Expression of Osteopontin in Gentamicin-Induced Acute Tubular Necrosis and Its Recovery Process

Kidney International, Vol. 59 (2001), pp. 959–974

Expression of osteopontin in gentamicin-induced acute tubular necrosis and its recovery process

YUANSHENG XIE,SHINICHI NISHI,SEITARO IGUCHI,NAOFUMI IMAI,MINORU SAKATSUME, AKIHIKO SAITO,MIKA IKEGAME,NORIAKI IINO,HISAKI SHIMADA,MITSUHIRO UENO, HIROYUKI KAWASHIMA,MASAAKI ARAKAWA, and FUMITAKE GEJYO

Department of Nephrology, Guangxi Zhuang Autonomous Region Nan Xi Shan Hospital, Guilin, China; Department of Medicine II, Niigata University School of Medicine, First Department of Oral Anatomy and Department of Pharmacology, Niigata University, Faculty of Dentistry, Niigata, Japan

Expression of osteopontin in gentamicin-induced acute tubular including the kidney, lung, liver, bladder, pancreas, and necrosis and its recovery process. breast [1–3]. Structurally, the OPN protein is roughly Background. There is controversy regarding the exact localization and roles of osteopontin (OPN), a multipotential che- globular, contains an arginine-glycine-aspartic acid (RGD) mokine, in renal injury. There is little information on the ex- sequence, and is highly phosphorylated on up to 28 serine pression and role of OPN in gentamicin-induced acute tubular residues [4]. necrosis (ATN) and its recovery process. Osteopontin is expressed in the normal kidney and Methods. A severe ATN model was made using male Wistar rats by injecting gentamicin (150 mg/kg/day) for five days and induced under various experimental pathological condi- limiting the provision of water. The expression and localization tions [5, 6], such as angiotensin II-induced tubulointer- of OPN mRNA and protein, ED1 as a macrophage marker, stitial nephritis [7], cyclosporine nephropathy [8], hydro- proliferating cellular nuclear antigen (PCNA), CD44 as an nephrosis caused by unilateral ureteral ligation [9], renal OPN receptor, megalin as a proximal tubule marker, and their relationships to each other were examined from the early tubu- ischemia [10, 11], cisplantin nephropathy (abstract; Iguchi lar necrotic period to the late recovery period by Northern et al, J Am Soc Nephrol 9:A3043, 1998), and crescentic blotting, in situ hybridization, and double immunohistochemi- glomerulonephritis [12, 13]. Despite this, little is known cal staining. about the expression of OPN in gentamicin-induced Results. In the gentamicin group, OPN mRNA and protein were expressed in only the PCNA-positive proliferating corti- acute tubular necrosis (ATN) and its recovery process, cal distal tubules, not in the necrotic proximal tubules, until including regeneration of renal tubular epithelial cells day 6 after the first administration, but were found markedly following ATN. In addition, the exact localization of in PCNA-positive regenerative proximal and distal tubules on days 10, 15, and 30. The localization of PCNA-positive cells OPN in the kidney has been controversial because the was almost always accompanied with the up-regulated expres- discrimination between the proximal and distal tubules sion of OPN using quantitative analysis (P Ͻ 0.01). CD44 becomes difficult in pathological conditions [14]. expression was markedly up-regulated in the renal cortical Despite the significant progress in understanding the tubular epithelium from days 6 to 30. In the control group, no expression of OPN and CD44 in the cortical area was found structure and topography of OPN, its functional role throughout the experimental period. remains insufficiently understood [15]. Based on the Conclusions. These results suggested that OPN is related to known activities of OPN in vitro and in vivo, there are the proliferation and regeneration of tubular epithelial cells currently two hypotheses for its role in renal pathologies after tubular damage. and pathophysiologies [6]. One hypothesis is that OPN is not only produced by macrophages and activated T Osteopontin (OPN) is a 44 kD secreted glycosylated cells, but also modulates their activity, both in migrating phosphoprotein. Originally isolated from bone, it has been to sites of injury and in responding to the activators shown to be expressed in a number of different tissues, present at such sites [6]. Another attractive hypothesis holds that in some settings, OPN may act as a cell survival Key words: acute renal failure, megalin, cell proliferation, regenera- factor for renal cells [15–17]. Recently, studies on the tion, macrophage, CD44. roles of OPN have focused on its renoprotective action [15–17], including repair and regeneration following re- Received for publication March 29, 2000 and in revised form August 14, 2000 nal injury, in addition to its chemoattractive function. Accepted for publication September 27, 2000 Gentamicin toxic nephropathy includes acute tubular  2001 by the International Society of Nephrology necrosis in the early period, infiltration of monocytes/

