Glomerular Injury and Tubular Loss in Adriamycin Nephrosis

J Am Soc Nephrol 12: 1391–1400, 2001 Glomerular Injury and Tubular Loss in Adriamycin Nephrosis

BASIT JAVAID, JEAN L. OLSON, and TIMOTHY W. MEYER Departments of Medicine, VA Palo Alto Health Care System and Stanford University, Palo Alto; and Department of Pathology, University of California, San Francisco, California.

Abstract. Glomerular injury manifested by sustained proteinuria NoRx, 85 Ϯ 18%; control, 1 Ϯ 2%). Glomeruli without tubules usually leads to tubule injury and reduction of the GFR. The were not, however, globally sclerosed. Disruption of the glomer- current study explored the link between these processes in rats ular tubular junction was associated with the presence of amor- with adriamycin nephrosis. One group of nephrotic rats received phous material separating damaged tubule cells from the base- a vasopressin V2 receptor blocker (V2X) from 4 to 16 wk after ment membrane. Serial sections revealed that this material spread injection of adriamycin, whereas a second group received no from extensive areas of adhesion between the glomerular tuft and treatment (NoRx). V2 receptor blockade increased urine volume capsule to invest the tubular neck. Reduction of the GFR was without affecting protein excretion. At 16 wk, both groups of strongly correlated with the fraction of glomeruli not connected to nephrotic rats exhibited a marked reduction in GFR in comparison normal tubules (r2 ϭ 0.82; P Ͻ 0.0001). V2 receptor blockade did with normal controls (V2X, 0.22 Ϯ 0.19 ml/min; NoRx, 0.20 Ϯ not preserve renal function or structure. These findings suggest 0.11 ml/min; control, 1.23 Ϯ 0.11 ml/min). Morphologic studies that local extension of glomerular injury to destroy the tubule neck revealed that the majority of glomeruli in nephrotic rats were no is an important cause of loss of renal function in adriamycin longer connected to normal tubule segments (V2X, 81 Ϯ 21%; nephrosis.

