Abstract
The persistence of NF-κB independent inflammatory signals in the cortical tubulointerstitium may explain the incomplete suppression of interstitial monocyte accumulation by the antioxidant NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), in nephrotic rats with established Adriamycin nephropathy (AN). Because PDTC is known to have anti-proteinuric effects, in this study we investigated whether earlier commencement, during the pre-nephrotic phase of AN, would be more effective in reducing interstitial monocyte accumulation. Male Wistar rats with AN received either vehicle or PDTC (50 mg/kg bd i.p.i.) from d7 until d30 (n = 8 per group). On d30, PDTC reduced renal cortical lipid peroxidation (43%), wet kidney weight and tubulointerstitial injury in AN, but did not decrease proteinuria. Accordingly, inhibition of interstitial ED-1 accumulation remained incomplete (52%). Interestingly, the early administration of PDTC in AN, induced polyuria and renal cortical NF-κB DNA-binding activity was reduced by only 35%. These results suggest that: (i) the combination of an anti-proteinuric agent with PDTC may be required to completely suppress interstitial monocyte cell accumulation in AN and, (ii) the timing and duration of PDTC therapy are an important determinant of its efficacy to reduce NF-κB activation, in vivo.
INTRODUCTION
Pyrrolidine dithiocarbamate (PDTC) is a metal chelator and antioxidant, with potent anti-inflammatory properties, in part due to, its ability to suppress nuclear factor (NF)-κB Citation[[1]]. The latter is an inducible transcription factor family, involved in the regulation of multiple genes that control inflammation, growth and survival Citation[[2]]. Previously, we reported that the therapeutic administration of PDTC (from day 14–30) to nephrotic rats with Adriamycin nephropathy (AN), completely suppressed renal cortical NF-κB activation (73%), but reduced interstitial monocyte infiltration by only 48% Citation[[3]]. Because PDTC did not alter proteinuria, the persistence of protein-induced NF-κB independent or PDTC-resistant inflammatory signals within the cortical tubulointerstitium could explain the incomplete inhibition of interstitial monocyte accumulation Citation[[4]]. PDTC is known to have anti-proteinuric effects in an immune-mediated model of experimental glomerulonephritis, when administered in the early stage of disease Citation[[5]]. Thus, we postulated that commencement of PDTC from day 7, during the pre-nephrotic stage of AN, may attenuate the progression of proteinuria and, thereby more completely suppress interstitial monocyte infiltration. The following study was undertaken to test this hypothesis.
MATERIALS AND METHODS
Animal Model and Experimental Design
Male Wistar rats (6 to 8 weeks old) were supplied by the Animal Care Facility, Westmead Hospital and allowed free access to rat food (19% protein, Glen Forrest Stock, Perth, Australia) and water. All animals were handled within the guidelines of the National Health and Medical Research Council of Australia. AN was induced in rats (n = 16) by a single injection of doxorubicin hydrochloride (7.5 mg/kg, David Bull Laboratories, Victoria, Australia) as previously described Citation[[3]]. On day 5, baseline 24 h urinary protein and creatinine excretions were measured. Venous blood for serum creatinine and albumin was collected at the end of the clearance period. On day 7, animals were stratified into two groups (n = 8 each) according to baseline UpV, endogenous creatinine clearance (CrCl) and body weight. Each group received either PDTC (50 mg/kg bd ip, 0600 and 1800 h) or an equivalent volume of vehicle (saline). We have previously shown that this dose and route of administration of PDTC is non-toxic to rats and reduces renal NF-κB activation and interstitial monocyte infiltration Citation[[3]]. Body weight was monitored daily and groups were pair-fed. Urinary protein and creatinine excretions were measured on days 10, 20 and 29. On day 30, animals were anesthetized, three hours after the final injection of PDTC, bilateral nephrectomies were performed and the kidneys were processed as described below.
Biochemical Parameters
Urine and blood protein, albumin and creatinine were analysed by the Institute of Clinical Pathology and Medical Research as previously described Citation[[3]]. Plasma, urinary and renal lipid peroxidation was determined by production of malondialdehyde (MDA) as previously described Citation[[3]].