959 960 Xie et al: OPN in tubular necrosis and recovery macrophages, and regeneration of renal tubular epithe- ing vector, introduced into E. coli super competent cells, lial cells during the recovery process [18, 19]. The rela- and a single colony of transfected cells was used to pre- tionship between these toxic pathological and patho- pare OPN cDNA. physiological processes and OPN expression is not The plasmid sample with the OPN cDNA was linear- understood. In this study, we used a severe ATN model ized by incubation at 37ЊC with Nco I as an antisense induced by gentamicin administration and dehydration probe and Sal I as a sense probe, respectively. The linear- and examined the expression and localization of OPN ization of the plasmid was confirmed by running the sam- in cortical tubules by Northern blot analysis, in situ hy- ples on a 1% agarose gel. The linearized plasmid was bridization, immunohistochemistry, and double staining extracted with phenol/chloroform and precipitated in with antimegalin antibodies as a proximal tubule marker. ethanol. Throughout the observation of the exact localization of The in vitro transcription of the cDNA was performed OPN, ED-1 as a monocyte/macrophage marker, prolifer- using a DIG RNA labeling kit (SP6/T7; Boehringer ating cellular nuclear antigen (PCNA), CD44 as an OPN Mannheim, Leverkusen, Germany). One microgram of receptor, and their relationship to each other from the linearized plasmid was used as a template and incubated early ATN period to the later recovery period, we tried with SP6 DNA-dependent RNA polymerase for two to clarify OPN’s role in severe gentamicin-induced ATN hours to obtain an antisense probe. A T7 promoter was and its recovery process. used to produce a sense probe, which was used as a negative control. The synthesized RNA probes were re- covered by phenol/chloroform extraction and ethanol METHODS precipitation and then redissolved in 100 ␮L diethyl Animal experiments pyrocarbonate H2O and 1 ␮L of an RNase inhibitor Experiments were performed on 40 seven-week-old (Wako, Osaka, Japan). male Wistar rats (Charles River Japan, Yokohama, Ja- pan), weighing between 230 to 270 g, that were divided Northern blot analysis into two groups: a gentamicin group and a control group. After removal of the kidneys, renal tissues were frozen The rats in the gentamicin group were given 150 mg/kg/ in liquid nitrogen and stored at Ϫ80ЊC until analysis. day of gentamicin sulfate solution (Sigma-Aldrich Co., After homogenization, the total RNA of the tissue sam- St. Louis, MO, USA) by subcutaneous injection in the ples was extracted using ISOGEN௣, following the manu- neck for five days, and the rats in the control group were facturer’s protocol (Nippon Gene, Tokyo, Japan). For given an equal volume of normal saline instead. All ani- Northern blotting, 20 ␮g total RNA was loaded per lane mals were fed a diet of standard laboratory chow and and electrophoresed on a 1.5% agarose gel containing allowed free access to water, but were deprived of water formaldehyde and transferred to a nylon membrane for 24 hours before the first gentamicin administration (Hybond-Nϩ; Amersham, Arlington Heights, IL, USA). and for 12 hours a day during gentamicin administration. In this study, the previously mentioned digoxigenin Five rats in each group were sacrificed under ether (DIG)-labeled OPN cRNA probe (50 ng/mL) was used. anesthesia on days 6, 10, 15, and 30 after the first gentami- Hybridization was performed at 68ЊC for 16 hours with cin injection, respectively. Blood was taken from the shaking according to the standard protocol. After hybrid- abdominal aorta for the assay of urea nitrogen and creati- ization, the filter was stringently washed and then incu- nine via urease ultraviolet absorption spectrophotometry bated at room temperature for 30 minutes in a buffer and alkali picric acid method, respectively. The bilateral with 0.1 ␮L/mL of polyclonal sheep anti-DIG Fab frag- kidneys of one rat were rapidly removed and bisected ments conjugated with alkaline phosphatase (Boehringer for Northern blot analysis. The other four rats were infused Mannheim). The excessive antibodies were washed off with 4% paraformaldehyde from the abdominal aorta, with the buffer. A color reaction was performed by incu- and the kidneys were removed and bisected for histologic bating the filter with 0.1 ␮L/mL NBT/BCIP color-sub- and immunohistochemical examination and in situ hy- strate solution at room temperature for two hours. bridization. All tissues were routinely processed, embedded in paraffin, and cut serially into 5 ␮m thick sections. In situ hybridization Renal tissues for in situ hybridization were fixed with Probe preparation 4% paraformaldehyde in 0.01 mol/L phosphate-buffered To obtain rat OPN cDNA, two primers, OPN5 (5Ј-TC saline (PBS; pH 7.4), dehydrated in an ethanol series, TGACGAATCTCACCATTC-3Ј) and OPN3 (5Ј-TCTT and embedded in paraffin under RNase-free conditions. CCTTACTCTTAGGGTCTA-3Ј), were designed and syn- Serial 5 ␮m thick sections were cut. Details of in situ thesized according to the rat OPN cDNA sequence to hybridization techniques used here were reported pre- amplify a 568 bp fragment. The PCR product was cloned viously [20]. In this study, a DIG-labeled OPN cRNA into a pGEM-T Easy (Promega, Madison, WI, USA) clon- probe (0.5 ␮g/mL) and an OPN sense probe as a negative Xie et al: OPN in tubular necrosis and recovery 961

