Abstract
Abstract. Five/six nephrectomy induces systemic and glomerular hypertension, glomerulosclerosis, proteinuria, and tubulointerstitial fibrosis. Polysulfate pentosan (PPS) decreases mesangial proliferation and extracellular matrix accumulation. The aim of this study was to determine whether PPS prevents glomerular hemodynamic changes and renal damage. Micropuncture studies were performed in three groups of eight male Wistar rats. Two groups included rats with 5/6 nephrectomy—one of which was treated with PPS in drinking water (100 mg/kg body wt) and the second of which received normal drinking water—and the third group consisted of normal rats that served as controls. Five/six nephrectomy produced systemic hypertension, a 50% reduction in GFR, and a 67% increase in single-nephron GFR due to elevated glomerular pressure and single-nephron plasma flow as well as proteinuria. Hypertension persisted in PPS-treated animals. Despite a similar reduction in GFR, PPS prevented the rise in single-nephron GFR, glomerular capillary hydrostatic pressure, and proteinuria. By morphometry, glomerular volume was increased by 46% and mesangial area by 94%. Fractional glomerular capillary area decreased by 24%. PPS prevented these changes. Tubular dilatation, epithelial cell atrophy, and increased interstitial area were largely prevented by PPS, as was the interstitial inflammatory infiltrate. These results suggest that the renal protection conferred by PPS was mediated both by prevention of glomerular hypertension as well as suppression of the inflammatory response. It was postulated that this was partly due to the preservation of a greater fraction of functional nephrons.
Subtotal renal ablation is a commonly used experimental model to study progressive renal damage in susceptible rat strains. Functional adaptive changes in the remaining nephrons, induced by an abrupt reduction of renal mass, eventually leads to progressive renal injury. Glomerular hypertension results from the transmission of systemic pressure to glomerular capillaries, as a consequence of afferent vasodilation. The rise in glomerular capillary pressure is thought to be the initiating factor that leads to proteinuria and progressive glomerular and tubulointerstitial injury (1,2). Indeed, maneuvers that reduce arterial and glomerular pressure and suppress the renin angiotensin system prevent the development of glomerulosclerosis (3,4,5,6).
It has been suggested that glomerular hypertension injures the glomerular endothelium and up-regulates cytokines and growth factors that promote monocyte/macrophage infiltration, mesangial proliferation, matrix expansion, and the development of glomerulosclerosis (1,2,7). Proteinuria has also been postulated to be a mediator of tubulointerstitial injury. Proteinuria induces the local release of chemokines and growth factors that promote tubular injury, which results in tubular atrophy and interstitial fibrosis (8).
Thus, the initial hemodynamic adaptive changes induce an inflammatory process that results in glomerular and interstitial fibrosis. Some of the inflammatory mediators are potent vasoactive agents that may enhance glomerular hemodynamic alterations. Previously, we (9) and others (10) demonstrated that suppression of the inflammatory process with mycophenolate mofetil retards progression of renal damage. In addition, a nonsteroidal anti-inflammatory drug has been shown to reduce renal inflammation and damage in this model (11). Moreover, Ichikawa et al. (12) and Purkerson et al. (13) demonstrated that subcutaneous nonanticoagulant heparin prevented glomerulosclerosis in the 5/6 nephrectomy model, despite persistent systemic hypertension.
More recently, polysulfate pentosan (PPS), a sulfated oligosaccharide with one fifteenth of the anticoagulant activity of unfractionated heparin, was demonstrated to reduce glomerular and tubulointerstitial fibrosis in experimental models such as streptozotocin-induced diabetes (14) in mice transgenic for bovine growth hormone (15) and in rats with cyclosporine A nephropathy (16). The protective effect of PPS is attributed to inhibition of mesangial and smooth muscle cell proliferation and decreased extracellular matrix accumulation (15,17). In addition, we recently found that PPS reduces macrophage activation in vitro and in vivo (18,19). Finally, the effect of PPS on glomerular hemodynamic changes had not been examined.
