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University of Groningen

Advancements in renal protectionWaanders, Femke

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CHAPTER 8

Femke Waanders1, Harry van Goor2, Gerjan Navis1

1 Department of Nephrology, 2 Department of Pathology and Medical Biology, UniversityMedical Center Groningen, University of Groningen, Groningen, the Netherlands.

Kidney Blood Press Res. Provisionally accepted.

ADVERSE RENAL EFFECTS OF THE AGE INHIBITOR PYRIDOXAMINEIN COMBINATION WITH ACEI IN NON-DIABETIC ADRIAMYCIN-INDUCED RENAL DAMAGE IN RATS

PF Hfdst 8:Proefschrift 18-08-2008 13:02 Pagina 175

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ADVERSE RENAL EFFECTS OF THE AGE-INHIBITOR PM IN COMBINATION WITH ACEI IN NON-DIABETIC RENAL DAMAGE IN RATS

Abstract

Background. Advanced glycation end products (AGEs) are involved in diabetic nephropa-thy. In non-diabetic proteinuria renal AGEs also accumulate. The AGE inhibitor pyri-doxamine (PM) is renoprotective in obese Zucker rats and experimental chronic allograftnephropathy supporting its renoprotective potential in non-diabetic renal damage.

Methods. To investigate whether PM is renoprotective in non-diabetic proteinuria, we stud-ied its effects in adriamycin nephropathy (AN, 1.5 mg/kg iv). Six weeks after disease in-duction, treatment started with vehicle (VEH), lisinopril (ACEi, 75 mg/L drinking water),PM (2 g/L drinking water) and PM plus lisinopril (PM/ACEi) (n =12 per group) for 18weeks. Age-matched healthy rats (n=6) served as controls (CON).

Results. ACEi reduced proteinuria, blood pressure and renal damage. PM gradually in-creased blood pressure and did not affect proteinuria. In PM/ACEi the antiproteinuricand blood pressure lowering effects of ACEi were abrogated during long-term treatment.Remarkably, serum creatinine, focal glomerulosclerosis and interstitial fibrosis were con-siderably increased in PM/ACEi. Pronounced hypercholesterolemia which occurred inboth PM treated groups, was accompanied by marked glomerular lipid deposition.

Conclusion. PM was not renoprotective in this model of non-diabetic proteinuria inducedrenal damage. By contrast, renal damage was aggravated when PM was combined withACEi. Despite the fact that there is no current evidence that these findings apply to thedrug as used in human diabetic nephropathy, we emphasize the importance of close mon-itoring of blood pressure, lipids and possible direct toxic effects in future studies with PMin renal patients, especially when combining PM with ACEi.


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Introduction

Several lines of evidence support a pathophysiological role for advanced glycation endproducts (AGEs) in diabetic nephropathy.1 First, accumulation of AGEs occurs inglomerular and tubulointerstitial compartments in proportion to the severity of renaldamage.2 Second, formation of AGEs precedes and correlates with early manifestationsof diabetic nephropathy.3 Moreover, pharmacological intervention in AGE formationwith pyridoxamine (PM) and aminoguanidine protects against renal structural lesions,proteinuria and renal function loss in experimental diabetes,4-6 supporting the therapeuticpotential of AGE inhibition. Interestingly, PM not only provides renoprotection in dia-betes, but also in experimental chronic allograft nephropathy7 and in normoglycemicobese Zucker rats,4;8 demonstrating that its beneficial effects are not limited to hypergly-caemia-related models of renal damage.

Involvement of renal AGE accumulation in proteinuria-induced non-diabetic renal dam-age is supported by several studies. Renal accumulation of AGEs has been shown in focalglomerulosclerosis, hypertensive nephrosclerosis and lupus nephritis.2 Mesangial accu-mulation of GA-pyridine, a novel glycolaldehyde-derived AGE, not only occurs in dia-betic nephropathy but also in the mesangium of chronic proteinuric renal diseases in man.9

In experimental renal disease, in adriamycin-induced nephropathy10 and in subtotallynephrectomized rats11 renal AGE accumulation occurs, which can be ameliorated byblockade of the renin-angiotensin aldosterone system (RAAS).10;11

Since AGEs are nephrotoxic12;13 and accumulate in adriamycin nephropathy (AN),10 wehypothesized that renal AGE accumulation, resulting from the primary renal insult, cancontribute to further progression of renal damage in non-diabetic proteinuria-inducedrenal disease. Intervention in AGE formation by PM may thus have renoprotective po-tential in non-diabetic proteinuria induced renal damage. Currently, standard treatment forproteinuria is RAAS blockade, both in experimental models and in human. In the presentstudy the effect of PM was therefore compared to that of the angiotensin converting en-zyme inhibitor (ACEi) lisinopril. In many patients, despite RAAS blockade, a certain resid-ual proteinuria persists, which predicts the rate of subsequent renal function loss.14-16

Thus, improvement of antiproteinuric treatment is advocated. Therefore, in the currentstudy we also investigated whether PM allows additional renoprotection in AN whencombined with the ACEi lisinopril.