control were used. The hybridization for OPN mRNA viously in this article. In immunohistochemical double was performed at 52ЊC for 16 hours. The alkaline phos- staining, the first immunohistochemical staining was per- phatase-conjugated anti-DIG-antibody was reacted with formed with a DAB peroxidase substrate solution, which the sections at 37ЊC for 30 minutes, and a coloring reac- showed a brown color reaction, followed by washing in tion with NBT/BCIP was performed at room tempera- PBS for 3 ϫ 5 minutes and treatment for 10 minutes in ture for 12 hours. The sections obtained from the cisplan- a microwave oven in a 0.01 mol/L citrate buffer (pH 6.0). tin nephropathy model (abstract; Iguchi et al, JAmSoc Then the second immunohistochemical staining was per- Nephrol 9:A3043, 1998) were used as a positive control. formed with a Fuchsin alkaline phosphatase substrate solution, which showed a red color reaction. Immunohistochemistry Immunohistochemical staining was carried out on 5 ␮m Quantitative analysis wax sections. The sections were first dewaxed and dehy- A point-counting method was employed to quantitate drated. Then they were incubated with 0.6% H2O2 in the OPN mRNA and OPN protein-positive fractional methanol for 30 minutes to eliminate endogenous perox- volume of renal cortical proximal (megalin-positive), idase activity and were treated with normal goat serum nonproximal (megalin-negative), and total tubular epi- (Chemicon, Temecula, CA, USA) at room temperature thelium on double-staining sections for OPN mRNA or for 30 minutes. Subsequently, they were incubated at OPN protein and megalin, respectively [21]. In each field, Њ 4 C for 24 hours with primary antibodies, which included 121 points (intersections) were counted on a 1 cm2 eye- the following: mouse monoclonal anti-OPN antibody piece graticule with 11 ϫ 11 equidistant grid lines. Under ␮ (1 g/mL; ARP, Belmont, MA, USA), rabbit polyclonal high magnification (ϫ400), 20 consecutive nonoverlap- ␮ antimegalin antibody (5 g/mL; kindly provided by Drs. ping fields per section, that is, a total of 2420 points of Robert A. Orlando and Marilyn G. Farquhar of the the renal cortex in each kidney, were observed. The per- University of California, San Diego, CA, USA), mouse centages of OPN-positive proximal, nonproximal, and ␮ anti-ED1 antibody (10 g/mL; Serotec, Oxford, UK), total tubules were calculated, while the points falling on mouse monoclonal antiproliferating cell nuclear antigen glomeruli, Bowman’s capsules, or tubular lumens were antibody (1:20; Dako, Glostrup, Denmark), and mouse excluded. monoclonal anti-CD44 antibody (1:100; Pharmingen, The quantitations for ED1-positive cells and PCNA- San Diego, CA, USA). The sections were incubated with positive cells were undertaken under high magnification a secondary antibody, either goat anti-mouse IgG conju- (ϫ400) in which 20 randomly selected cortical graticule gated with the alkaline phosphatase or the peroxidase fields per section were examined. The ED1-positive cells or anti-rabbit IgG conjugated with the peroxidase at located in the tubulointerstitial area, including renal tu- room temperature for 30 minutes. After washing with bular lumens and the PCNA-positive cells located in the PBS, the sections were incubated with a Fuchsin alkaline renal tubular epithelium, were counted, and the numbers phosphatase substrate solution (Dako, Carpinteria, CA, were averaged for each field. USA) with an endogenous alkaline phosphatase inhibitor (Dako, USA), or a DAB peroxidase substrate solu- The correlation analysis for OPN and PCNA was un- tion (Nichirei, Tokyo, Japan). The cellular nuclei of the dertaken on double-staining immunohistochemistry sec- sections were counterstained with hematoxylin. The cis- tions for the OPN protein and PCNA. plantin nephropathy model sections (abstract; Iguchi et Statistical analysis al, J Am Soc Nephrol 9:A3043, 1998) were used as a positive control. Statistical analysis was performed using the statistical software StatView Version 5. A P value of less than Double staining 0.05 was considered significant. Data are expressed as Ϯ Double staining was performed on the same tissue mean SD. section, which included a combination of in situ hybridization for OPN mRNA and immunohistochemistry for RESULTS megalin as a proximal tubule marker, a combination of immunohistochemistry for OPN protein and megalin, a Biochemical data combination of OPN protein and ED-1 as a monocyte/ The level of blood urea nitrogen (BUN) and serum macrophage marker, and a combination of the OPN creatinine (SCr) in the gentamicin group rats increased protein and PCNA as a cell proliferation or regeneration significantly on day 6 after the first administration (P Ͻ marker. For the in situ hybridization and immunohisto- 0.01), reached its peak on day 10 (P Ͻ 0.05), and then chemistry tests, the sections were washed in PBS for 3 ϫ gradually decreased and almost returned to normal by 5 minutes after in situ hybridization, and then immuno- day 30. Two rats in the gentamicin group died on days histochemical staining was performed as described pre- 13 and 15, respectively (Table 1). 962 Xie et al: OPN in tubular necrosis and recovery

