Hypoperfusion of Peritubular Capillaries Induces Chronic Hypoxia before Progression of Tubulointerstitial Injury in a Progressive Model of Rat Glomerulonephritis
*Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan; Department of Urology, Nagoya University School of Medicine, Nagoya, Japan; Institute of Medical Sciences, Tokai University School of Medicine, Kanagawa, Japan.
Correspondence to Dr. Masaomi Nangaku, Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113–8655, Japan. Phone: 81-3-5800-8648; Fax: 81-3-5800-8806; E-mail: mnangaku-tky{at}umin.ac.jp
ABSTRACT. Chronic hypoxia likely plays a pivotal role in chronicrenal disease, but the specifics of its involvement remain unclear.To elucidate how chronic hypoxia occurs and whether hypoxiaparticipates in the progression of renal disease, the authorsestablished an irreversible glomerulonephritis model inducedby uninephrectomy and repeated anti-Thy-1 antibody injections.Glomerulosclerosis with microvascular obliteration was completeat 2 wk after antibody injection and was not restored until11 wk. Tubulointerstitial injury was mild at 2 wk and was graduallyexacerbated until 11 wk, a pattern that was in accordance withthe loss of peritubular capillaries. Immunohistochemical analysisusing pimonidazole revealed the augmentation of hypoxia in thecortex before the aggravation of tubulointerstitial injury andsubsequent peritubular capillary loss. The preexistence of hypoxiaimplies that it had substantial participation in the progressionof tubulointerstitial injury. To test whether blood flow wasinhibited in diseased kidneys, capillaries with intact bloodflow were identified by tail vein injection of biotinylatedlectin specific to endothelial cells. The renal microvasculaturewas well recognized by lectin in the controls, whereas lectinbinding to peritubular capillaries was strikingly decreasedin diseased kidneys, suggesting a disturbance of blood flow.Intravital microscopy analysis confirmed that blood flow inperitubular capillaries was decreased by approximately 40% inthe disease group compared with the controls. In conclusion,stagnation of blood flow in peritubular capillaries inducedchronic hypoxia at an early stage in this model, which was followedby progressive tubulointerstitial injury and a loss of peritubularcapillaries.
Considerable evidence from both clinical and experimental studiesindicates that the progressive loss of renal function that occursin various glomerular diseases correlates better with structuraldamage in the renal tubulointerstitium than with that in theglomeruli (1–3). Following from this, tubulointerstitialinjury is considered a final common pathway to end-stage kidneydisease. A number of findings have suggested a link betweenheavy proteinuria and subsequent tubulointerstitial injury;more recent reports, however, have illuminated the role of chronichypoxia in the tubulointerstitium as a second unifying mechanismin the progression of renal disease, on the basis that hypoxiapromotes renal scarring via its pro-fibrogenic response. Experimentsin animals (4–6) have shown that hypoxia predisposes thekidney to fibrosis (7, 8).
Renal chronic hypoxia may occur as a consequence of impairmentof blood flow or oxygen delivery (or both) to the tubulointerstitium.Recent studies have shown a correlation between a decline inthe density of the renal microvasculature and the developmentof glomerular and tubulointerstitial injury in several kidneydisease models (9–14) and progressive renal diseases inhumans (15). These studies suggest that insufficient oxygenationresulting from peritubular capillary loss pivotal role in thepathogenesis of renal diseases, although the mechanism by whichdeterioration in renal function ensues from hypoxia remainsunknown.
Glomerulosclerosis, a landmark feature of renal pathophysiology,is also thought to impair peritubular blood flow and thus oxygensupply. Anti-Thy-1 nephritis is a model of reversible mesangialproliferative glomerulonephritis (16). Previous reports showedexacerbation of this model by various boosters such as uninephrectomyor repeated injection of pathogenic antibodies to result inmore severe damage and irreversible glomerular sclerosis (12,17,18).We induced an irreversible progressive model of anti-Thy1 nephritisby repeated anti-Thy-1 antibody injection after uninephrectomyin rats. This characteristic makes this model valuable in determiningthe relationship among impeded blood flow due to glomerulosclerosis,renal hypoxia, and progressive tubulointerstitial injury.