In human renal disease, sustained heavy proteinuria is usually anesthesia provided by thiopental (50 mg/kg intraperitoneally). Six accompanied by rapid loss of renal function. Rats that are given additional rats received normal saline by the same route and served as adriamycin provide an experimental model of the association normal controls. After 4 wk, animals that were given adriamycin were between proteinuria and loss of function, as described by Bertani divided into two groups, matched for their mean body weight and et al. (1,2) and Okuda et al. (3). These animals develop heavy urinary protein-to-creatinine ratio. One group then received the V2 proteinuria within a few weeks after adriamycin administration. receptor blocker SR121463 (V2X), whereas the other group received no treatment (NoRx). Rats in the current study received SR121463B, Protein excretion rates remain high thereafter, and the GFR de- which is a close congener of SR121463A, a nonpeptide V2 receptor clines gradually. Previous studies showed that the appearance of blocker that has been shown to be active in several in vitro and in vivo proteinuria is associated with injury to the glomerular visceral models (7). All rats had free access to water and powdered rat chow. epithelial cells and that the subsequent decline in GFR is associ- SR121463 was administered by mixing it in the chow. On the basis of ated with both glomerular sclerosis and widespread tubulointer- preliminary studies in control rats, the initial mix provided 1.5 mg of stitial injury (1–6). The current study examined further the struc- SR121463 in 20 g of chow. The mix was changed to 2.0 mg of tural basis for the decline in GFR that accompanies sustained SR121463 in 20 g of chow after 4 wk of treatment and then to 2.5 mg proteinuria in adriamycin nephrosis. Its major aim was to explore of SR 121463 per 20 g of chow after 8 wk of treatment. These the link between proteinuric glomerular injury and subsequent adjustments were made to maintain the urine volume of treated tubular atrophy. The serial section technique was used to deter- nephrotic rats two to three times greater than that of untreated ne- mine whether the decline in GFR was associated with the loss of phrotic rats. The duration of SR121463 treatment was 12 wk, so that glomerular connections to normal tubules. A recently developed the study lasted a total of 16 wk after adriamycin or saline injection. vasopressin V2 receptor blocker was used to test whether increas- Urine was collected every 4 wk, except that no collection was made at 16 wk in two rats in each nephrotic group. Urine protein was ing urine flow rate and reducing urine concentration could pre- measured by the Coomassie blue method, and urine creatinine was serve tubule structure and limit loss of function. measured by use of a Beckman Creatinine 2 analyzer (Palo Alto, CA). Systolic BP was measured by tail cuff at 10 and 14 wk. One rat in the Materials and Methods untreated nephrotic group died before the study was completed. Eighteen male Munich Wistar rats weighing 250 to 290 g received At 16 wk, GFR was measured under anesthesia provided by inac- a single dose of adriamycin (3 mg/kg) by tail-vein infusion under tin. A dose of 100 mg/kg intraperitoneally was given to normal rats, and a reduced dose of 45 mg/kg intraperitoneally was given to nephrotic rats. A PE-50 tubing catheter was inserted in the left femoral Received September 12, 2000. Accepted January 10, 2001. artery to record arterial pressure and to obtain arterial blood samples. Correspondence to Dr. Timothy W. Meyer, Palo Alto VA Medical Center, After tracheotomy, catheters were inserted in the jugular veins for Nephrology 111R, 3801 Miranda Avenue, Palo Alto, CA 94303. Phone: 650-852- infusion of rat plasma, saline, and radiolabeled inulin, and a PE-10 5000 x3314; Fax: 650-849-0213; E-mail: [email protected] catheter was installed into the left ureter for collection of urine. 1046-6673/1207-1391 Plasma was infused in an amount equal to 1% body weight over 40 to Journal of the American Society of Nephrology 45 min, followed by reduction of the infusion rate to 0.4 ml/h for the Copyright © 2001 by the American Society of Nephrology duration of the study. Saline was infused at 2.4 ml/h throughout the 1392 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 1391–1400, 2001 study. After 60 min, tritiated methoxy-inulin was added to the saline remnants of glomerular capillaries but to exclude other parts of the to achieve an infusion rate of approximately 9 ␮Ci/h after a loading adhesion. The fractional volume of cortical components was deter- dose of approximately 4 ␮Ci, and clearance measurements were mined by point counting with the use of a 6 ϫ 6-point eyepiece carried out over two to three 30-min periods. No GFR value was reticule grid and a magnification of ϫ400. In each case, a minimum obtained in one rat. of 500 points in four sections spaced 120 ␮m were counted. The After GFR measurements, kidneys were fixed by retrograde aortic tubular cell category included points falling on both proximal and perfusion with 2.5% paraformaldehyde and 0.1% glutaraldehyde in distal tubular cells as well as on cells whose original type was difficult 0.1 M cacodylate buffer. Kidneys were sliced perpendicular to the to ascertain because of tubular injury. Amorphous material in conti- long axis at approximately 2-mm intervals. Slices from the midportion nuity with tuft adhesions was counted as part of the interstitium when of the kidney were fixed further in 10% formalin for embedding in it was surrounding the tubular cells and as Bowman’s capsule when it paraffin and in 1.25% glutaraldehyde in 0.1 M cacodylate for embed- was found surrounding the glomerulus. Volume fractions were calcu- ding in Epon. Periodic acid-Schiff–stained and Alcian blue–stained lated as the number of points falling on each component divided by sections were prepared from paraffin-embedded slices. For each kid- the number of points evaluated. A glomerulus with representative ney, a small block of tissue extending from the capsule into the injury was selected for examination by electron microscopy. This was medulla was embedded in Epon. These blocks were sectioned serially accomplished by saving the alternate 3-␮ sections between those at 3-␮ intervals so that glomeruli at all levels of the cortex were stained with Epon in one case. The section after the stained section included in the sections. Every other serial section was mounted and that contained the tubular junction of the selected glomerulus was then stained with toluidine blue so that the tissue could be examined at 6-␮ trimmed to isolate the area of interest and reembedded for thin intervals. Sixty-five to 100 consecutive sections were examined in sectioning. each rat, to yield 26 to 30 glomeruli contained completely within the Studies were carried out in a separate group of three rats to examine tissue sections. Every fourth mounted section was photographed, and renal injury at an earlier time point in the course of nephrosis. These a series of prints were prepared to use as maps of the serial sections. rats received a single dose of adriamycin and no other treatment. These maps served as a guide to identification of the individual Proteinuria was measured at 4 wk, and GFR measurements and glomeruli, as described elsewhere (8). Glomeruli were classified as morphologic studies were performed at 6 wk by use of the methods being either connected to a normal proximal tubule, connected to an described above. atrophic proximal tubule, or without any tubular connection. No correction was made for the effect of glomerular volume on the Statistical Analyses relative prevalence of complete glomeruli of different types, because ANOVA was used to assess the significance of differences between preliminary calculation indicated that corrected values would be groups. The paired t test was used to assess the significance of within 0.9 to 1.1 times observed values. The proximal tubular segment differences within each group. Values are expressed as the mean Ϯ connected to the glomerulus was considered atrophic when there was SD throughout. P Ͻ 0.05 was considered to denote statistical thinning or disruption of tubular cells accompanied by narrowing or significance. loss of the tubular lumen at the glomerular-tubular junction. The volumes of the glomerular tuft and of Bowman’s capsule was calcu- lated from the areas of their midsections, by use of the maximal planar Results area method (9). Tuft adhesions to the capsule were frequent, and in Values for the urine protein to creatinine ratio and for urine these areas the line delimiting the tuft was drawn to include all visible volume are depicted in Figure 1. As expected, rats that were