Thus, the aims of the present study were to determine (1) whether PPS prevents glomerulosclerosis, interstitial inflammation, and fibrosis induced by subtotal renal ablation and (2) whether the protective effect of PPS is associated with prevention of glomerular hemodynamic and morphologic alterations in 5/6 nephrectomy rats.
Materials and Methods
Subtotal nephrectomy (5/6 Nx) was performed in male Wistar rats weighing 200 to 250 g, by right nephrectomy and ligation of two or three branches of the left renal artery. The rats were randomly divided into two experimental groups of eight rats each. One received tap water, and the second was given PPS (Ivax, Miami, FL) in the drinking water (100 mg/k per d) for 28 d. Values of daily water intake were similar in 5/6 Nx—treated and — untreated rats (45.8 ± 1.4 and 51.2 ± 1.0 ml/d, respectively); however, to ensure that all 5/6 Nx-treated rats received the same dose, PPS concentration was adjusted in the drinking water to compensate daily variations. For this purpose, the dose of PPS was given in the volume of water that the rats had drunk the day before. Because there were no differences in food intake and body weight in both 5/6 Nx groups, the rats were not pair fed. In addition, eight normal rats were included as a control group.
Systolic BP was measured by a noninvasive tail cuff method at the first and third week. Individual 24-h urine samples were collected by placing animals in metabolic cages. All samples were collected under 1 ml of mineral oil. Urinary protein excretion was measured weekly by the trichloracetic acid turbidimetric method (20). Renal hemodynamic and histologic studies were performed in eight rats from each group.
Micropuncture Studies
Hemodynamic studies were performed in eight rats of each group. Rats were anesthetized with sodium pentobarbital (30 mg/kg, intraperitoneally), and supplemental doses were instilled as required. The rats were placed on a thermoregulated table, and temperature was maintained at 37°C. The trachea, both jugular veins, the femoral arteries, and the left ureter were catheterized with polyethylene tubing PE-240, PE-50, and PE-10. The left kidney was exposed, placed in a lucite holder, sealed with elastomer (Xantropen, Bayer, Pittsburgh, PA), and covered with Ringer's solution. Mean arterial pressure was monitored with a pressure transducer (Model p23 db, Gould, Hato Rey, Puerto Rico) and recorded on a polygraph (Grass Instruments, Quincy, MA). Blood samples were taken periodically and replaced with blood from a donor rat.
Rats were maintained under euvolemic conditions by infusion of 10 ml/kg of body wt of isotonic rat plasma during surgery, followed by an infusion of 25% polyfructosan, at 2.2 ml/h (Inutest; Laevosan-Gesellschaft, Gesellschafft, Austria). After 60 min, five to six 3-min collection samples of proximal tubular fluid were obtained to determine flow rate and polyfructosan concentration. Intratubular pressure under free flow and stop flow conditions and peritubular capillary pressure were measured in other proximal tubules with a servo-null device (Servo Nulling Pressure System, Instrumentation for Physiology and Medicine, Inc., San Diego, CA), as described previously (21). Polyfructosan was measured in plasma samples. Glomerular colloid osmotic pressure was estimated in protein from blood of the femoral artery and surface efferent arterioles. Polyfructosan concentrations were determined by the technique of Davidson et al. (22). Tubular fluid volume was estimated as described previously (21). Concentration of tubular polyfructosan was measured by the method of Vurek and Pegram (23). Protein concentration in afferent and efferent samples was determined according to the method of Viets et al. (24).
Mean arterial pressure (MAP) and GFR were measured. Single-nephron GFR (SNGFR) and tubular free flow and stop flow pressure were determined. From these values, glomerular capillary hydrostatic pressure (PGC), single-nephron filtration fraction, single-nephron plasma flow, afferent and efferent resistances, as well as the ultrafiltration coefficient, and oncotic pressure (π), were calculated according to equations previously described (25).