Histologic changes during the experimental period (Fig. 3). OPN mRNA Periodic acid-Schiff (PAS) staining showed that renal expression was shown simultaneously in the renal tu- tubular necrosis apparently developed on day 6 after the bules of the cisplantin nephropathy model as a positive first administration in the gentamicin group. Superficial control (figure not shown). The sense control for OPN cortical tubules revealed severe degeneration or necrosis mRNA was negative (figure not shown). of epithelial cells. On day 10, there were lots of desqua- The expression of the OPN protein was recognized as mated epithelial cell debris in the renal tubular lumens. a punctate distribution in the cortical tubular epithelium Mononuclear cells infiltrated interstitial lesions from the from days 6 to 30, with a peak on day 15 in the gentamicin superficial cortex to the corticomedullary zone. New cel- group. In the control group, the expression of OPN pro- lular lines that were thought to be regenerative tubular tein was not detected in the cortical tubules during the epithelial cells appeared along the tubular basement experimental period (figure not shown). The localization membranes. The regenerative tubular epithelial cells be- of the OPN protein was positive in the medullary tubules came larger, and the renal tubular epithelium became in both groups from days 6 to 30, but its distribution in thicker on day 15. On day 30, most of the cortical tubular the gentamicin group was wider than the control on structures almost returned to the normal architecture, days 6, 10, and 15 except for day 30. Bowman’s capsule but spotty infiltration of mononuclear cells, atrophy of epithelial cells showed positive OPN protein expression renal tubules, and fibrosis of the interstitium remained. in only the gentamicin group (Fig. 4). OPN protein ex- The control group showed no significant histologic pression was shown simultaneously in the renal tubules changes throughout the experimental period. of the cisplantin nephropathy model as a positive control Based on the immunohistochemical staining of mega- (figure not shown). Double staining of in situ hybridization for OPN lin and cellular nuclear staining, it was evident that the mRNA and immunohistochemistry for the megalin pro- necrotic tubules were proximal tubules. The distal tu- tein used as a proximal tubule marker clearly revealed bules showed some proliferation changes on day 6. The that expression of OPN mRNA was present in only distal regenerative changes occurred mainly in the proximal tubules, but not in the proximal tubules of the renal tubules, but also in the distal tubules from day 10 to day cortex during the ATN period on day 6 after the first 15. By combination with PAS staining, it was shown that administration. However, in the recovery period on days focal atrophic tubules existed in both the proximal and 10, 15, 30, the expression of OPN mRNA was present distal tubules on day 30 (Fig. 1). not only in the distal tubules, but also in the proximal Expression and localization of OPN mRNA tubules showing regenerating changes in the renal cortex. and protein Double-staining immunohistochemistry for the OPN protein with megalin showed that the localization of the Compared with the control group, Northern blot anal- OPN protein was coincident with that of OPN mRNA ysis showed markedly up-regulated expression of OPN in the renal cortex (Fig. 5). mRNA in the kidneys of the gentamicin group from days Quantitative analysis showed that the percentage of 6 to 30 (Fig. 2). OPN mRNA-positive tubules in the proximal (megalin- In the gentamicin group, histologic localization of positive) tubules was highest on day 15 in the gentamicin OPN mRNA was shown in the cortical tubules by in situ group, while that in the nonproximal (megalin-negative) hybridization from the early ATN period on day 6 to tubules, for example, the distal tubules, was highest on the later recovery period on day 30. OPN mRNA expres- day 10 (Fig. 6). The location and level of OPN protein sion was most widely distributed on day 10 and day 15, expression was almost the same as that of OPN mRNA and decreased on day 30 in the cortical region. In the in the renal cortical tubules (data not shown). control group, OPN mRNA was not detected in the cortical region throughout the experimental period, ex- Demonstration of ED1-positive cells and cept for a weak expression in the descending thin limb the relationship between ED1 and OPN of the loop of Henle (not shown). The expression of To examine the relationship between OPN and mono- OPN mRNA in the gentamicin group was also positive cyte/macrophage infiltration, we performed immunohis- in the medullary tubules from days 6 to 30 and was tochemistry for ED1 (a monocyte/macrophage marker) stronger than that of the control. The OPN mRNA sig- and double staining for OPN and ED1. In the gentamicin nals in the medulla were strongest on day 6 and then group, only a few ED1-positive cells were present in decreased gradually and almost returned to normal on the cortical tubulointerstitium on day 6, but many were day 30 in the gentamicin group. Intraglomerular expres- present in the cortical interstitium and within the tubular sion of OPN mRNA was not detected in either group, lumens on days 10 and 15, with the most on day 10 after and only parietal epithelial cells of Bowman’s capsule the first administration. On day 30, they were focally showed weak positive signals in the gentamicin group present in an atrophic, thick tubular epithelium and its Fig. 1. Renal histologic changes in gentamicin-induced acute tubular necrosis (ATN) and recovery. (A–E and F–J) Serial sections, respectively. (A–E) PAS staining. (F–J) Immu- nohistochemical staining of megalin (a proximal tubule marker, brown) and cellular nuclear staining. (A and F) Normal cortical renal tubules. Note that megalin is positive in only the proximal tubules (arrow), but not in other tubules (arrowhead, F). (B and G) ATN on day 6 after the beginning of gentamicin injection (150 mg/kg/day ϫ 5). Note the flattened, desquamated epithelium, denuded basement membranes, and intraluminal cellular debris (arrow, B). Tubular necrosis occurs only in proximal tubules (arrow, G), and evidence of proliferation in distal tubules is shown (arrowhead, G) as compared with control (arrowhead, F). (C) Recovering renal cortical tubules on day 10. Renal tubules are lined by regenerating cells (arrow). Some tubules are filled with amorphous debris, while others have cleared. Mononuclear cells aggregate in the renal interstitium (arrowhead). (D) Recov- ering renal cortical tubules on day 15. The renal tubular epithelium becomes thick, but the microvilli are relatively short and simple (arrow). (H and I) Regenerating changes oc- cur not only in proximal tubules (arrow), but also in distal tubules (arrowhead) on day 10 (H) and day 15 (I). (E) Cortical renal tubules on day 30. Most of the cell structure is compa- rable to controls, but a small focus of atrophic tubules (arrow), interstitial fibrosis, and mononuclear cells remained (arrowhead). (J) Focal atrophic tubules also include proximal (arrow) and distal tubules (arrowhead) on day 30. Original magnification ϫ100. 964 Xie et al: OPN in tubular necrosis and recovery

