincreased (data not shown). There was a tendency for urinaryHCO3

excretion rates to be increased, but the difference wasnot statistically significant at the 3- to 6-h time point. Table 1summarizes these results.

In contrast to the lack of sustained effects of ischemia-reperfusion on Na and K excretion, ischemia-reperfusioninduced significant and persistent decreases in urinary ammo-nia excretion (Fig. 1). Ischemia-reperfusion injury appears tospecifically alter ammonia metabolism through mechanismsindependent of changes in water, sodium, and potassium ex-cretion.

Effect of ischemia-reperfusion injury on specific nephronsegments. Multiple renal segments contribute to ammoniametabolism (10, 31). To begin identifying which specificnephron segments were involved in altered ammonia metabo-lism, we examined the effect of ischemia-reperfusion injury onthe cortical proximal convoluted tubule, proximal straighttubule in the outer stripe of the outer medulla, the medullary

thick ascending limb of the loop of Henle in the inner stripeof the outer medulla, and the collecting duct in the outer medulla.Table 2 summarizes these results. Ischemia-reperfusion injuryresulted in significant damage, identified by cell swelling andvacuolization, brush-border loss, nuclear condensation, and thedevelopment of karyolysis, karyorrhexis, and cell sloughing ineach of the nephron segments identified compared with con-

Fig. 2. Effect of ischemia-reperfusion injury on collecting duct morphology.A: control kidney. Representative example from the outer medulla of a controlkidney. Tissue morphology is normal. A collecting duct is shown in the middleof the panel and is denoted with the asterisk (*). Sections were stained withhematoxylin and eosin. B: ischemia-reperfusion kidney. Hematoxylin andeosin-stained section demonstrating representative area of outer medulla froma kidney exposed to ischemia-reperfusion injury. The asterisk (*) labels thelumen of an outer medullary collecting duct (OMCD). Collecting duct cells inthe process of detaching from the basement membrane (arrows) and intralu-minal cells (arrowhead) are evident.




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trol, sham-operated rat kidneys. In particular, there was nosignificant damage, as identified by any of these criteria, in anyof these segments in control rat kidneys. Thus, in addition topreviously identified effects of ischemia-reperfusion injury inthe proximal tubule and the thick ascending limb of the loop ofHenle, there is also significant damage to the collecting duct,particularly in the outer medulla.

Further examination of kidneys from ischemia-reperfusionkidneys showed that a subset of cells was detached from thebasement membrane and appeared to be in the process of beingextruded into the tubule lumen (Fig. 2). No detached cells werevisible in sham-operated control kidneys. Detached cells, whenexamined using hematoxylin and eosin staining, had increased

cytoplasmic density, suggesting they were intercalated cells.These changes were most dramatic in the outer medulla. In theinner medulla fewer cells were in the process of being extrudedinto the tubule lumen. In the cortex, there was no evidence ofcellular damage in the collecting duct, connecting segment, ordistal convoluted tubule.

Identification of damaged collecting duct cell-type. To iden-

tify which of the different cell types present in the collectingduct were damaged in ischemia-reperfusion injury, we per-formed immunohistochemical examination using a variety ofintercalated cell- and principal cell-specific markers. Antibod-ies directed against the vacuolar H-ATPase labeled cells inthe outer medullary collecting duct which appeared to be in the

Fig. 3. H-ATPase and AE1 immunoreac-tivity in the OMCD. A: control kidney. In-tercalated cells in the OMCD (arrow) exhibitapical H-ATPase (blue) immunoreactivityand basolateral AE1 (brown) immunoreac-tivity. B: ischemia-reperfusion injury kidney.Several intercalated cells in the OMCD inthe ischemia-reperfusion injury kidney areeither in the process of detaching from thebasement membrane (black arrowhead) orhave completely detached and are present inthe tubule lumen (white arrowhead). C: noprimary control: control kidney. No nonspe-cific immunoreactivity was present in controlkidney sections in which the primary anti-body was omitted. The asterisk (*) identi-fies the lumen of an OMCD. D: no primarycontrol: ischemia-reperfusion injury kid-ney. No nonspecific immunoreactivity waspresent in ischemia-reperfusion injury kid-ney sections in which the primary antibodywas omitted. Arrows indicate detached orintraluminal OMCD cells and verify the ab-sence of nonspecific immunoreactivity indamaged cells.