Histologic Analyses
After micropuncture, remnant kidneys were perfused with phosphate buffer, through the femoral catheter at the pressure corresponding to the MAP of each animal. After the kidney was blanched, the perfusate was replaced by 10% freshly prepared buffered paraformaldehyde, and the perfusion was continued until fixation was completed. The kidneys were bisected longitudinally, embedded in methyl methacrylate, and sectioned. Sections were stained with periodic acid—Schiff (American Histolab, Gaithersburg, MD).
Images were recorded by use of a one-chip CCD (640 × 480 pixel) video (Bunton Instruments, Rockville, MD) mounted on an Olympus BH-2 light microscope (Olympus Optical Co., Tokyo, Japan). The total cortical area was examined, excluding a 2-mm-wide zone in the vicinity of ischemic polar extremities of the remnant kidney. Glomerular images were recorded by moving the slide from the outer to the inner cortex in a random fashion to obtain noncrossing sample fields.
Images were processed by use of Photoshop software (Adobe System Inc., San Jose, CA). A total of 50 glomeruli were randomly selected from each kidney, and morphologic analysis was performed. Glomerular tufts were encircled, and the enclosed area was copied to create a new image (26). The number of pixels (independently of their individual density) of this image gave the surface area density of the tuft. According to DeHoff's equation for the measurement of spheroids of differing size (27,28), the harmonic mean of glomerular areas (Svm) was used to calculate the mean glomerular volume (GlmVm) for each animal, by use of the following formula: GlmVm = 4/3(Svm)(3/2). From the glomerular image, the entire amount of periodic acid—Schiff—positive material, except for the peripheral basement membranes, was selected automatically by use of the properties of color recognition of the software and was manually completed by the inclusion of nuclei. The number of pixels in this area was considered to represent the mesangial area and was expressed as a fraction of the tuft surface area. Finally, the luminal surface of tuft capillaries was individually selected by use of the color recognition properties of the software. The individual processing allowed exclusion of artifacts and noncapillary clear spaces. The number of pixels selected was considered to represent the capillary surface area and was expressed as a fraction of tuft surface area. The morphologic changes were analyzed without knowledge of the experimental groups or micropuncture study results.
Statistical Analyses
Comparison among the three groups, for continuous data, used ANOVA. When ANOVA showed a statistically significant difference, a group-by-group comparison was performed by use of a t test with a Bonferroni's correction for multiple comparisons. Results of hemodynamic studies are represented as mean ± SEM, whereas histologic results are expressed as mean ± SD. The results were considered to be statistically significant if P < 0.05. All analyses were performed by use of Statview 5 software (SAS Institute Inc., Cary, NC).
Results
BP and Proteinuria
Systolic BP was elevated at week one in both groups of rats and remained elevated throughout the course of the study, although a small reduction of systolic BP was observed after the third week in PPS-treated rats (Figure 1). A significant increase in proteinuria was observed in the untreated 5/6 Nx group 1 wk after subtotal renal ablation. PPS treatment resulted in marked reduction of proteinuria throughout the study. Of note is that the reduction in proteinuria occurred despite a persistently elevated systolic BP in the PPS-treated group.
Effect of polysulfate pentosan (PPS) on systolic BP and proteinuria. ○, control; [UNK], 5/6 nephrectomy (5/6 Nx); and [UNK], 5/6 Nx + PPS. *P < 0.05 versus control, †P < 0.05 versus 5/6 Nx.
Hemodynamic Measurements
Subtotal renal ablation produced the expected functional compensatory changes in remnant nephrons after 4 wk (Table 1 and Figure 2). GFR decreased by 45%, despite the fact that 80% of the renal mass was ablated. Micropuncture studies revealed elevated glomerular plasma flow (107.8%) and glomerular pressure (10.9% higher), which resulted in a 67% elevation of the single-nephron filtration rate. MAP was also significantly elevated.