Fig. 2. Northern blot analysis in gentamicin- induced ATN and recovery.

Fig. 3. Osteopontin (OPN) mRNA expression from in situ hybridization in renal cortical tubules treated by gentamicin. (A) Up-regulation of OPN mRNA only in cortical tubules having no necrosis, but not in necrotic tubules on day 6 after the ﬁrst gentamicin administration. (B and C) Marked up-regulation of OPN mRNA in regenerative renal tubules on day 10 (B) and day 15 (C). (D) Up-regulation of OPN mRNA focally in some areas of the renal cortex on day 30. Original magniﬁcation ϫ100. Xie et al: OPN in tubular necrosis and recovery 965

Fig. 4. Expression of the OPN protein with immunohistochemistry in renal cortical tubules treated by gentamicin. (A) Up-regulation of OPN protein in cortical tubules having no necrosis, but not in necrotic tubules on day 6 after the first gentamicin administration. (B and C) Marked up-regulation of the OPN protein as a punctate distribution in regenerative renal tubules on day 10 (B) and day 15 (C). (D) Up-regulation of the OPN protein in the focal area of renal tubular atrophy and interstitial fibrosis on day 30. Original magnification ϫ100.

peripheral interstitium (Figs. 7 and 8). No distinct macro- Demonstration of PCNA-positive cells and phage infiltration was found in the control group (not colocalization of PCNA and OPN shown). Double staining showed that the location of To clarify the relationship between OPN expression and OPN expression had no distinct relationship with the site cellular proliferation/regeneration, we carried out immu- of monocyte/macrophage accumulation in most areas of nohistochemistry for PCNA and double staining for OPN the cortex, but in some areas of the cortex, the location and megalin, OPN and PCNA. There were few PCNA- of OPN expression was almost coincident with the site positive cells in the rat kidneys of the control group. In of monocyte/macrophage infiltration in the gentamicin contrast, in the gentamicin group, the PCNA-positive group (Fig. 9). cells were observed in great numbers in the distal tubules Fig. 5. Localization of OPN mRNA and the OPN protein in renal cortical tubules treated by gentamicin. OPN mRNA (blue) is shown by double staining of in situ hybridization for OPN and immunohistochemistry for megalin (a proximal tubule marker, brown; A–E). OPN protein (red) is shown by immunohistochemical staining of OPN and megalin (F–J). A–E and F–J are serial sections, respectively. (A and F) Normal renal tubules showing no OPN mRNA or OPN protein. (B and G) Up- regulation of OPN mRNA and the OPN protein in cortical distal tubules (arrow), but not in necrotic proximal tubules on day 6 after the first gentamicin administration. (C and H) Marked up-regulation of OPN mRNA and the OPN protein not only in cortical distal tubules (arrow), but also in proximal tubules (arrowhead) on day 10. (D, E, I, and J) Up-regulation of OPN mRNA and the OPN protein mainly in cortical proximal tubules (arrow), but also in distal tubules on day 15 (D and I) and on day 30 (E and J). Double staining showed that the localization of the OPN protein was coincident with that of OPN mRNA in the renal cortex (arrow). Original magnification ϫ100. Xie et al: OPN in tubular necrosis and recovery 967