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process of being extruded into the tubule lumen. Colocalizationof H-ATPase with AE1 demonstrated that these cells ex-pressed both H-ATPase and AE1, identifying these as A-typeintercalated cells (Fig. 3, A and B). In control experiments

performed without primary antibody, there was no nonspecificimmunoreactivity (Fig. 3, C and D).

Because ischemia-reperfusion injury decreased ammoniaexcretion, we examined expression of the ammonia transporter

Fig. 4. Rh B-glycoprotein (Rhbg) expres-sion in ischemia-reperfusion injury and con-

trol kidneys. A: low-power micrograph ofouter medulla from control kidney. Basolat-eral Rhbg expression in intercalated cells inthe OMCD is evident. No immunoreactivityis present in other tubular or epithelial struc-tures. B: high-power micrograph from con-trol kidney. Basolateral Rhbg immunoreac-tivity in intercalated cells (arrow) in thisOMCD is evident. No detached or intralumi-nal Rhbg-positive cells are present. C and

D: low-power and high-power micrographs,respectively, of the outer medulla in ische-mia-reperfusion injury kidney. MultipleRhbg-positive cells with either loss of baso-lateral Rhbg polarization (white arrow) ordetached from the basement membrane andpresent in the luminal space (white arrow-

head) are present.

Fig. 5. Rh C-glycoprotein (Rhcg) immuno-reactivity in the OMCD of control and is-chemia-reperfusion kidney. A and B: low- andhigh-power micrographs, respectively, ofRhcg immunoreactivity in the outer medullaof control kidney. Both apical and basolat-eral Rhcg immunoreactivity is present inboth intercalated cells and principal cells inthe OMCD. Intercalated cells (B, black ar-rows) exhibit more intense apical and baso-lateral immunoreactivity, whereas principalcells (B, black arrowhead) exhibit weakerRhcg immunoreactivity. No detached or in-

traluminal Rhcg-positive cells are present.C and D: low- and high-power micrographs,respectively, of Rhcg immunoreactivity inthe outer medulla of ischemia-reperfusioninjury kidney. Cells with intense Rhcg im-munoreactivity that are either in the processdetaching from the tubular epithelium (whitearrow) or have detached and are present inthe tubule lumen (white arrowhead) areshown. Principal cells (black arrowhead) areneither detached nor present in the tubulelumen.




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family members, Rhbg and Rhcg (68, 70). As shown inFigs. 4 and 5, collecting duct cells in the process of beingextruded expressed intense Rhbg and Rhcg immunoreactivity,consistent with their identification as A-type intercalated cells.

The majority of cells in the collecting duct are principalcells. However, principal cells, identified by AQP-2 immuno-reactivity, appeared histologically normal, were not detachedfrom the basement membrane, and were not identified in the

tubule lumen (Fig. 6). Thus ischemia-reperfusion injury ap-pears to damage collecting duct intercalated cells but notprincipal cells.

To determine whether all of the detached cells present in thetubule lumen were collecting duct cells, we used double-labeling with Rhbg, which labels collecting duct intercalatedcells, and AQP-2, which labels collecting duct principal cells.Both Rhbg-positive, AQP-2-negative collecting duct interca-

Fig. 6. Aquaporin-2 (AQP-2) immunolabel

in control and ischemia-reperfusion kidneys.A: control kidney. AQP-2 immunoreactivityin principal cells (black arrow) in the OMCDof control kidney is shown. Cells appearhistologically normal and are neither de-tached nor present in the tubule lumen.

B: ischemia-reperfusion injury kidney. AQP-2immunoreactivity is apparently normal inOMCD principal cells (black arrow). Detach-ing (black arrowhead) and intraluminal (whitearrowhead) cells do not express AQP-2 immu-noreactivity.