Effect of pentosan polysulfate (PPS) on hemodynamic changes by 5/6 nephrectomya
Effect of PPS on glomerular hemodynamic changes induced by 5/6 Nx. □, control; ▪, 5/6 Nx; and [UNK], 5/6 Nx + PPS. *P < 0.05 versus control, †P < 0.05 versus 5/6 Nx. QA, single-nephron plasma flow; RA, afferent resistance; and RE, efferent resistance.
Although PPS treatment largely prevented the glomerular hemodynamic changes, MAP was only slightly reduced, compared with that in untreated rats. In fact, MAP remained markedly elevated compared with normal controls. Glomerular plasma flow was 37.4% lower than in untreated 5/6 Nx rats, and PGC was not different from that in normal controls. As a result, SNGFR was 23.1% lower in PPS-treated than in untreated 5/6 Nx rats but was still 28.7% higher than that in normal controls. These glomerular hemodynamic findings were calculated to be the result of a 61% higher afferent arteriolar resistance. Thus, in the face of persistent systemic hypertension, PPS treatment preserved the autoregulatory capacity of preglomerular vessels to prevent hyperfiltration and glomerular hypertension in remnant nephrons. It is interesting to note, however, that, despite a significantly lower SNGFR in PPS-treated rats, whole-kidney GFR was quite similar to that in untreated 5/6 Nx rats, which suggests that a larger number of nephrons was preserved by PPS treatment.
Histologic Changes
There was extensive dilatation of the tubules and tubular atrophy in the remnant kidneys of untreated 5/6 Nx rats. In addition, the tubules were separated by areas of interstitial widening, which contained many inflammatory cells (Figure 3A). Mesangial sclerosis was also present (Figure 3C). PPS-treated animals had considerably less tubular dilatation and tubular atrophy. Interstitial infiltration and fibrosis were nearly completely absent (Figure 3B). The glomerular lesions also appeared to be much less marked in PPS-treated rats (Figure 3D).
Renal cortex (periodic acid—Schiff [PAS]). (A) There are extensive tubular atrophy and dilatation in 5/6 Nx rats. Areas of interstitial infiltration separate tubules. (B) representative cortical area in a rat from Nx + PPS group. There is minor dilation of tubules. Mild epithelial cell atrophy is present in occasional tubules. Note the decrease in interstitial inflammatory cell infiltration. Representative glomeruli (PAS). (C) Representative glomerulus from the 5/6 Nx group. This large glomerulus contains conspicuous mesangial sclerosis, which is PAS positive. The number of mesangial nuclei is increased. (D) Representative glomerulus and adjacent arteriole from Nx + PPS group with no obvious abnormalities. Magnifications: ×250 for A and B; ×400 for C and D.
Morphometric Analysis of Glomerular Changes
Five/six nephrectomy induced a significant increase in glomerular volume (+46%; P < 0.001 5/6 Nx versus normal group), which was prevented by PPS treatment (Figure 4A). To normalize for differences in glomerular volume, mesangial and capillary areas were expressed as a fraction of the overall tuft area (Figure 4, B and C). There was a 94% increase (P < 0.001) in fractional mesangial area in the 5/6 Nx rats. PPS treatment largely prevented this increase (+26%; P < 0.05 versus normal; P < 0.001 versus Nx). Capillary area was decreased in the untreated Nx group (-24%; P < 0.01 versus normal), and this was also prevented by PPS.
(A) Glomerular volume (μm3) in 2K rats, 5/6 nephrectomy, or 5/6 Nx rats treated with PPS (5/6 Nx + PPS). ***P < 0.001 when compared with normal group; ††P < 0.01 compared with 5/6 Nx group. (B) Fractional changes in mesangial and (C) fractional changes in capillary areas after 5/6 Nx or 5/6 Nx + PPS treatment compared with 2K rats. *P < 0.05; **P < 0.01; and ***P < 0.001 compared with 2K rats; †††P < 0.001 compared with 5/6 Nx rats.