To confirm the existence of tubulointerstitial hypoxia in associationwith glomerular damage and to examine how hypoxia occurs, weclarified the mechanisms of hypoxia in uninephrectomized anti-Thy-1nephritis. Furthermore, as a first step in addressing whetherhypoxia accelerates renal disease, we also analyzed the sequenceof events from tubular hypoxia in the early stage to severetubulointerstitial injury in the late stage.
Experimental Protocol
All experiments were conducted in accordance with the Guidefor Animal Experimentation, Faculty of Medicine University ofTokyo, Japan. Six-week-old male SD rats (Nippon Seibutsu ZairyoCenter Co., Ltd., Saitama, Japan) weighing 160 to 200 g receivedrepeated intravenous injection of IgG (OX-7) mouse monoclonalanti-Thy1.1 antibody (1.2 mg/kg body wt) or vehicle at 1 and2 wk (weeks –1 and 0) after right nephrectomy (week –2).Rats were housed in metabolic cages for overnight collectionof urine, and blood samples were obtained via the tail veinfor determination of renal functions. A sham operation, consistingof laparotomy and manipulation of the right renal pedicle withoutnephrectomy, was performed as a control.
In the first set of experiments, we analyzed physiologic andhistologic changes in nephrectomized anti-Thy-1 rats (NX Thy-1;n = 13), nephrectomized control rats (NX; n = 13), and sham-operatedrats (sham; n = 11). For tissue analyses in the early phaseof the disease, animals of each group were randomly selected(n = 6) and sacrificed at 2 wk after the final OX-7 injection(week 0). This time point was chosen because obvious glomerulardamage and mild tubulointerstitial injury were observed in ourpreliminary studies. Two rats each from the NX and NX Thy-1groups were sacrificed at week 1 to evaluate oxygenation ofthe kidney at an earlier time point, as described below. Theremaining rats (n = 5) were sacrificed at week 11 to evaluatehistologic changes in the late phase. No rats died during theexperimental period. Physiologic and histologic data of thesham group were closely similar to those of the NX group (datanot shown).
The second set of experiments investigated peritubular capillaryblood flow in 10 NX Thy-1, 10 NX and five sham rats. Physiologiclectin perfusion was done by utilizing NX Thy-1, NX, and shamrats (n = 5 for each group). A separate set of experiments wasperformed to study NX Thy-1 and NX rats (n = 5 for each group)by intravital microscopy as described below. Physiologic andhistologic data of animals of the second set were equivalentto those of the rats of the first.
Renal Histology Analysis
Tissues were fixed in methyl Carnoy’s solution and paraffin-embedded.Three-micrometer sections were stained with periodic acid-Schiff(PAS) and counterstained with hematoxylin. An indirect immunoperoxidasemethod was used to identify the following antigens; aminopeptidaseP of microvascular endothelial cells with murine monoclonalIgG1 antibody JG-12 (19) (Bender MedSystems, San Bruno, CA);monocytes/macrophages with murine monoclonal IgG1 antibody ED-1(Chemicon, Temecula, CA); vimentin with murine monoclonal IgGantibody V9 (Dako, Carpinteria, CA); and thiol-binding pimonidazolederivatives with murine monoclonal pimonidazole antibody (Chemicon).
Semiquantitative Analysis of Renal Histology Features
Quantification was performed in a blinded manner using 30 randomlyselected glomeruli or more than 15 randomly selected fieldsof cortex per cross section. Glomerulosclerosis, defined assynechiae formation by PAS staining with focal or global obliterationof capillary loops, was graded as follows: 0, normal; 1, 0%to 25% of glomerular area affected; 2, 25% to 50% affected;3, 50% to 75% affected; and 4, 75% to 100% affected (20). Tubulointerstitialinjury was graded (0 to 5+) on the basis of the percentage oftubular cellularity, basement membrane thickening, cell infiltration,dilation, atrophy, sloughing, or interstitial widening as follows:0, no change; 1, <10% tubulointerstitial injury; 2, 10% to25% injury; 3, 25% to 50% injury; 4, 50% to 75% injury; and5, 75% to 100% injury (21,22). Tubules that were vimentin-positiveor were surrounded by vimentin-positive cells and ED-1-positivecells were counted in 20 randomly selected cortical fields witha x20 objective.