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Methods

Animals, experimental groups and treatmentFifty-four adult male Wistar rats (± 300 g) were studied (Hsd.Cpb. Wu; Harlan Inc., Zeist,the Netherlands). Rats were housed in a temperature-controlled room of 18-20°C witha 12h-light/dark cycle. The rats had free access to standard chow and drinking water. Thelocal animal ethics committee at the University of Groningen approved all experimentalprocedures (Committee protocol number: D4091A) and the Principles of LaboratoryAnimal Care (NIH publication no. 85-23) were followed.After two weeks of acclimatization and blood pressure measurement training, we inducedadriamycin-nephropathy (AN) by injection of 1.5 mg/kg adriamycin in the tail vein underlight isoflurane/O2 anaesthesia. Healthy control rats were injected with correspondingvolumes of saline (0.9 % NaCl). Rats were sacrificed at week 24. Rats were randomly divided into five groups, four groups with AN (n=12 per group) andone age-matched healthy control group (CON, n=6). Six weeks after the injection of adri-amycin, when proteinuria was stabilized, treatment started. AN rats were treated for 18weeks, until week 24 with vehicle (VEH), lisinopril (ACEi, 75 mg/L drinking water), py-ridoxamine (PM, (PM(HCl)2, 2 g/L drinking water) and with the combination treat-ment of PM and lisinopril (PM/ACEi). The dose of PM (2 g/L) was based on previous studies as performed in our lab in Lewis andFisher rats with allograft nephropathy and isograft controls7 and by others in non-diabeticSprague-Dawley rats and Zucker obese rats.4;8 In these studies, PM was well tolerated by allstrains of rats and did not induce adverse effects on renal function and renal morphology.

Blood pressure and body weight measurementsWe trained blood pressure measurements daily for two weeks, prior to the induction ofnephrotic syndrome. Body weight was measured weekly and systolic blood pressure (SBP)was measured every other week. An automated multichannel system was used with tailcuffs and photoelectric sensors to detect the tail pulse (Apollo 179; IITC Life Science.Woodland Hills, CA, USA). The rats were placed in a test chamber in restrainers whiletemperature was maintained at 27 to 29 ºC. For each rat, the value was calculated from themean of two consecutive measurements.

Proteinuria, creatinine, urea, electrolyte and cholesterol measurementsEvery other week, we put the rats in metabolic cages (BioquantTM; Merck, Darmstadt,


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Germany) to collect 24 hour urine samples. Urinary protein excretion was measured by thepyrogallol red molybdate method. Blood samples were collected at the end of the experiment, immediately prior to sacrifi-cation of the rats. Concentrations of creatinine, urea, sodium, potassium and cholesterolwere all analyzed on a multi-test analyzer system (Merck Mega, Darmstadt, Germany)with Ecoline®MEGA® reagents (Diasys Diagnostic Systems, Holzheim, Germany). Cre-atinine concentrations in urine and serum were determined with the Jaffé method. Serumvalues of urea were determined with the urease-GLDH method and concentrations ofpotassium and sodium were measured with indirect potentiometry. Cholesterol was de-termined enzymatically.

Sacrifice and assessment of renal morphologic damageAt the end of the studies (week 24) rats were anaesthetized with isoflurane/O2, kidneyswere perfused in situ with saline and rats were sacrificed. One part of kidney tissue was snapfrozen in liquid nitrogen. Another part of kidney tissue was fixed in 4% paraformaldehydeand processed for paraffin embedding.17 Paraffin sections (4μm) were stained with perio-dic acid-Schiff (PAS) and examined by a qualified pathologist in a blinded fashion by lightmicroscopy to evaluate focal glomerulosclerosis (FGS) and interstitial fibrosis (IF). To assess the degree of FGS, a blinded pathologist semi-quantitatively scored 50 glome-ruli on a scale of 0 to 4. FGS was scored positive when collapse of capillary lumens, me-sangial matrix expansion, hyalinosis, and adhesion formation were present in the samequadrant. If 25% of the glomerulus was affected, a score of 1 was given, 50% was scored as2, 75% as 3 and 100% as 4. The degree of IF was scored similarly in 30 consecutive visual fields. IF was defined as ex-pansion of the interstitial space, with or without the presence of atrophied and dilated tu-bules and thickened tubular basement membranes. Medullary tissue, glomeruli and vesselswere excluded from the calculated areas of fibrotic involvement. A score of 0 was givenwhen no interstitial fibrosis was present in a field, 1 for 0 25% with IF, 2 for 25-50%, 3 for 50-75% and 4 for 75-100% of the field showing IF. To obtain the final score, we multiplied the degree of change by the percentage of glo-meruli (for FGS) or visual fields (for IF) with the same degree of injury and added thesescores, rendering a theoretical range of 0 to 400.