Table 1. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels in rats after gentamicin administration

BUN mg/dL SCr mg/dL Group Day 6 Day 10 Day 15 Day 30 Day 6 Day 10 Day 15 Day 30 Gentamicin 112.78Ϯ32.69a 229.41Ϯ134.40a 44.80Ϯ15.32 29.98Ϯ7.63 3.10Ϯ0.96a 5.66Ϯ3.65b 0.63Ϯ0.22b 0.40Ϯ0.18 Control 22.92Ϯ1.39 23.16Ϯ3.78 21.12Ϯ3.75 23.52Ϯ2.89 0.20Ϯ0.00 0.20Ϯ0.00 0.28Ϯ0.05 0.20Ϯ0.06 Data are means Ϯ SD. Gentamicin group rats were given 150 mg/kg/day gentamicin sulfate solution for 5 days by subcutaneous injection. Control group rats received an equal volume of normal saline instead. a P Ͻ 0.01 compared with Control b P Ͻ 0.05 compared with Control

in the deep cortex, corticomedulla, and medulla in the acute proximal tubular necrotic period on day 6 and in the proximal and distal tubules of the cortex, as well as medullary tubules, in the recovery period on days 10, 15, and 30, with the most seen on day 10 after the ﬁrst administration (Figs. 10 and 11; double staining for OPN and megalin not shown). The double staining for OPN and PCNA showed that PCNA-positive cells were mainly present in the tubular epithelium with up-regulated expression of OPN (Fig. 12). The correlation analysis showed that PCNA-positive tubular epithelial cells were associated with the expression of OPN in the gentamicin-induced ATN period and the following recovery period (r ϭ 0.966, 0.913, 0.850, and 0.956 on days 6, 10, 15, and 30, respectively, P Ͻ 0.01).

Expression of CD44 The immunohistochemistry for CD44 showed that expression of CD44 was markedly up-regulated in the cortical tubular epithelium and inﬁltrating cells in the cortical interstitium and tubular lumens from the tubular necrotic period on day 6 to the recovery period on days 10, 15, and 30 after the ﬁrst administration in the gentamicin group. In the control group, no expression of CD44 in the cortical tubular epithelium was found throughout the experimental period (Fig. 13).

DISCUSSION Osteopontin is a secreted glycoprotein that has broad tissue distribution and diverse biological functions [3, 6]. Its kidney expression has been demonstrated in normal and a number of renal injury models [5, 6]. There is one report regarding the expression of OPN in a gentamicin- induced acute renal failure model [22]. In that report, rats were given subcutaneous injections of gentamicin (40 mg/kg) daily for 10 days and were sacrificed for analysis on the 10th day of gentamicin administration. In Fig. 6. Quantitative analysis of cortical tubular OPN mRNA expres- the model, the serum creatinine and blood urea nitrogen sion in gentamicin-induced ATN and recovery. Quantitation was based levels revealed abnormal elevation from the eighth day. on double-staining sections of in situ hybridization for OPN mRNA and immunohistochemistry for megalin as a proximal tubule marker The expression of OPN mRNA was not enhanced in using a point-counting method. (A) Proximal (megalin-positive) tu- that mild model by Northern blot analysis or in situ bules. (B) Cortical nonproximal (megalin-negative) tubules. (C) Corti- hybridization. We successfully made a severe ATN model cal total tubules. GM6, GM10, GM15, and GM30 show days 6, 10, 15, and 30 after the first administration in the gentamicin group, respec- by injecting a high dose of gentamicin (150 mg/kg/day) tively. *P Ͻ 0.01; #P Ͻ 0.05; NS, no significant difference. for five days and limiting the provision of water to the Fig. 7. Demonstration of ED1-positive cells in gentamicin-induced ATN and recovery. (A) A few ED1-positive cells are present in the cortical tubular epithelium and interstitium on day 6 after the first gentamicin administration. (B and C) Many ED1-positive cells are present in the cortical interstitium and within tubular lumens on day 10 (B) and day 15 (C). (D) Quite a few ED1-positive cells are focally present in some tubular epithelia and interstitia on day 30. Original magnification ϫ100.