Fig. 7. Double labeling for Rhbg andAQP-2. A and B: control kidney. All OMCDcells exhibit either basolateral Rhbg immu-noreactivity (black arrowhead, brown immu-noreactivity, intercalated cell marker) or api-cal AQP-2 immunoreactivity (black arrow,blue reaction product, principal cell marker).No detached or intraluminal cells are presentin the OMCD in the normal kidney. C and

D: ischemia-reperfusion kidney. Multiple de-tached Rhcg-positive intraluminal cells (whitearrow) are evident. Occasional intraluminalAQP-2- and Rhbg-negative, non-collectingduct cells (white arrowhead) are also seen.




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lated cells and Rhbg-negative, AQP-2-negative, non-collecting

duct cells were present in the tubule lumen (Fig. 7). No

Rhbg-negative, AQP-2-positive collecting duct principal cells

were identified in the tubule lumen. Thus the intraluminal cells

present in the collecting duct in ischemia-reperfusion injury is

a mixed population of cells that comprises both collecting duct

intercalated cells and non-collecting duct cells. The latter

probably includes proximal tubule and thick ascending limb ofthe loop of Henle cells. Accurate counting of the number of

non-collecting duct cells could not be performed because they

frequently were either fragmented or formed large clumps,

preventing accurate enumeration.

TUNEL staining. Ischemia-reperfusion injury can cause

cellular loss through either cellular necrosis or through

induction of apoptosis (57). Histological aspects of apopto-

sis were examined using standard TUNEL histology com-

bined with double-labeling with H-ATPase to identify

whether ischemia-reperfusion injury induces apoptosis of

H-ATPase-positive collecting duct intercalated cells.

Many TUNEL-positive cells were observed in the ischemic

kidneys both in the proximal tubule and the thick ascendinglimb of the loop of Henle (not shown), as well as in A-type

intercalated cells in the outer medullary collecting duct and the

inner medullar collecting duct (Fig. 8). TUNEL-positive, H-

ATPase-negative collecting duct cells, i.e., apoptotic principal

cells, were, in general, not observed. Table 3 quantifies the

number of TUNEL-positive and TUNEL-negative intercalated

cells in ischemia-reperfusion injury and sham-operated control

kidneys. At least a component of the damage to medullary

collecting duct intercalated cells in response to ischemia-

reperfusion injury is due to apoptosis.

Transmission electron micrographic examination of ischemia-reperfusion injury kidneys. To confirm the presence of apop-tosis, we examined kidneys using transmission electron mi-croscopy. In ischemia-reperfusion injury kidneys, cells in theouter medulla that were either partially or completely detachedfrom the basement membrane had intact cellular and intracel-lular membranes and were not necrotic (Fig. 9). We also

observed irregularly shaped nucleus and nuclear condensation,consistent with cells undergoing apoptosis.

Caspase-3 immunoreactivity. Caspase-3 is a key enzymeinvolved in the apoptotic pathway in some, but not all, cells(47). Activated caspase-3 immunoreactivity was observed inproximal tubule, but not in the collecting duct, of theischemia-reperfusion kidney. To confirm expression in theproximal tubule, but not the collecting duct, we used colo-calization with H-ATPase to identify collecting duct inter-calated cells (Fig. 10). No caspase-3 immunoreactivity wasobserved in the control kidney. To confirm specificity ofcaspase-3 immunoreactivity, we used HL60 promyelocyticleukemia cells in which apoptosis was induced by pretreatmentwith 0.5 g/ml actinomycin D (Fig. 10E) (14). Preincubatingthe antibody to cleaved caspase-3 with a blocking peptideblocked immunoreactivity (Fig. 10F). Thus the cellular mech-anisms underlying apoptosis appear to differ in the proximaltubule and the collecting duct.