Figure 1. Metabolic cage studies. (A) Urine protein-to-creatinine ratio. Values in rats that received an injection of adriamycin and received the V2 antagonist (F) and in rats that received an injection of adriamycin and received no treatment (NoRx; ⅜) were increased to a similar degree above control (□). *, P Ͻ 0.05 versus control. (B) Twenty-four-h urine volume. V2 receptor blockade (F) increased urine volume above values seen in rats that received an injection of adriamycin and received no treatment (⅜) and in control rats (□). *, P Ͻ 0.05 versus control; †, P Ͻ 0.05 versus NoRx. J Am Soc Nephrol 12: 1391–1400, 2001 Tubule Loss in Adriamycin Nephrosis 1393 given adriamycin developed heavy proteinuria, reflected by a similar reduction of the hematocrit in both groups of nephrotic marked increase in the urine protein-to-creatinine ratio. Treat- rats (V2X, 33 Ϯ 7%; NoRx, 31 Ϯ 4%; control, 45 Ϯ 2%). ment with a V2 receptor antagonist beginning at 4 wk led to Loss of renal function in rats with sustained nephrosis was increased urine volume without affecting the urine protein-to- associated with widespread structural abnormalities. Consis- creatinine ratio. The protein-to-creatinine ratio was used to tent with the finding of functional studies, no difference was assess proteinuria because of concern that differences in urine noted between untreated nephrotic animals and animals that volume might influence the completeness of urine collection in received the V2 receptor antagonist. Interstitial volume was metabolic cages and thereby affect measured 24-h protein increased, and the number of tubule profiles that appeared to be excretion. Values for protein excretion, however, proved to normal was markedly reduced. The widened interstitium as have only a slightly different pattern than the protein-to-creat- noted by separation of the tubules from one another was due to inine ratios. Values in rats treated with the V2 receptor antag- both fibrosis and edema. In many cases, tubule profiles exhib- onist were slightly greater than those in untreated rats, but the ited atrophic changes characterized by thickening of the base- difference was statistically significant only at 16 wk (V2X ment membrane, loss of brush border, and attenuation of cell versus NoRx: 8 wk, 599 Ϯ 140 versus 488 Ϯ 213 mg/d; 12 wk, cytoplasm. In some cases, the atrophy was so severe as to have 586 Ϯ 143 versus 574 Ϯ 163 mg/d; 16 wk, 652 Ϯ 124 versus resulted in the loss of the lumina. In other cases, tubule lumina 474 Ϯ 214 mg/d). Values obtained in control rats ranged from were filled by casts. There were occasional lymphoid aggre- 27 Ϯ 6to45Ϯ 15 mg/d during the same interval. gates. Glomeruli showed severe injury, manifested by occlu- Nephrotic rats exhibited impaired growth, but there was no sion of capillary lumina by hyalinosis or foam cells and by difference between the treated and untreated groups at any time extensive loss of podocytes. Such loss of podocytes was asso- point. Final body weight averaged 281 Ϯ 41 g in nephrotic rats ciated with adhesion of the tuft to Bowman’s capsule, accom- that received the V2 receptor antagonist, 264 Ϯ 41gin panied by the accumulation of amorphous material between the untreated nephrotic rats, and 375 Ϯ 15 g in normal rats. tuft and the capsular basement membrane, the structure of Systolic BP measured by the tail-cuff method was not different which could often not be delineated clearly (Figure 2). The among the three groups at 10 wk (V2X, 140 Ϯ 12 mmHg; appearance of the amorphous material on toluidine blue– NoRx, 147 Ϯ 12 mmHg; control, 144 Ϯ 11 mmHg) or at 14 wk stained sections varied from near lucent to approximately the (V2X, 146 Ϯ 14 mmHg; NoRx, 152 Ϯ 17 mmHg; control, 138 density of basement membrane, and in some areas it exhibited Ϯ 8 mmHg). delicate stranding. These strands stained lightly with periodic Studies performed at 16 wk showed loss of renal function in acid-Schiff. Alcian blue also stained the amorphous material. nephrotic rats. Single-kidney GFR values in treated and un- Parietal epithelial cells reflected back onto the tuft at the edges treated nephrotic rats averaged only 0.22 Ϯ 0.19 and 0.20 Ϯ of adhesions, and the amorphous material extended circumfer- 0.11 ml/min, respectively. These values were not different entially beyond the adherent segments, separating the parietal from each other but were markedly lower than the single- epithelium from the capsular basement membrane. In many kidney GFR of 1.23 Ϯ 0.11 ml/min in normal control rats. areas, a layer of small, spindled cells rimmed the outer aspect Mean arterial pressure under anesthesia in treated rats (139 Ϯ of Bowman’s capsule. 23 mmHg) but not in untreated rats (131 Ϯ 22 mmHg) was Global glomerular sclerosis rarely was seen. Serial sections significantly greater than that in normal rats (115 Ϯ 11 showed, however, that many glomeruli were no longer con- mmHg); the values in treated and untreated rats were not nected to normal tubules. In most cases, atubular glomeruli significantly different. Renal insufficiency was associated with exhibited extensive adhesion of the tuft to the capsule with