Semiquantitative Analysis of Glomerular and Peritubular Capillary Loss
Glomerular or peritubular capillary loss was assessed by immunostainingfor renal microvascular endothelium with JG-12 antibody. Lossof glomerular capillary loops was graded as follows: 0, no negativeglomerular tuft staining for endothelium; 1, 1% to 25% of glomerulartufts negative for endothelium; 2, 25% to 50% negative; 3, 50%to 75% negative; and 4, 75% to 100% negative (23). Peritubularcapillary loss was analyzed using a previously reported rarefactionindex of peritubular capillary sparseness, which was calculatedas the percentage area with no capillaries identified with JG-12antibody (9). Briefly, this index was determined by countingthe number of squares in 10 x 10 grids that did not containJG-12-positive peritubular capillary staining in at least 10non-overlapping sequential fields, at x200 magnification. Theminimum possible capillary rarefaction index is 0, i.e. everysquare in the grid contains a JG-12-positive peritubular capillary,whereas the maximal score is 100, i.e. JG-12-positive peritubularcapillaries are absent from every square in the grid.
Assessment of Renal Hypoxia
Nitroimidazole compounds, which bind to thiols of cellular macromoleculesat low oxygen concentrations, have been used to detect hypoxia(<10 mmHg) in a variety of tissues (24). In this study, pimonidazole(Chemicon) was used to detect renal hypoxia as previously reported(8,25,26). To investigate the existence of renal hypoxia, animalsat week 2 (n = 3, 6, and 6 of sham, NX, and NX Thy-1 groupsfrom the first set of experiments, respectively) were injectedwith pimonidazole (60 mg/kg) via the tail vein. Two hours afteradministration of pimonidazole, the kidneys were excised andfixed for subsequent histologic studies. Pimonidazole bindingwas detected by immunohistochemistry with a specific antibodyas described above. For semiquantitative analysis of renal hypoxia,the number of pimonidazole-positive tubules per field was countedin more than ten randomly selected cortical areas per crosssection. To verify the presence of renal hypoxia at an earliertime point, nephrectomized Thy-1 rats (n = 2) and nephrectomizedcontrol rats (n = 2) were injected at week 1 with pimonidazole,followed by sacrifice and tissue processing in the same manneras described above.
Evaluation of Microvasculature with Intact Circulation by Lectin Perfusion
To identify vessels with intact blood flow, renal vasculaturewas identified with lectin that binds uniformly to the luminalsurface of endothelial cells as described previously (27). Animalsat week 2 were intravenously injected with biotinylated Lycopersiconesculentum lectin (Vector Laboratories, Burlingame, CA) at adose of 250 µg/animal. After 2 min, the renal vasculaturewas perfused with phosphate-buffered saline to wash out residualnonbinding lectin for 3 min at a pressure of 120 mmHg via ablunt 22-gauge needle inserted into the abdominal aorta justbelow the renal artery. The left kidney was dissected, fixed,and paraffin-embedded for immunohistochemical analysis by indirectimmunoperoxidase methods.
Measurement of BUN and Urinary Protein Excretion
BUN level was determined colorimetrically with a commercialkit that employed the urease-indophenol method (Wako Pure ChemicalIndustries, Tokyo, Japan). Urinary protein excretion was measuredusing a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules,CA).