Fig. 8. Quantitative analysis of cortical ED1-positive cells in gentamicin-induced ATN and recovery. ED1-positive cells in the tubules, interstitium, and tubular lumens of the renal cortex reached a peak on day 10. GM6, GM10, GM15, and GM30 show days 6, 10, 15, and 30 after the first administration in the gentamicin group, respectively. *P Ͻ 0.01; #P Ͻ 0.05. Xie et al: OPN in tubular necrosis and recovery 969 experimental rats. In this study, the up-regulated expres- ogy have been insufficiently understood. In most studies, sion of OPN mRNA and protein was shown distinctly a correlation existed between the magnitude of OPN by Northern blot analysis, in situ hybridization, and im- expression in areas of tubulointerstitial injury and infil- munohistochemistry in the renal cortex from days 6 to tration with macrophages, suggesting that OPN could 30 after the first gentamicin administration. The OPN participate directly in renal macrophage infiltration in mRNA levels of the whole kidney by Northern blot vivo [35–37]. In OPN knockout mice, it was reported analysis on day 30 in the gentamicin group were almost that the macrophage influx after unilateral ureteral ob- the same as the control, possibly because the OPN struction was significantly reduced as compared with con- mRNA signals in the medulla almost returned to normal trol mice (abstract; Ophascharoensuk et al, J Am Soc and OPN expression was restricted only in the focal area Nephrol 8:A481, 1997). Further evidence that OPN may of the cortex on day 30. modulate macrophage function comes from wound-heal- Studies of histologic localization in the normal kidney ing experiments done by Liaw et al, in which the in- and sources of OPN have given conflicting results [14]. creased cell debris in the healed wounds in OPN-defi- In an early immunohistochemical study in the rat, OPN cient mice was thought to represent a reduction of was found only in the proximal tubules [23]. Because macrophage activity [38]. However, there are some op- immunostaining was observed in components of the vac- posing reports. For example, in the renal cortex of strep- uolar-lysosomal system, it was concluded that OPN was tozotocin-induced diabetic rats, increased levels of OPN absorbed from the tubule fluid [14]. Subsequent studies were not associated with accumulation of monocyte/mac- using both in situ hybridization and immunohistochemis- rophages as identified by a cell type-specific monoclonal try demonstrated OPN mRNA and protein expression antibody ED1 [39]. In human crescentic glomerulone- in only the distal tubular cells of the normal rat kidney phritis, the majority of strongly OPN-positive cells were [24], whereas other investigators found that OPN was monocyte/macrophages in a number of crescents, but expressed in the descending thin limb of the loop of interstitial monocyte/macrophages did not express OPN Henle and in the papillary and pelvic epithelium of the [40]. In our current study, the location of OPN expression renal fornix in the normal rat kidney [25]. Under renal had no close relationship with the site of monocyte/mac- disease conditions, the OPN expression of some models, rophage accumulation in most areas. Only in some areas, such as renal stone formation, was found mainly in the the location of OPN expression was almost coincident distal tubular cells [22], but that of other models, such with the site of monocyte/macrophage infiltration in the as ischemia-induced acute renal failure, was noted not gentamicin group, as shown in Figure 9. These contrasting only in the distal tubular cells, but also in the proximal results suggest that OPN may work as a chemotactic pro- tubular cells [5]. These different results may be mainly tein in certain areas and time phases, and additionally due to the different causes and regions of damage in may play other roles as a multifunctional chemokine in different disease models. On the other hand, they may other areas and time phases in renal injury and its re- also be due to different investigators having different covery process. opinions on the same renal tubule segment. In fact, it is Recently, OPN has emerged as an important growth very difficult to distinguish proximal tubules clearly from promoter of vascular smooth muscle cells and cardiac the loop of Henle, distal tubules, and collecting ducts in fibroblasts [41, 42]. In the kidney, OPN has been shown some pathological conditions, as shown in Figure 1. In to play a role in the regeneration of renal proximal tu- this study, we confirmed the exact localization and source bule cells after acute severe hypoxic or ischemic injury of OPN in renal cortical tubules using serial sections and [43, 44]. In addition, OPN is up-regulated during the double staining of OPN mRNA and megalin, as well as repair process of glomerular injury in the anti-Thy-1 double staining of the OPN protein and megalin. Megalin model of glomerulonephritis (abstract; Prols et al, JAm served as a renal proximal tubule marker because it is Soc Nephrol 7:A2585, 1996). Hypoxia stimulated the re- thought to exist only in proximal tubular and glomerular lease of OPN, and anti-OPN antibodies completely pre- epithelial cells, but not in distal tubules, the loop of vented hypoxia-induced proliferation in cultured mesan- Henle, or collecting ducts of the kidney [26–31]. Because gial cells and LLC-PK1 tubular epithelial cells (abstract; OPN mRNA and OPN protein were expressed in the Sahai et al, J Am Soc Nephrol 8:A1983, 1997, and JAm same region as renal cortical tubules, as shown in Fig- Soc Nephrol 9:A2280, 1998). These results suggest that ure 5, it was clear that the OPN protein of renal cortical OPN may mediate the proliferation of mesangial and tubules resulted from renal cortex tubular epithelial cell LLC-PK1 cells induced by hypoxia [45]. In our study, production, but not from resorption of renal tubular OPN mRNA and protein were only expressed in the fluid. Several in vitro studies have demonstrated that proliferating distal tubules of the rat kidney, but not cultured renal epithelial cells have the ability to secrete in necrotic proximal tubules in the gentamicin-induced OPN [32–34]. ATN period on day 6 after the first of administration, The roles of OPN in renal pathology and pathophysiol- whereas during the recovery process on days 10, 15, Fig. 9. Relationship between ED1 and OPN in gentamicin-induced ATN and recovery. Double staining for OPN (brown) and ED1 (red) show the location of OPN expression had no distinct relationship with the site of ED1-positive cell accumulation in some areas of the cortex on day 10 (A) and day 15 (B and C). However, in some areas of the cortex on day 30 (D), the location of OPN expression was almost coincident with the site of ED1- positive cell infiltration in the gentamicin group. Original magnification ϫ100.