Figure 2. Representative glomerular injury. (A) Glomerular profile showing extensive adhesion of the tuft to capsule with paraglomerular accumulation of amorphous material (*). Hypertrophied parietal epithelial cells reflect onto the tuft at the limits of the adhesion (arrowheads). (B) Glomerular profile showing Alcian blue staining of amorphous material in the areas of tuft-to-capsule adhesion and disrupted proximal tubule (asterisks). Magnification, ϫ240 (toluidine blue in A; Alcian blue in B). 1394 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 1391–1400, 2001

accumulation of amorphous material as described above (Fig- ure 3A). A smaller number of atubular glomeruli exhibited reduction of tuft volume with less extensive capsular adhesions (Figure 3B). Occasionally, the shrunken tuft occupied a small portion of Bowman’s space, giving the glomerulus the appearance of a cyst containing a tuft remnant (Figure 3C). In addition to atubular glomeruli, there were many glomeruli that were connected to atrophic tubule segments (Figure 4). In these cases, the tubule neck was almost invariably surrounded by amorphous material, which was in continuity with the site of a tuft to capsule adhesion. Where this material extended along the tubule, tubule epithelial cells either were missing entirely or exhibited damage characterized by attenuation, the presence of intracellular vacuoles, and the loss of brush border. The site of the tubular junction sometimes was marked only by individual cells, which often had a stellate appearance, floating in amorphous material that seemed to occupy most of the space delimited by the former tubule basement membrane (Figure 4A). Injury extended for a variable distance away from the glomeruli along the tubules. In some cases, accumulation of amorphous material and tubule cell injury extended only a short length or even extended only part way around the tubule at its junction with the glomerulus (Figure 4B). In other cases, the damage extended further. Remarkably, in some of these cases, tubule structure returned nearly to normal at a considerable distance from the glomerulus (Figure 5), whereas in other cases, the tubule could be traced only as an atrophic structure without any lumen. Examination of sections from a subset of 14 individual nephrons indicated that in approximately half of the cases in which the tubule was atrophic at the glomerular junction, more normal structure was preserved at some distance from the glomerulus. Electron microscopy was used to examine a glomerulus that showed representative injury. On light microscopy, the glomerular tuft proximate to the tubule junction was embedded in amorphous material that extended along the tubule neck. Elec- tron microscopy revealed that this material was variably com- posed of delicate strands of basement membrane–like material, cell debris, and granular material resembling proteoglycan and was in some areas devoid of any substance (Figure 6). Similar material dissected down the tubule neck so that normal tubule cell attachment to the basement membrane was disrupted. Injury to tubule cells in contact with the amorphous material was manifested by loss of the brush border, intracellular edema, autophagosomes, and attenuation of the cell cytoplasm. An abrupt transition was noted with attachment of a damaged tubule cell to a cell without apparent injury. The amorphous material around the tubule neck contained lymphocytes, mac- rophages, and occasional spindled cells. Spindled cells that Figure 3. Examples of atubular glomeruli. (A) Typical appearance of showed features suggestive of myofibroblasts also were the midsection of an atubular glomerulus. There is extensive adhesion present in the interstitium along the outer aspect of the tubule of the tuft to the capsule with paraglomerular accumulation of amor- basement membrane and Bowman’s capsule. phous material, but the tuft is not collapsed. (B) Less-common appearance of an atubular glomerulus. The tuft is smaller and has Quantitative measures of the extent of structural abnormal- shrunken capillary loops, whereas the area of adhesion is less exten- ities are summarized in Table 1. As expected, almost all sive. (C) An unusual atubular glomerulus with a small tuft remnant glomeruli in control rats were connected to normal tubules. within an enlarged Bowman’s space, giving a cystic appearance. There were a few very small atubular glomeruli and no glo- Magnifications: ϫ290 in A and; ϫ240 in C (toluidine blue for all). meruli connected to atrophic tubule segments. In contrast, only J Am Soc Nephrol 12: 1391–1400, 2001 Tubule Loss in Adriamycin Nephrosis 1395