Discussion
We found that PPS prevented proteinuria and structural lesions, despite persistent systemic hypertension. This was associated with a reduction of PGC due to higher afferent resistance, which indicates that the autoregulatory function of afferent arterioles was preserved. Total GFR remained equal in the PPS-treated and 5/6 Nx—untreated rats. However, this was accomplished in different ways. In the untreated group, SNGFR was increased, whereas it was unchanged in the PPS-treated group. This suggested that a greater proportion of nephrons was preserved by PPS treatment.
Progressive nephron loss results at least from two processes, glomerulosclerosis and tubulointerstitial injury. Glomerular hypertension is thought to be the initiating factor.
Numerous studies have demonstrated that decreasing glomerular pressure with angiotensin-converting enzyme inhibitors and angiotensin AT1 receptor antagonists prevents progression of renal injury in the remnant kidney model (29,30). Renal protection by these agents is also due to their effect of blocking the angiotensin II—induced elevation of cytokines and growth factors involved in progressive renal injury (31,32).
On the other hand, renal injury in 5/6 nephrectomized rats can also be prevented with drugs that suppress the inflammatory process (9,10,11,12,13). In addition, protection of renal injury in several experimental models was induced by PPS, a sulfated oligosaccharide; its effect was attributed to inhibition of mesangial cell proliferation and extracellular matrix accumulation (14,15,16). Furthermore, we have preliminary evidence that this compound inhibits tumor necrosis factor—α—induced activation of macrophages in vitro (17); thus, it also has anti-inflammatory properties.
The aim of the present study was to evaluate the effect of PPS treatment on glomerular hemodynamics and on renal injury in 5/6 Nx. We found that PPS prevented proteinuria and glomerular hemodynamic alterations implicated in the initiation and progression of chronic renal injury, including inhibition of mesangial proliferation and inflammatory cell infiltration, despite the fact that systolic BP continued to be significantly elevated.
Fuhihara et al. (10) reported that treatment of 5/6 nephrectomized Munich-Wistar rats with mycophenolate mofetil prevented structural injury. The rats had persistent systemic hypertension, proteinuria, and glomerular hypertension. The absence of histologic lesions, in the face of persistent glomerular hypertension and proteinuria, is contrary to the findings of others who have used antihypertensive regimens (29,30,33) and our preliminary data from a study that used mycophenolate mofetil in Sprague-Dawley rats (34). Although the differences remain unexplained, possibilities for this discrepancy include differences between rat strains, gender, and the time periods studied.
In this study, morphometric evaluation of structural changes revealed that PPS treatment, which preserved glomerular pressure, also prevented the increased glomerular volume and mesangial fractional area that accompanies subtotal renal ablation. In addition, PPS treatment also largely prevented tubulointerstitial lesions.
The capillary lumen area represented 40.4 ± 4.6% of the tuft area in normal rats, a value close to that observed by Lee et al. (35) using electron microscopy in a similar model. Fractional capillary lumen area decreased by 25% in Nx rats, similar to that observed 15 d after ablation in Sprague-Dawley rats (35). However, others found that the fractional capillary volume remained stable 6 and 26 wk after Nx (36,37). In the latter reports, the studies were performed in WKY rats, with and without systemic hypertension. In addition, the authors reported a high frequency of capillary aneurysms, which could have resulted in an increase in absolute capillary volume. In the present study, capillary aneurysms were uncommon, whereas capillary thromboses were numerous in damaged glomeruli, a feature also observed in Sprague-Dawley rats (38). This discrepancy could result from the fact that (1) susceptibility to develop glomerular lesions after 5/6 Nx varies between rat strains (39) and (2) we studied rats at an earlier time (30 d) than some others.
In the 5/6 Nx group, the increase in fractional mesangial area was accompanied by a decrease in the fractional capillary area. The latter finding is characteristic of most models of glomerulosclerosis. Subtotal nephrectomy is also associated with severe morphologic alterations in the cortical tubulointerstitial region. Interestingly, PPS totally prevented the increase in glomerular volume and tubulointerstitial lesions. A factor that complicates the assessment of this change was that we were not able to determine whether the size distribution coefficient of glomerular volume was changed by the treatment. This may have introduced a bias in the calculations.