DISCUSSION

The present studies are the first to examine the effects ofacute renal injury induced by ischemia-reperfusion injury onrenal ammonia excretion. In this model of acute renal injury,there was decreased urinary ammonia excretion associatedwith specific loss of A-type intercalated cells, predominantly

Fig. 8. TUNEL and H-ATPase double la-beling. A: OMCD of control kidney. Interca-lated cells, identified by apical H-ATPaseimmunoreactivity (blue reaction product,black arrowheads) are histologically normaland do not show TUNEL reactivity (brownreaction product). B: OMCD of ischemia-reperfusion injury kidney. Many TUNEL-positive cells (black arrow, brown reactionproduct) are present. The presence of H-ATPase immunoreactivity (blue reactionproduct) identifies these cells as intercalatedcells. A TUNEL-positive interstitial cell(white arrowhead) is also evident. C: controlkidney: inner medulla. Histologically normal

intercalated cells (black arrowhead), identi-fied by apical H-ATPase immunoreactivity(blue reaction product), and inner medullarycollecting duct (IMCD) cells (absence ofH-ATPase immunoreactivity) are present.TUNEL-reactivity is observed in neitherIMCD intercalated cells nor IMCD cells.

D: inner medulla of ischemia-reperfusioninjury kidney. Multiple TUNEL-positive(brown reaction product) intercalated cells(black arrows) with apical H-ATPase im-munoreactivity (blue reaction product) areevident. IMCD cells, identified by the ab-sence of apical H-ATPase immunoreactiv-ity, are TUNEL negative.




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from the outer medullary collecting duct, but also involvingloss in the inner medullary collecting duct. The cellular dam-age was highly specific for intercalated cells and did notinvolve principal cells. Intercalated cells that were damagedappeared to be undergoing apoptosis and were in the process ofbeing detached from the basement membrane and extruded intothe lumen where they could be shed into the urine. Theseeffects of ischemia-reperfusion injury on the collecting ductmay contribute to the metabolic acidosis that frequently ac-companies acute renal injury.

Acute renal injury is a commonly observed complication inhospitalized patients that results frequently in metabolic aci-dosis (27, 28, 43). The acidosis can be severe and can result insubstantial complications, including compromised cardiac con-tractility, increased susceptibility to arrhythmias, and compro-mised cellular functions (1, 58, 60). Despite the well-knownassociation of metabolic acidosis with acute renal injury, themechanism by which the acidosis occurs has not been exten-

sively studied. One proposed mechanism is an acute increase inmetabolic acid production (32). However, as the present studyshows, other mechanisms also contribute. Urinary ammoniaexcretion is decreased and likely contributes to the metabolicacidosis occurs in acute renal injury. Although ischemia-reperfusion injury frequently damages the proximal tubule andthe loop of Henle, decreased bicarbonate reabsorption is un-likely to be the only cause of acidosis, as there were nosignificant changes in urinary bicarbonate excretion.

A second major finding of these studies is that ischemia-reperfusion injury damages the collecting duct. These effectsare most prominent in the outer medulla, which is consistentwith previous studies showing that the predominant effect ofischemia-reperfusion injury occurs in outer medulla. Our ob-

servation of collecting duct damage is consistent with previousobservations in humans where acute renal injury was associ-ated with both cell loss from the collecting duct and thepresence of collecting duct cells in urine (46, 54). Furthermore,in humans with acute renal injury, the number of collectingduct cells in the urine exceeds the number of loop of Henlecells and is 50% of the number of proximal tubule cells (54).The present study is both consistent with these previous ob-servations and extends them by showing that the damagedcollecting duct cells, at least in the acute renal injury modelused in the present study, are intercalated cells. Moreover, thepresent study shows that the intratubular cellular casts ob-served in the collecting duct in acute renal injury (45, 59) can

consist of both collecting duct intercalated cells and cells frommore proximal epithelial sites, most likely the proximal tubuleand the loop of Henle. Why other studies have not routinelyidentified the presence of collecting duct damage in ischemia-reperfusion injury is not clear.