Figure 4. Glomeruli attached to atrophic proximal tubule segments. (A) Advanced atrophy. The tubule is reduced to islands of cells (arrowheads) surrounded by amorphous material that is continuous with paraglomerular amorphous material. There is circumferential adhesion of the tuft to the capsule. (B) Early atrophy. The amorphous material extends only a short distance along the tubule, which retains a lumen. Tubule cells are flattened where they are in contact with the amorphous material (arrowhead) but have a normal appearance immediately downstream. Magnification, ϫ290 (toluidine blue). the minority of glomeruli in rats that remained nephrotic for tubule cell volume, and fractional tubule cell volume was, in Ͼ3 mo remained connected to normal proximal tubule seg- turn, correlated with the portion of glomeruli attached to nor- ments. In these animals, approximately half of the glomeruli mal tubule segments. Fractional interstitial volume, as mea- were no longer connected to tubules, and approximately one sured by the current technique, was somewhat less strongly third were connected to atrophic tubule segments. Treatment correlated with the portion of glomeruli attached to normal with the V2 receptor antagonist, which failed to limit reduction tubule segments and was only weakly correlated with the GFR. of the GFR, also failed to limit reduction in the number of A small group of animals were killed at 6 wk after infusion glomeruli connected to normal tubules. The average volume of of adriamycin, to confirm that the tubule loss described above glomeruli connected to normal-appearing tubule segments was develops only with sustained nephrosis. At 4 wk after infusion not significantly greater in nephrotic rats than that in control of adriamycin, these animals exhibited a urine protein-to- rats, but the range of volume values encountered in nephrotic creatinine ratio of 65 Ϯ 6, which was similar to that observed rats was larger than that in normal rats. Within the nephrotic in the groups of rats studied for 16 wk. In accord with reports rats, connection of the glomerulus to an atrophic tubule seg- elsewhere, kidneys perfused at 6 wk showed only mild inter- ment was not associated with a significant change in glomer- stitial widening and relatively little tubule injury. Glomeruli ular volume. Atubular glomeruli in nephrotic rats were reduced showed epithelial cell injury with bleb formation. There were in size but were not as small as the few atubular glomeruli in focal, segmental adhesions. The extent of these adhesions, normal rats. Of note, neither atubular glomeruli nor glomeruli however, and of the accumulation of amorphous material be- connected to atrophic tubules were distinguished by marked tween the tuft and capsule was much less than that at 16 wk. expansion of Bowman’s space reflected by a reduction in the Examination of serial sections revealed that the adhesions tuft-to-capsule volume ratio. A few glomeruli did have a cystic developed in all sectors of the tuft and had no tendency to be appearance, as described above and illustrated in Figure 3C. In localized to the area of the vascular pole. Examination of 30 these glomeruli, the tuft-to-capsule volume ratio was as low as glomeruli in each of three rats disclosed no glomeruli that were 0.05. The number of glomeruli with cystic appearance and very connected to atrophic tubule segments or were atubular. low tuft-to-volume ratios, however, was small. Average values for the tuft-to-capsule volume ratio were only modestly re- Discussion duced in atubular glomeruli, and this reduction was significant Previous studies have shown that reduction of the GFR in only in the untreated group. chronic adriamycin nephrosis is associated with development Correlations between structural parameters and renal func- of both glomerular sclerosis and tubulointerstitial injury (1–6). tion are summarized in Table 2. Overall, the GFR was nega- The major finding of the current study is that reduction of the tively correlated with various features of injury, and these GFR is closely correlated with the extent to which glomeruli features of injury were positively correlated with each other. are no longer connected to normal tubules. Most glomeruli The strongest correlation was that of the GFR with the portion examined in the current study exhibited injury characterized by of glomeruli attached to normal tubule segments, as illustrated tuft-to-capsule adhesions, epithelial cell injury, and segmental in Figure 7. GFR also was strongly correlated with fractional occlusion of capillary loops. However, glomerular volume 1396 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 1391–1400, 2001