Intravital Microscopy and Analysis of Peritubular Blood Flow
Animals at week 2 were anesthetized with ketamine hydrochloride(50 mg/kg). Peritubular capillaries were visualized with a pencil-lensprobe, a charge-coupled videomicroscope device with a tip diameterof 1 mm, as described previously (28). The probe had a magnificationof x520, depth of field <60 µm, and spatial resolutionof 0.86 µm, permitting identification of individual erythrocytes.Illumination was provided by a concentric set of optical fiberstransmitting light from a xenon AC 100-V light source. Videosignals were digitized with an analog-to-digital converter andfed into a digital videocassette recorder DVCAM (Sony, Tokyo,Japan) interfaced with a computer. Analysis was performed usingthe NIH Image program combined with Matlab or specifically writtenprograms. Peritubular capillary blood flow was recorded witha pencil-lens microscope brought into direct contact with thedecapsulated renal surface. Consecutive images of blood flowwere collected at a rate of 30fps for 60 min. Images were analyzedusing the freeze-frame mode. The velocity of red blood cells(RBC) in individual segments of the peritubular capillarieswas analyzed using a specifically designed adaptation of a previouslydeveloped algorithm. Specifically, a line segment was set alonga capillary bed in sequentially videotaped images, and a spatiotemporalimage was constructed (the line-shift method), allowing us todiscern differences in gray level during the passage of RBC.The angle of a line-shift striped pattern was estimated to computethe erythrocyte velocity vector.
Statistical Analyses
All data are reported as mean ± SE. Statistical analyseswere performed using the t test. Nonparametric data were analyzedwith the Mann-Whitney test when appropriate. Differences withP values < 0.05 were considered significant.
Renal Function of Uninephrectomized Anti-Thy-1 Nephritis Rats
Urinary protein excretion in the disease group at week 1 wassignificantly higher than that in the NX group and continuedto increase gradually until the end of the study. BUN levelsshowed biphasic increases, as shown in Figure 1.
Figure 1. Changes in urinary protein excretion (A) and blood urea nitrogen BUN (B) in uninephrectomized anti-Thy-1 nephritis. Urinary protein excretion in the disease group (NX Thy-1) increased gradually until the end of the study. BUN levels in the disease group were higher than those in the control group undergoing uninephrectomy only (NX) during the experimental course. BUN levels showed biphasic increases. * P < 0.05. **P < 0.01.
Development of Irreversible Glomerulosclerosis in Uninephrectomized Anti-Thy-1 Nephritis Rats Associated with Glomerular Capillary Destruction
Glomerulosclerosis occurred in the NX Thy-1 group at week 2,and the degree of the injury remained severe up to week 11,as confirmed by semiquantitative analysis (Figure 2 and Table 1).Almost all glomeruli were damaged to various degrees, with50% of those investigated classified as grade 4 as early asat week 2 (grade 0, 2.9 ± 0.9%; grade 1, 10.0 ±3.8%; grade 2, 12.8 ± 3.6%; grade 3, 24.3 ± 4.0%;grade 4, 50.0 ± 7.4%). Glomerular capillaries identifiedwith JG-12 antibody were disrupted in association with glomerulosclerosisat both weeks 2 and 11 (Table 1).
Figure 2. Changes in glomerular lesions in uninephrectomized anti-Thy-1 nephritis. Glomeruli in the NX group were intact at weeks 2 (A) and 11 (B), whereas glomerulosclerosis was already apparent in the NX Thy-1 group at week 2 (C) and showed no improvement up to week 11 (D). Periodic acid-Schiff staining. Magnification, x400.
Aggravation of Tubulointerstitial Injury from Week 2 until the End of the Study
Mild diffuse tubulointerstitial damage was observed in NX Thy-1at week 2, and these lesions were aggravated at week 11 (Figure 3and Table 1). Expression of vimentin as a marker of tubulointerstitialinjury (22,29) was increased in diseased kidneys at weeks 2and 11. Semiquantitative analysis showed that the tubulointerstitialinjury in the NX Thy-1 group tended to progress from week 2to week 11, although the difference did not reach statisticalsignificance (vimentin-positive tubules at week 11 versus week2, P = 0.069) (Figure 4 and Table 1). The number of infiltratingmacrophages in the cortical tubulointerstitium was higher inthe diseased group than in the controls. Macrophage infiltrationin the cortex was significantly and gradually increased in theNX Thy-1 group from weeks 2 to 11 (Table 1).