The collecting duct intercalated cell damage present inresponse to ischemia-reperfusion injury likely mediates, at

least in large part, the decreased rate ammonia excretionobserved in these studies. A majority, 7080%, of theammonia present in the urine is secreted by the collecting duct(20, 52). Ammonia secretion involves parallel proton andammonia transport, and intercalated cells are the primary siteof proton secretion (16, 19). Collecting duct ammonia transportappears to involve both diffusive and transporter-mediatedcomponents (22, 23); the transporter-mediated componentsappear to be mediated by Rhbg and Rhcg (22, 23), which areexpressed in highest concentrations in intercalated cells (11,21, 49, 55, 56, 65). The damage to collecting duct intercalatedcells likely impairs secretion of both H and NH3, therebyresulting in decreased net acid excretion without substantialchanges in urinary pH, as is observed clinically.

Although it is possible that altered ammonia metabolism inother renal epithelial cells contributes to the decreased ammo-nia excretion, this is unlikely, at least in the time course thesestudies examined. Ammonia is produced in the proximal tu-bule, where it is preferentially secreted into the luminal fluid,

Fig. 9. Transmission electron microscopy in the outer medulla. A: low-powermicrograph of OMCD. A detaching intercalated cell (IC; black arrow) isshown. Plasma membrane and cytoplasmic structures are intact and there is noevidence of necrosis. An adjacent principal cell (PC) is histologically normal.

B: a high-power micrograph of an OMCD intercalated cell with an eccentricnucleus and nuclear condensation, a characteristic finding of apoptosis, isshown (black arrow).

Table 3. Proportion of intercalated cells withTUNEL-positive staining

Condition

Sham-Operated Control

(n 4), %Ischemia-Reperfusion Injury

(n 4), %

Cortex 0.00.0 0.30.03OSOM 0.00.0 3.50.5*ISOM 0.20.2 10.80.7*Inner medulla, initial 0.00.0 20.72.2*

Values are mean SE and represent mean percentage of intercalated cells(identified by H-ATPase immunoreactivity) with TdT-mediated dUTP nick-end labeling (TUNEL)-positive immunoreactivity; n represents no. of kidneysexamined. At least 60 intercalated cells in each region in each kidney werequantified. OSOM, outer stripe of the outer medulla; ISOM, inner stripe of theouter medulla. *P 0.001 vs. sham-operated control.




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and is then reabsorbed by the thick ascending limb of the loopof Henle (20, 31). We are unaware of studies reportingproximal tubule ammoniagenesis in response to ischemia-reperfusion injury, but in another model of acute renal injury,glycerol-induced acute renal injury, renal ammoniagenesis isunchanged (48). Ischemia-reperfusion injury decreasesNa/H exchanger-3 (NHE-3) and Na-K-2Cl cotrans-

porter (NKCC-2) expression, the transporters involved in prox-imal tubule and thick ascending limb of the loop of Henleammonia transport (17, 34, 66, 67), suggesting ammonia trans-port may be decreased in these segments. However, this cannotbe the entire cause of the acute decreased renal ammoniaexcretion. If it were, then intrarenal ammonia content would bedecreased, whereas direct measurements have shown increasedintrarenal ammonia in ischemia-reperfusion injury (15, 35, 71).This increase in intrarenal ammonia in ischemia-reperfusioninjury likely reflects decreased collecting duct ammonia secre-tion due to intercalated cell damage.

Another important finding in the present study is that theeffects of ischemia-reperfusion on the collecting duct were

maximal in the outer medulla and the initial portion of the innermedulla and were highly specific for intercalated cells. Thepredominance of effects in the outer medulla is consistent witha wide variety of studies examining the proximal tubule and theloop of Henle (25, 26, 37, 63), and extends these studies byalso demonstrating effects in the collecting duct in this region.Our observation that ischemia-reperfusion injury preferentially

affected intercalated cells, with no detectable damage to adja-cent principal cells, however, has not been reported previouslyto our knowledge. Since one mechanism underlying ischemia-reperfusion injury involves generation of oxygen free radicals,it is possible that there are higher rates of oxygen utilization byintercalated cells than in principal cells that may underlie thegreater sensitivity of intercalated cells to ischemia-reperfusioninjury. Consistent with this possibility is that the mitochondriadensity in intercalated cells is greater than in principal cells(39, 40).