Renal Histology
A mid-coronal slice of the right kidney was immersion-fixed in 10% neutral buffered formalin for 24 h. Paraffin sections, 3 microns in thickness, were stained with periodic-acid Schiff (PAS). Cortical tubulointerstitial injury was assessed by a semi-quantitative scoring system. Twenty random cortical fields were viewed at × 200 magnification and graded according to the percentage area occupied by tubular injury (tubular atrophy, dilatation) and interstitial inflammation (0 = normal; 1 = less than 25%; 2 = 25–50%; 3 = 50–75%; 4 = 75–100%). The number of interstitial ED-1 positive cells was assessed by immunohistochemistry, on acetone-fixed frozen sections as previously described Citation[[3]]. The number of positive interstitial cells were quantitated in ten non-overlapping cortical fields (× 400, measuring 0.075 mm2 each).
Renal Cortical NF- B Activation
Extraction of nuclear protein and electrophoretic mobility for NF-κB were performed as described previously Citation[[3]].
Statistical Methods
All data are expressed as mean ± SEM. Comparisons between experimental groups were performed using the independent t-test and Mann-Whitney test for parametric and non-parametric data respectively. A P value less than 0.05 indicated a significant difference between groups.
RESULTS
No animals died during the study. Body weight and food intake were similar in both groups (). The data for renal function and proteinuria are shown in . By design, baseline (day 5) proteinuria and CrCl, were similar in both groups. Treatment with PDTC had no effect on the increase in proteinuria or reduction in CrCl in AN. Of note, PDTC-treated rats had a significant increase in urine volume, beginning as early as 3 days after the commencement of PDTC (day 10). The urine from PDTC-treated animals had reduced specific gravity and was negative for glucose. PDTC treatment reduced renal cortical MDA production by 43%, but had no effect on plasma or urinary MDA (). PDTC treatment reduced kidney weight (AN + vehicle: 1.68 + 0.12; AN + PDTC: 1.25 + 0.10 g; P < 0.05) cortical tubulointerstitial injury (tubulointerstitial injury score: AN + vehicle: 2.7 + 0.3; AN + PDTC: 1.5 + 0.2; P < 0.05). and decreased interstitial ED-1 infiltration incompletely (52%) (AN + vehicle: 33.6 + 3.6; AN + PDTC: 16.1 + 1.7 cells/hpf; P < 0.05). Interestingly, renal cortical DNA binding activity of NF-κB was reduced by 35% with PDTC treatment (), whereas previously we had reported that this was up to 73%, when PDTC was commenced on day 14.
Figure 1. Mean body weight and food intake of the experimental groups (n = 8 per group). The lower section of the graph shows the experimental design of the study (Day 0: injection with doxorubicin hydrochloride; Day 5: measurement of baseline proteinuria and creatinine clearance; Day 7: randomisation and commencement of either vehicle or PDTC; Day 30: end of study).
Table 1. Effect of Early Pyrrolidine Dithiocarbamate (PDTC) Administration on Renal Function in Adriamycin Nephropathy (AN)
Table 2. Effect of Early Pyrrolidine Dithiocarbamate (PDTC) Administration on Renal, Urinary and Plasma Malondialdehyde Production in Adriamycin Nephropathy (AN)
Figure 2. DNA-binding activity of nuclear factor (NF)-κN (5′-AGT TGA GGG GAC TTT CCC AGC-3′) in renal cortical nuclear extracts from rats with Adriamycin nephropathy treated with either vehicle or PDTC from day 7 to day 30 (A). Representative autoradiographs. Cold NF-κB is unlabeled probe plus nuclear protein; (B) Mean densitometry (n = 8 per group). *P < 0.05 vs AN.
DISCUSSION
In this study, the administration of PDTC during the pre-nephrotic stage of AN failed to reduce proteinuria. Sakura and colleagues had previously reported that prophylactic commencement of PDTC in rats with immune-mediated glomerulonephritis, attenuated proteinuria and NF-κB activation Citation[[5]]. The exact mechanisms of the anti-proteinuric effect and its relation to NF-κB inhibition in the latter model are not known, but it may relate to the potent antioxidant properties of PDTC Citation[[1]]. Presumably the lack of efficacy of PDTC as an anti-proteinuric agent in AN, reflects the relative irreversibility of established podocyte damage in this model.