often was only moderately reduced, and many capillary loops remained patent even after the connection to the proximal tubule had been obliterated. These findings suggests that nephron function often ceased because of tubule loss before glomerular injury was far enough advanced to preclude filtration. The question of how tubules are lost remains unsettled. In adriamycin nephrosis, tubule injury generally is considered to be secondary to glomerular injury and proteinuria, although a toxic effect of adriamycin on tubule cells has not been completely excluded. Obstruction by casts is one mechanism by which proteinuria could injure tubules, as was originally noted by Bertani et al. (1,2). The precise site at which casts begin to form and the conditions governing their formation are not known, but casts have been localized mostly to the distal nephron in adriamycin nephrosis (1,2). We therefore considered it possible that reducing urine concentration with a V2 receptor antagonist would limit cast formation and consequent tubule injury in nephrotic rats. Results showed, however, that V2 receptor antagonism in a dose sufficient to increase urine flow approximately threefold had no effect on the course of injury. It should be noted that studies elsewhere have suggested other mechanisms by which V2 receptor antagonism could lessen renal injury. In particular, Okada et al. (10) found that V2 receptor antagonism lessened proteinuria in rats given adriamycin, a finding that was not confirmed in the current study. Other workers have found that a forced increase in water intake reduces both glomerular and tubulointerstitial injury in rats subjected to renal ablation (11,12). An increase of water intake and urine excretion of approximately the same magni- tude induced by V2 blockade in nephrotic rats had no benefi- cial effect in the current study. There are a number of other mechanisms whereby proteinuric glomerular injury could cause tubule injury. The most frequently proposed is that tubule uptake of filtered proteins or protein-bound substances injures proximal tubules (13–17). Recent studies have shown that tubule injury is as extensive in analbuminemic rats as in normal rats given adriamycin, although the analbuminemic rats excrete less total protein and no albumin (18). These findings suggest that if filtered protein causes tubular injury, albumin cannot be the major protein involved. A second hypothesis is that diminished perfusion downstream from injured glomeruli leads to tubule ischemia (19,20). Morphologic studies are consistent with the hypothesis that ischemic injury leads to tubule loss in renal artery disease (21). Clear evidence of tubule cell ischemia in proteinuric renal disease, however, has not been obtained. Recently, Kriz et al. Figure 5. Resumption of normal tubule structure at a distance from an (22,23) proposed an additional mechanism whereby protein- injured glomerulus. (A) Tubule cells are atrophic and separated from uric glomerular injury can lead to tubule loss. They found that the basement membrane by amorphous, nearly lucent material (*) at visceral epithelial cell injury and adhesion of the tuft to capsule the level of the glomerular tubular junction. (B) At 30 ␮ from A, the is followed by accumulation of amorphous material in a para- tubule has a lumen but is again atrophic and separated from the basement membrane by amorphous material (*). (C) At 48 ␮ from A, glomerular space that sometimes extends along the capsule to amorphous material (*) is observed along one aspect of the tubule, and invest the neck of the tubule. Tubule degeneration was ob- tubule cells are atrophic where this material separates them from the served where the material extended down the outside of the basement membrane. As the tubule is followed in a distal direction, tubule, separating the tubule cells from the tubule basement however, this material is no longer seen, and tubule structure returns membrane. These changes were demonstrated most exten- to normal (ϩ). Magnification, ϫ290 (toluidine blue). sively in Fawn hooded and Milan normotensive rats but were J Am Soc Nephrol 12: 1391–1400, 2001 Tubule Loss in Adriamycin Nephrosis 1397