The precise mechanism by which PPS prevented glomerular hypertension in this model is not well defined. It is conceivable that increased glomerular pressure may be partially mediated by the inflammatory process that accompanies progressive renal injury. Local release of inflammatory mediators, some of which are also potent vasoactive agents, may contribute to enhanced glomerular hypertension by constriction of efferent arteriole and/or afferent arteriolar vasodilation. The fact that histologic examination of the kidneys from PPS-treated rats showed a marked decrease in inflammatory cells in the interstitial space lends support to this hypothesis.
Another possible explanation is that prevention of the inflammatory process and proteinuria by PPS allowed the survival of a greater proportion of nephrons. This would decrease the requirement for vasodilation and overperfusion in the remnant nephrons to compensate for the lost renal mass. This point is emphasized by the fact that micropuncture studies conducted at different stages after subtotal renal ablation suggested that the rise of glomerular pressure is determined by the number of remaining functioning nephrons. After 4 to 6 wk, structural changes are minor, and mean PGC values are 61 mm Hg (4,12,40,41). In contrast, after 8 to 16 wk, extensive renal damage is present, and a significant proportion of glomeruli appear to be sclerotic. At this stage, glomerular pressures can be as high as 65 to 82 mm Hg (40, 42,43,44). Accordingly, if the number of nephrons is not continually decreased, the glomerular capillary pressure and filtration rate required to compensate for nephron loss will be lower. Thus, in the PPS group, the lower glomerular pressure could result from preservation of the residual nephrons in a functional state.
In fact, SNGFR was significantly lower in PPS-treated rats, although total GFR was identical to that of the untreated group. This suggested that there were more functional nephrons remaining in the PPS group.
In the course of 5/6 nephrectomy, nephron loss occurs as the consequence of at least two processes: initial glomerular capillary endothelial damage and increasing hydraulic pressure and shear stress. These may up-regulate the synthesis of cytokines and growth factors, as well as stimulate inflammatory cell accumulation, mesangial cell proliferation, and matrix production. In the present study, morphometric evaluation demonstrated a 15% increase of mesangial fractional area but not total sclerosis of glomerular tufts.
The second process that leads to nephron loss is tubulointerstitial injury. In fact, several observations of human biopsies suggested that tubulointerstitial injury scores correlated better with renal function than did glomerular injury (45,46). Gandhi et al. (47), using serial sections obtained at 10 and 25 wk after ablation, showed that a significant number of glomeruli had lost their corresponding tubule or were connected to an atrophic tubule segment. Such glomeruli would no longer contribute to total renal function. Histologically, these glomeruli were smaller but otherwise normal and could not be identified by routine examination. After 10 wk, up to 20% of glomeruli were found to be atubular or connected to an atrophic tubule segment. Those authors suggested that tubular injury starts to cause loss of remnant nephrons function early after ablation and before sclerosis is severe.
In summary, PPS treatment prevented renal injury in rats with 5/6 nephrectomy. Despite the persistence of significant elevated arterial pressure, PPS prevented proteinuria, glomerular hypertension, and hyperfiltration. Histologic evaluation showed a marked decrease in glomerular lesions, interstitial fibrosis and inflammatory cell infiltration, and tubular changes. Given that the rise in glomerular pressure is determined to a great extent by remaining functional renal mass, we postulate that prevention of glomerular hypertension by PPS was associated with decreased glomerular and tubulointerstitial injury that resulted in preservation of a larger number of nephrons.
Acknowledgments
This work was supported by research Grant 28668 to N.A.B. from the Mexican Council of Science and Technology (CONACYT) and NIH Grants R01AG19366 and R01AG17170. Parts of this work were presented at the 31st and 32nd Meetings of the American Society of Nephrology, Philadelphia, PA, 1998 and Miami, FL, 1999.
- © 2001 American Society of Nephrology
References