Figure 3. Progressive tubulointerstitial injury. No injury was observed in the NX group at weeks 2 (A) or 11 (B). In the NX Thy-1 group, tubulointerstitial injury was mild at week 2 (C) and progressed at week 11 (D). Periodic acid-Schiff staining. Magnification, x200.
Figure 4. Immunohistochemical analysis of vimentin, a marker of tubulointerstitial injury, at weeks 2 (A and C) and 11 (B and D) in the NX and NX Thy-1 groups, respectively. Vimentin-positive cells were clearly increased in diseased kidneys in comparison with control kidneys. Magnification, x200.
Association of Peritubular Capillary Loss with Tubulointerstitial Injury
With regard to peritubular capillary density, an important factorin tubulointerstitial injury, the rarefaction index score wasslightly but significantly higher in NX Thy-1 than NX grouprats at 2 wk. Simultaneous scoring of tubulointerstitial injuryand rarefaction index in individual subject fields at 2 wk showeda correlation between tubulointerstitial injury and damage tothe microvasculature in the corresponding region. Cortical areaswith no peritubular capillaries showed further expansion atweek 11 compared with those at week 2 (Figure 5 and Table 1).
Figure 5. Changes in peritubular capillaries in uninephrectomized anti-Thy-1 nephritis. Peritubular capillaries in the NX group were preserved at weeks 2 (A) and 11 (B). In the NX Thy-1 group, glomerular capillary density started to decrease at week 2 (C) and the decline was obvious at week 11 (D). Endothelial cells were stained with JG-12 antibody. Magnification, x200. Rarefaction index score was determined by counting the number of squares in 10 x 10 grids that did not contain JG-12-positive peritubular capillary staining. Simultaneous scoring of tubulointerstitial injury and rarefaction index per respective subject fields at week 2 (E).
Exposure of Renal Tubules to a Hypoxic Environment before Progression of Tubulointerstitial Injury
To verify tubular hypoxia is present before the progressionof tubulointerstitial injury, renal local oxygen tensions wereevaluated at week 2 utilizing pimonidazole, which is incorporatedinto hypoxic cells and serves as a hypoxic marker. In the shamand NX groups, pimonidazole incorporation in tubular epithelialcells occurred mainly in the medulla and medullary rays, whereborderline hypoxia is present even under physiologic conditions(30). Sham operation did not change the staining pattern ofpimonidazole. In the NX Thy-1 group, in contrast, pimonidazolestaining was widely distributed from the medulla to the outercortex, and the number of tubules with pimonidazole uptake washigher than in the NX or sham animals (percentage of hypoxictubules against total tubules per cortical field: 58.5 ±5.45%, 26.2 ± 6.34%, and 18.0 ± 5.58%, respectively;NX Thy-1 versus NX; P < 0.01) (Figure 6). Additional experimentsat week 1 showed that renal cortical hypoxia was already augmentedat a very early time point (NX Thy-1, 43.3 ± 1.74; NX,29.1 ± 0.04%).
Figure 6. Immunohistochemical analysis of pimonidazole, a hypoxic marker, at week 2 in the sham (A), NX (B), and NX Thy-1 (C) groups. Pimonidazole accumulation was intense and ubiquitous in the cortex of NX Thy-1 rats compared with both control groups. Magnification, x200.
Decrease in Lectin Binding to Peritubular Capillary Endothelial Cells in the Early Stage of Disease
While hypoxic tubules were located over a wide range in thecortex, peritubular capillary loss around damaged tubules waslimited, as described above. This observation prompted us totest the possibility that a disturbance in blood flow in tubulointerstitialcompartments might cause tubular hypoxia. For this purpose,we identified capillary endothelium by physiologic perfusionwith biotinylated lycopersicon esculentum lectin, which is specificfor N-acetyl-D-glucosamine oligomers of vessel luminal surfaces.Lectin bound to all endothelial cells of both glomerular andperitubular capillaries in the NX group in a more diffuse andintense manner than in the sham group. However, significantlyless binding of lectin to capillaries was seen in the NX Thy-1group than in either the NX and sham groups (rarefaction indexscore of lectin binding capillaries: sham, 16.4 ± 2.5%;NX, 6.8 ± 1.9%; NX Thy-1, 31.3 ± 3.2%; NX Thy-1versus sham, P < 0.01). Furthermore, the number of endothelialcells identified by lectin perfusion was less than that identifiedby JG12 in the disease group at the same time point (rarefactionindex score of lectin-binding capillaries: 31.3 ± 3.2%;that of staining with JG-12, 12.1 ± 0.40%; P < 0.01,NX Thy-1 group at week 2). Double staining with lectin and pimonidazoleshowed that low oxygenation co-localized with poor lectin perfusionin diseased kidneys (Figure 7).