᭤ Fig. 10. Demonstration of proliferating cell nuclear antigen (PCNA)-positive cells in cortical tubules in gentamicin-induced ATN and recovery. (A) There are very few PCNA-positive cells in normal cortical tubules. (B) The PCNA-positive cells are present in tubules having no necrosis on day 6 after the first gentamicin administration (a lot of desquamated epithelial cell debris in the PCNA-positive tubular lumens can be seen). (C) There are many PCNA-positive cells in the cortical tubules on day 10. The new regenerative cells show flat cellular nuclei. (D) The PCNA-positive cellular nuclei become larger on day 15. (E) PCNA-positive cells are focally present in the cortical tubules and interstitium on day 30. Original magnification ϫ100.

Fig. 12. Colocalization of PCNA and OPN in gentamicin-induced ATN and recovery. Double staining for OPN (brown) and PCNA (red) shows that the PCNA-positive cells are mainly present in the tubular epithelium with up-regulated expression of OPN on days 6 (A), 10 (B), 15 (C), and 30 (D) after the first gentamicin administration, although not all cells in the OPN-positive area are PCNA-positive, especially on day 15 (C). Original magnification ϫ100. Fig. 11. Quantitative analysis of cortical tubular PCNA-positive cells in gentamicin-induced ATN and recovery. The number of cortical tubular PCNA-positive cells reached a peak on day 10, but there was no significant difference compared with that on day 15. GM6, GM10, GM15, and GM30 show days 6, 10, 15, and 30 after the first administration in the gentamicin group, respectively. *P Ͻ 0.01. NS, no significant difference. Fig. 13. Expression of CD44 in gentamicin-induced ATN and recovery. Immunohistochemistry for CD44 shows CD44 staining is negative in the cortical tubules in the control group (A), but CD44-positive staining is markedly present in the cortical tubular epithelium and infiltrating cells in the cortical interstitium and tubular lumens on days 6 (B), 10 (C), 15 (D), and 30 (E) after the first administration in the gentamicin group. Original magnification ϫ100. Xie et al: OPN in tubular necrosis and recovery 973 and 30, OPN was found markedly in the regenerative 43rd Annual Meeting of the Japanese Society of Nephrology (Nagoya, Japan, 2000). We thank Drs. Robert A. Orlando and Marilyn G. Far- proximal and distal tubules of the renal cortex. Double quhar for kindly providing the antimegalin antibody. staining showed that PCNA-positive cells were mainly present in the tubular epithelium with up-regulated ex- Reprint requests to Shinichi Nishi, M.D., Department of Medicine pression of OPN at any time during the necrotic period (II), Niigata University School of Medicine, 1-757 Asahimachi-dori, Nii- gata 951-8510, Japan. on day 6 or the recovery period on days 10, 15, and 30, E-mail: [email protected] and at any site in the proximal or the distal tubules. Correlation analysis showed that the number of PCNA- REFERENCES positive tubular epithelial cells was associated signifi- 1. Prince CW, Oosawa T, Butler WT, et al: Isolation, characteriza- cantly with the expression of OPN. These results suggest tion, and biosynthesis of a phosphorylated glycoprotein from rat that OPN may be related to the proliferation and regen- bone. J Biol Chem 262:2900–2907, 1987 eration of renal tubular epithelial cells following ATN 2. 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