Figure 6. Electron micrograph showing material accumulated beneath the glomerular parietal epithelium (P) in a nephrotic rat. The material includes delicate strands of basement membrane–like substance (arrowhead), cell debris, and small granules with the appearance of proteoglycans (arrow). The underlying basement membrane is grossly thickened and laminated. Magnification, ϫ5000. also observed in several other rodent models and in a number circumferentially along the capsule in a paraglomerular space of human cases (23). beneath a layer of fibroblast-like cells. Tubule injury was The findings of the current study seem to be consistent with present where the amorphous material reached the tubule neck the hypothesis of Kriz et al. (23). Small tuft-to-capsule adhe- and dissected between the tubule cells and basement mem- sions were observed early in the course of adriamycin nephro- brane. Indeed, the lesions seen in the current study looked very sis. Subsequently, these adhesions enlarged and were associ- similar to those illustrated by Kriz et al. (23). There were some ated with accumulation of amorphous material that spread differences, which may have been accounted for in part by

Table 1. Morphometric findings at 16 wka

Normal Nephrotic, No Nephrotic, V2 Finding Control Treatment Blockade

Kidney weight (g) 1.51 Ϯ 0.14 1.28 Ϯ 0.21 1.26 Ϯ 0.31 Prevalence of glomeruli (%) attached to normal tubule 99 Ϯ 216Ϯ 15b 19 Ϯ 21b attached to no tubule 1 Ϯ 249Ϯ 17b 58 Ϯ 20b attached to atrophic tubule 0 Ϯ 034Ϯ 12b 23 Ϯ 18b Volume of glomeruli (106 mm3) attached to normal tubule 1.64 Ϯ 0.15 1.94 Ϯ 0.72 1.80 Ϯ 0.71 attached to no tubule 0.40 Ϯ 0.16c 1.06 Ϯ 0.26b,c 1.15 Ϯ 0.23b attached to atrophic tubule — 1.50 Ϯ 0.35c 1.92 Ϯ 0.70 Vg/Vc ratio for glomeruli attached to normal tubule 0.77 Ϯ 0.03 0.83 Ϯ 0.04 0.80 Ϯ 0.17 attached to no tubule 0.70 Ϯ 0.12 0.67 Ϯ 0.11c 0.72 Ϯ 0.01 attached to atrophic tubule — 0.82 Ϯ 0.06 0.84 Ϯ 0.06 Fractional volume (%) of the interstitium 12 Ϯ 238Ϯ 8b 39 Ϯ 4b of tubular cells 56 Ϯ 533Ϯ 10b 31 Ϯ 8b

a Vg/Vc ratio, the ratio of the tuft volume to the volume enclosed by the capsule. Kidney weight is weight of left kidney. b P Ͻ 0.05 versus normal control. c P Ͻ 0.05 versus glomeruli with normal tubules in the same group. 1398 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 1391–1400, 2001