Figure 7. Determination of hypoperfused areas by physiologic lectin perfusion. In the NX group, glomerular and peritubular capillaries were stained with lectin (brown), which was associated with less pimonidazole staining (blue) (A). In contrast, lectin binding to capillary endothelial cells was strikingly decreased in the NX Thy-1 group, in which the number of hypoxic tubules was increased (B). Magnification, x200.
Direct Demonstration of a Decline in Blood Flow in Peritubular Capillaries of Uninephrectomized Anti-Thy-1 Nephritis Rats by Intravital Microscopy
Our findings on the hypoperfusion of lectin in diseased kidneyssuggested that blood flow stagnates in this renal disease model.To test this hypothesis, RBC velocity was directly measuredby intravital videomicroscopy. RBC velocity in peritubular capillariesof the NX group averaged 0.476 ± 0.047 mm/s, whereasthat in the NX Thy-1 group decreased to 0.296 ± 0.048mm/s (NX Thy-1 versus NX group, P < 0.05). In some capillaries,flow switched from orthograde to retrograde; in others, RBCdisplayed a pulsatile behavior with periodic stagnation followedby the resumption of flow in the orthograde direction. Thesefindings are similar to those previously observed in rat renalischemia reperfusion (28).
In this study, we demonstrated that chronic hypoxia occurs evenin the early stage of disease in the uninephrectomized anti-Thy-1nephritis model with irreversible glomerulosclerosis. Bloodflow in peritubular capillaries at this time was also shownto be decreased. In the late stage, deterioration of tubulointerstitialdamage proceeded and was associated with severe peritubularcapillary loss.
Repeated injection of anti-Thy-1 antibody after uninephrectomyled to severe and irreversible glomerulosclerosis within 2 wk.In contrast, tubulointerstitial scarring with this treatmentwas mild at 2 wk and progressed chronically up to 11 wk, whensevere tubulointerstitial injury was observed. Mild increasesin some injury markers such as macrophage infiltration and peritubularcapillary loss from week 2 to week 11 in the control group couldbe attributed to aging effects, as previously reported by Thomaset al. (31). BUN response was biphasic; we speculate that thefirst peak was due to the primary glomerular injury inducedby the antibody injections and the second to the progressionof tubulointerstitial disease. Similar findings were observedin a uninephrectomized anti-Thy1 model induced by a differentanti-Thy1 monoclonal antibody, 1–22–3 (Sogabe H,Tomita M, Kawachi H and Shimizu F; personal communication).
In this study, we confirmed that hypoxia is present from week1, at a time when the overall structure of tubules was stillmaintained. This finding implicates low oxygenation as an importantmediator of the progression of tubulointerstitial injury. Thechanges showed a heterogeneous pattern of hypovascularity andtubulointerstitial injury, including extracellular matrix (ECM)accumulation, while a distribution of hypoxic tubules was ubiquitous.Because of the diffuse development of glomerulosclerosis inthe early phase of this model, we hypothesized that the destructionof glomerular capillaries may disturb postglomerular microcirculationin peritubular capillaries.