Acute ischemic and nephrotoxic insults to the kidney cancause cellular damage due to either necrosis or apoptosis. Ingeneral, necrosis is the result of overwhelming and severe

Fig. 10. Activated caspase-3 immunoreac-tivity. Tissues were double-labeled using an-tibodies to cleaved (activated) caspase-3(brown) and to H-ATPase (blue immuno-reactivity). A: low-power micrograph of con-trol kidney showing absence of cleavedcaspase-3 immunoreactivity. Normal H-ATPase immunoreactivity is present in col-lecting duct intercalated cells. B: low-powermicrograph of ischemia-reperfusion kidneyshowing activated caspase-3 immunoreactiv-ity in separate cell populations from collect-ing duct intercalated cells expressing H-ATPase immunoreactivity. C: high-powermicrograph of proximal tubule S3 segment

demonstrating activated caspase-3 immunore-activity. D: high-power micrograph of OMCDfrom ischemia-reperfusion kidney showing de-tached and damaged intercalated cells, identi-fied by preserved H-ATPase immunoreactiv-ity (blue) but no detectable activated caspase-3immunoreactivity. E: positive control showingactivated caspase-3 immunoreactivity in HL60cells in which apoptosis was induced by pre-treatment with 0.5 g/ml actinomycin D.F: preincubation of the antibody with blockingpeptide blocks immunoreactivity.




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cellular ATP depletion and results in diffuse cellular dam-age and an acute inflammatory reaction in the surroundingtissue that is easily detected morphologically (3, 36). None ofthese were observed in the present study, consistent with therelatively short period, 30 min, of renal ischemia used beforerenal reperfusion. Instead, widespread evidence of apoptosis,both in the proximal tubule and loop of Henle, as previously

reported (7, 44), and in the collecting duct was observed inthese studies. In general, apoptotic cells can be identified foronly short periods of time, generally 13 h, before undergoingphagocytosis and destruction (5, 50). The relatively short time,6 h after reperfusion, at which tissue was obtained in thepresent study probably enabled identification of the apoptoticcollecting duct cells. Although this is the first identification, toour knowledge, of apoptotic intercalated cells in response toacute renal injury, apoptosis is important in the remodeling ofcollecting duct intercalated cells during embryogenesis andearly renal development (30). However, it is important to notethat apoptosis is involved in the developmental removal ofB-type intercalated cells, but not A-type intercalated cells (30).Thus it is likely that the mechanisms involved in apoptoticremoval of the B-type intercalated cell during developmentdiffer from those involved in apoptotic removal of medullarycollecting duct A-type intercalated cells in response to ische-mia-reperfusion injury.

In the kidney, apoptosis is important in renal tubular injuryfrom a variety of causes and contributes both to abnormal fluidand electrolyte transport, and possibly to the tubular repairafter induction of ischemia-reperfusion injury (36, 62).Caspases are cytosolic enzymes belonging to a large proteinfamily that are final mediators of apoptosis (42). Caspase-3 isone of several downstream executioner caspases and is impor-tant in many models of renal injury (61). The present study, byshowing that ischemia-reperfusion injury activates caspase-3 in

the proximal tubule but not in collecting duct intercalated cells,suggests that the cellular mechanisms of apoptosis differ inthese two renal epithelial cell populations. This differential roleof caspase-3 in different cell populations in the same tissue issimilar to findings previously observed in other models of renalinjury (6) and in ischemic injury in other tissues (8). Moreimportantly, the present study suggests that the cellular mech-anisms of ischemia-reperfusion injury-induced apoptosis maydiffer in different renal populations.

Acid-base and ammonia transport in the collecting duct areregulated by a wide variety of mechanisms. Changes in aproteins subcellular localization with trafficking from intra-cellular sites to plasma membrane is important for manyproteins involved in acid-base and ammonia transport, partic-

ularly H

-ATPase and Rhcg (2, 4, 56, 64). Protein-proteininteractions are also important in the regulation of acid-basetransport, such as demonstrated for H-ATPase-mediated H

secretion (38, 53). It is possible that these, and other regulatorymechanisms, may also contribute to altered ammonia metabo-lism in ischemia-reperfusion injury.