Table 2. Correlations among functional and structural the great majority of cases, glomeruli that had lost tubules did measurements not have a cystic appearance. Thus, the high prevalence of atubular glomeruli and glomeruli attached to severely atrophic Parameter r2 P tubules could be detected only by examination of serial GFR (ml/min) against sections. glomeruli with normal tubules (%) 0.82 0.0001 It should be noted that separation of degenerated epithelial fractional volume of tubular cells (%) 0.67 0.0001 cells from the tubular basement membrane by amorphous fractional volume of the interstitium (%) 0.15 0.14 material at the tubule neck is not simply a common feature of Glomeruli with normal tubules against advanced tubule injury. Atubular glomeruli were first de- fractional volume of tubular cells (%) 0.74 0.0001 scribed by Marcussen et al. in rats given lithium (24) and in fractional volume of the interstitium (%) 0.28 0.03 rats given cisplatin (25). Toxic injury in these models led to Fractional volume of tubular cells against 0.58 0.0004 tubule loss and glomerular shrinkage without the tubule neck fractional volume of the interstitium (%) lesions observed in the current study of nephrotic injury. We recently observed large numbers of atubular glomeruli and glomeruli attached to atrophic tubules in rats recovering from acute ischemic injury (26). Again, severe tubule atrophy in this setting was not associated with accumulation of amorphous material around the tubule neck, as observed in the current study of nephrosis. It also should be emphasized that the amorphous material investing the tubule neck in nephrotic rats was continuous with sites of glomerular tuft adhesion. To- gether, these observations suggest that most tubule loss in the current study was caused by the local extension of glomerular injury rather than by the toxic action of filtered macromole- cules, by ischemia, or by cast obstruction at some point further along the nephron. We presume that one or more of these latter processes were responsible for tubule injury in the smaller portion of nephrons in which tubule loss was not associated with prominent tuft adhesion and paraglomerular accumulation of amorphous material. It is, of course, likely that some nephrons were subject to more than one form of injury, and it is possible that amorphous material accumulated around some glomeruli after the attached tubule had been damaged by another process. Figure 7. Correlation of the GFR with the fraction of glomeruli Important questions concerning the paraglomerular lesion attached to normal-appearing proximal tubule segments. Values are described above remain to be addressed. One is the source of from the nephrotic rats that received the V2 receptor blocker (F)orno the amorphous material that seems to spread outward from treatment (⅜). Regression: y ϭ 0.008x ϩ 0.06; r2 ϭ 0.82; P Ͻ sites of adhesion. Kriz et al. (23) hypothesized that this mate- 0.0001. rial accumulates as a result of misdirected filtration from capillary loops that are denuded of epithelial cells and adherent to the capsule. It also seems possible that contact of denuded differences in the animal models studied. In Fawn hooded rats, capillaries with the capsule evokes a reaction by parietal epi- Kriz et al. (22) found that adhesions began most often in the thelial cells and other surrounding cell types, which then are area of the vascular pole. It has been speculated that this stimulated to produce a variety of growth factors and matrix pattern may be typical of injury of hemodynamic origin. In any constituents (27–29). The Alcian blue staining of paraglomeru- case, spread of injury from the vascular pole in the Fawn lar material suggests that it could contain acid mucous sub- hooded rat usually resulted in global collapse of the tuft before stances such as chondroitin sulfate and hyaluronic acid. Further the tubule junction was obliterated. Marked dilation of Bow- studies clearly are needed to define better its composition. man’s space was thought to occur in the lesser number of Further studies also are needed to elucidate how penetration of glomeruli in which the tubule was lost before tuft was de- the amorphous material into the paratubular space injures tu- stroyed, so that the primary tubule loss was reflected by the bule cells. Mechanical compression of the tubule seems not to appearance of cystic glomeruli. In adriamycin nephrosis, we be required, because injury is observed where the material found that tuft-to-capsule adhesions were not localized primar- comes in contact with only part of the tubule circumference. ily around the vascular pole. As the adhesions and the associ- One possibility is that the material disrupts contacts between ated capsular lesions spread, tubule loss often preceded col- the tubule epithelial cells and underlying basement membrane, lapse of the entire tuft, which suggests that tubule neck injury which are essential for maintenance of normal cell structure was a major contributor to the reduction in GFR. Moreover, in (30). J Am Soc Nephrol 12: 1391–1400, 2001 Tubule Loss in Adriamycin Nephrosis 1399

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