Both NF-κB-dependent and independent inflammatory signals are induced in the renal cortical tubulointerstitium, in response to proteinuria Citation[[4]], Citation[[6]]. For example, in vitro and in vivo, proteinuria induces the vasoactive peptide, endothelin-1 which is NF-κB-independent, and also mediates tubulointerstitial injury Citation[[4]]. Because of the previously demonstrated anti-proteinuric effect of PDTC Citation[[5]], we had expected that early administration would enhance its ability as an anti-inflammatory agent in AN. However, as no anti-proteinuric effect occurred, the reduction in interstitial monocyte infiltration was incomplete (52%), and similar to what we had previously observed when PDTC was commenced on day 14 (48%) Citation[[3]]. Thus, the combination of PDTC with an anti-proteinuric drug (such as angiotensin converting enzyme inhibitors) is probably required to completely suppress interstitial monocyte infiltration in AN Citation[[7]].
Early PDTC treatment also caused a significant increase in urine volume in AN. This has not been reported previously and was not observed when PDTC was commenced on day 14 in AN Citation[[3]]. Interestingly, Okada and colleagues found that an increase in urine volume and administration of anti-diuretic hormone antagonists was correlated with a reduction in renal injury in chronic AN Citation[[8]]. PDTC is not known to affect tubular function or anti-diuretic hormone secretion. Further studies are needed to determine the mechanism of PDTC-induced polyuria and its possible protective role in the tubulointerstitium.
Finally, the early commencement of PDTC was associated with a significant reduction in renal cortical NF-κB DNA binding activity, but which was lower in magnitude than that we observed previously when the compound was started on day 14 (35% vs 73%) Citation[[3]]. The reasons for this are not readily apparent, but the reduction in NF-κB activation in the present study was sufficient to attenuate tubulointerstitial injury. Nevertheless, these data suggest that the timing and duration of anti-NF-κB therapy are important determinants of the efficacy to reduce NF-κB activation, in vivo. Using different end-points, similar effects have also been noted with other pharmaceutical agents Citation[9-10]. For example, the renoprotective effect of diltiazem in chronic AN, was paradoxically more effective when administered in established disease, compared to if it was given in the early stage of the model Citation[[9]].
ACKNOWLEDGMENT
This study was supported by project grant no. 97021 from the National Health and Medical Research Council of Australia (to D.C.H.) and a medical scholarship from the Australian Kidney Foundation (to G.K.R.).
REFERENCES
- Schreck R, Meier B, Mannel D N, Droge W, Baeuerle P A. Dithiocarbamates as potent inhibitors of NF-κB in intact cells. J Exp Med 1992; 175: 1181–1194
- Guijarro C, Egido J. Transcription factor-kB (NF-κB) and renal disease. Kidney Int 2001; 59: 415–424
- Rangan G K, Wang Y, Tay Y-C, Harris D CH. Inhibition of NF-κB activation reduces tubulointerstitial injury in proteinuric rats. Kidney Int 1999; 56: 118–134
- Ruggenenti P, Remuzzi G. The role of protein traffic in the progression of renal diseases. Annu Rev Med 2000; 51: 315–327
- Sakurai H, Hisada Y, Ueno M, Sugiura M, Kawashima K, Sugita T. Activation of transcription factor NF-κB in experimental glomerulonephritis in rats. Biochim Biophys Acta 1996; 1316: 132–138
- Donadelli R, Abbate M, Zanchi C, Corna D, Tomasoni S, Benigni A, Remuzzi G, Zoja C. Protein traffic activates NF-κB gene signaling and promotes MCP-1 dependent interstitial inflammation. Am J Kidney Dis 2000; 36: 1226–1241
- Remuzzi G, Zoja C, Gagliardini E, Corna D, Abbate M, Benigni A. Combining an anti-proteinuric approach with mycophenolate mofetil fully suppresses progressive nephropathy of experimental animals. J Am Soc Nephrol 1999; 10: 1542–1549
- Okada H, Suzuki H, Kanno Y, Saruta T. Evidence for the involvement of vasopressin in the pathophysiology of Adriamycin-induced nephropathy in rats. Nephron 1996; 72: 667–672
- Podjarny E, Bernheim J L, Pomeranz A, Rathaus M, Pomeranz M, Green J, Bernheim J. Effect of timing of antihypertensive therapy on glomerular injury: comparison between captopril and diltiazem. Nephrol Dial Transplant 1993; 8: 501–506
- Ding Z K, Sumrani N, Hong J H. Effect of timing of cyclosporine administration on recovery from renal ischemia in rats. J Surg Res 1991; 51: 341–343