In the present study, ischemia-reperfusion injury caused anacute increase in urinary sodium excretion rates that thenreturned to normal. Similar observations have been reportedpreviously (17, 63). The increase in urinary sodium excretionis likely related to decreased proximal tubule and thick ascend-ing limb of the loop of Henle sodium absorption involvingchanges in Na-K-ATPase, NHE-3, NKCC-2, and the thia-

zide-sensitive cotransporter (17). Since GFR was decreased inresponse to ischemia-reperfusion injury, the fractional excretionof sodium, and potassium, was increased (data not shown).Whether this reflects impaired single-nephron sodium and potas-sium transport or reflects physiological decreases in single-nephron reabsorption to maintain solute homeostasis cannot bedetermined from the present studies. However, the rates of

intravenous sodium and potassium administration were identi-cal in ischemia-reperfusion injury and control animals. Thissuggests that the appropriate renal response would be identicalsodium and potassium excretion rates, which would neces-sitate increased fractional excretion in animals with ischemia-reperfusion injury-induced decreases in GFR. Thus, whetherthe increased fractional excretion of sodium and potassiumrepresents impaired transport or physiologically regulated de-creased transport cannot be determined at present.

Ischemia-reperfusion injury decreased GFR but did not sig-nificantly alter urine volume, which suggests that distal waterreabsorption was decreased. Supporting this is the observationthat urinary urea nitrogen and creatinine concentrations were

significantly decreased in ischemia-reperfusion compared withsham-operated control rats (data not shown). These findingsare consistent with previous observations of decreased collect-ing duct arginine vasopressin-dependent osmotic water perme-ability (25) and decreased AQP-2 and AQP-3 expression (13,17, 29, 33). The present study adds to these previous studies bydemonstrating that these changes in collecting duct watertransport are not due to loss of principal cells and insteadappear to reflect ischemia-reperfusion-induced changes in prin-cipal cell-mediated water transport.

Renal net acid excretion includes, in addition to ammonia,urinary bicarbonate and titratable acid excretion. Althoughthere was a tendency for urinary bicarbonate excretion toincrease, this did not reach statistical significance. Titratable

acid excretion comprises only 20 40% of total net acid excre-tion, and the primary urinary buffer comprising titratable acidsis phosphate (20). An almost universal finding in acute renalfailure, including ischemia-reperfusion injury-induced acuterenal failure, is hyperphosphatemia due to decreased urinaryphosphate excretion (9, 41, 51). Thus titratable acid excretionis likely to be decreased in parallel with ammonia excretion inresponse to ischemia-reperfusion injury and therefore to alsocontribute to the development of metabolic acidosis.

In summary, acute renal injury induced by ischemia-reperfusiondecreases urinary ammonia excretion. This appears to be due toselective loss of A-type intercalated cells from the outer med-ullary collecting duct and, to a lesser extent, the inner medul-

lary collecting duct. This cellular loss is specific to the inter-calated cell and does not involve adjacent principal cells.Intercalated cells in these regions are predominantly lost bydetachment from the basement membrane and extrusion intothe luminal space. These observations provide a cellular ex-planation for the metabolic acidosis commonly seen with acuterenal injury.

ACKNOWLEDGMENTS

We appreciate the secretarial assistance of Gina Cowsert and the technical

assistance of In-A Hwang, Nam-Sik Kim, and Jung-Mi Han. Tissue processing

for immunohistochemical studies was performed by the University of FloridaCollege of Medicine Electron Microscopy Core Facility.




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GRANTS

These studies were supported by funds from the National Institutes ofHealth (Grants DK-45788 and NS-47624), the Department of Veterans AffairsMerit Review Program, the Korea Research Foundation (Grant KRF-2006-311-E00005 to K.-H. Han), and the Anandamahidol Foundation (to S.Reungjui), and by an International Society of Nephrology Fellowship Award(to H.-Y. Kim).

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