Capillary binding of lectin in the NX Thy-1 group was significantlydecreased compared with the two control groups. Furthermore,the number of lectin-positive capillaries was much lower thanthat of JG-12-positive vessels even in the same kidneys of theNX Thy-1 group. Although we cannot exclude the possibility thatchanges in the endothelial glycocalyx lead to decreased lectinbinding, the loss of binding in these capillaries was more likelydue to a decrease in blood flow conveying lectin to the correspondingvessels, because the expression of N-acetyl-D-glucosamine oligomersspecific for lycopersicon esculentum lectin is known to be relativelyindependent of pathologic conditions (32). In addition, ourintravital videomicroscopy analysis also demonstrated the stagnationof blood flow in peritubular capillaries in diseased kidneys.
The mechanism of the impairment of peritubular capillary bloodflow may be multifactorial. For example, narrowing of glomerularcapillaries as a result of morbid mesangial matrix accumulationdue to sclerosis could decrease perfusion in postglomerularcapillaries. In addition to anatomical damage in glomerulartufts, imbalance of vasoactive substances in the kidney mayreduce peritubular capillary blood flow. We recently observedthat inappropriate activation of the renin-angiotensin systemreduced circulation in peritubular capillaries in the earlyphase of a remnant kidney model (33). Furthermore, Oyanagi-Tanakaet al. (34) reported that glomerular injury in anti-Thy1 nephritiswas associated with suppression of red blood cell velocity inglomerular capillaries, which may also have contributed to thestagnation of peritubular capillary blood flow in our model.
A number of studies have shown that hypoxia leads to pro-fibrogenicresponses in tubular epithelial cells and renal fibroblasts(4,35). We previously demonstrated that hypoxia promoted epithelial-mesenchymaltransdifferentiation of tubular cells (36). These phenotypicchanges of resident cells in the tubulointerstitium might disturbthe homeostasis of ECM, and subsequent accumulation of ECM mayblock oxygen diffusion and exaggerate hypoxia. We have alsoshown that hypoxia increases apoptotic cell death of proximalepithelial cells (37) as well as endothelial cells (38). Wespeculate that the tubular hypoxia found in the early stageof this study may play a crucial role in the progression ofinterstitial injury via these multiple pathways. In addition,the notable decline in peritubular capillary density in thelate stage likely renders both the lesion itself and the surroundingarea hypoxic, instituting a vicious cycle to end-stage renalfailure.
What is the clinical relevance of these findings? Recent researchhas focused on the development of novel therapeutic approachesto tubulointerstitial hypoxia. Administration of VEGF was effectivein renal disease models associated with the loss of peritubularcapillaries (10,39). Induction of hypoxia-activated genes asa treatment modality is another current topic in kidney research,and Eckardt and colleagues have demonstrated the usefulnessof this strategy (40,41). We also demonstrated that cobalt rendersthe kidneys resistant to hypoxia by activating hypoxia-inducedgenes (42). Our findings in the current study suggest that,once available for clinical use, these therapeutic modalitieswill be effective even in the early phase of kidney disease.
In summary, we have provided a direct demonstration that tubularhypoxia is present in the early stage of disease in a progressiveglomerulonephritis model. Our findings confirm that hypoxiais attributable not only to hypovascularity associated withtubulointerstitial injury but also to a decrease in blood flowdue to glomerular injury. To our knowledge, this is the firststudy to demonstrate that destruction of the glomerular capillariesleads to hypoperfusion of peritubular capillaries and the correspondinginterstitial area, and that this precedes the development oftubulointerstitial scarring. These results suggest that renalchronic hypoxia is not merely a result of tubulointerstitialinjury but plays a pivotal role in the progression of renaldisease.
Acknowledgments
We acknowledge research grants from the Japanese Ministry ofHealth, Labor and Welfare, KIRIN brewery pharmaceutical researchlaboratory (Japan), and NOVARTIS Foundation (Japan) for thePromotion of Science. We thank Dr. Takamoto Ohse, Dr. KrissanapongManotham (University of Tokyo), Dr. Kiyoshi Kurokawa (TokaiUniversity), and Dr. Takeyoshi Yamashita (Kirin Brewery Co.,LTD) for many helpful advices and generous supports.
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Received for publication November 10, 2003.
Accepted for publication March 26, 2004.
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Abstract
Introduction