ImmunohistochemistryDeparaffinized and rehydrated sections (4 μm) were subjected to heat-induced antigen


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retrieval by overnight incubation in a 0.1 M Tris/HCl buffer (pH 9.0) at 80°C. Endoge-nous peroxidase was blocked with 0.3% H2O2 in phosphate-buffered saline (PBS) for 30minutes. Sections were incubated with an ED1-antibody (Serotec, Oxford, UK) or ananti-pentosidine antibody (16.9 μg/mL)7;10 for 60 minutes at room temperature. Bindingof the antibody was detected using sequential incubations with peroxidase (PO)-labelledrabbit anti-mouse and PO-labelled goat anti-rabbit antibodies; both for 30 min. Peroxidaseactivity was developed using 3,3’-diaminobenzidine tetrachloride (DAB) for 10 min. Sec-tions were counterstained with haematoxylin. Macrophages were assessed in fifty glomeruli.

Lipid stainingTo assess renal accumulation of neutral fats we used the Oil red O staining on frozen tis-sue sections (4μm) that were air dried and fixed in 2% paraformaldehyde in PBS at 4ºC. Forcolocalization of intrarenal neutral fats with macrophages, we performed a double stai-ning with ED1 and Oil red O. Frozen tissue sections (4μm), air dried and fixed in 2% pa-raformaldehyde in PBS at 4ºC, were first stained for ED1 and then for Oil red O using thesame protocols as described above.

Renal ACE activityRenal ACE activity was determined using a method previously described in detail.18 Inshort, we homogenised tissue in a 50 mM L-1 K2PO4 buffer at pH 7.5 and transferred100 μl of this homogenate to a 0.5 M L-1 K2PO4 buffer. Then we added the ACE substrateHippuryl-His-Leu (12.5 nM L-1, Sigma Zwijndrecht, the Netherlands) and incubated thesamples at 37ºC for exactly 10 minutes. To stop the conversion of the substrate 1.45 ml of280 mM L-1 NaOH was added. Thereafter, 100 μl of 1% phtaldialdehyde was added tolabel the free His-Leu product. The amount of labelled His-Leu was determined fluori-metrically at excitation-emission wavelengths of 364/486 nm. Control samples were in-cluded in which the conversion of substrate was prevented by adding NaOH before thesubstrate Hippuryl-His-Leu. Moreover, the substrate was added after the incubation pe-riod in these control samples.

Data analysisData are expressed as mean ± standard error. Statistical analysis of group differences was per-formed by a Kruskal-Wallis ANOVA on ranks and Mann-Whitney U tests. Statistical ana-lyses were performed using SPSS version 14.0 (SPSS Inc., Chicago, IL, USA). Statisticalsignificance was assumed at the 5% level (two-tailed).


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Results

Body weight and mortalityThroughout the experiments, food and water intake was similar in all groups. The PMand the PM/ACEi treated groups had a significantly lower body weight than the othergroups (Table 1). Because of declined condition some rats had to be sacrificed prelimi-nary. At the end of the study data were available in 11/12 rats from the VEH group, 11/12rats from the ACEi group, 12/12 from the PM group, 10/12 from the PM/ACEi groupand 6/6 from the CON group.

Blood Pressure and Proteinuria Systolic blood pressure remained stable after induction of nephrosis until start of treat-ment. ACEi significantly reduced blood pressure, whereas blood pressure was unaffectedby VEH. A gradual, significant rise in blood pressure was observed in the PM group. In thePM/ACEi group blood pressure initially decreased, with a gradual escape afterwards. Inthe CON group blood pressure remained stable throughout the study (Figure 1A). Proteinuria developed rapidly during the four weeks after adriamycin injection and sub-sequently levelled off and stabilized. At onset of treatment mean proteinuria in the adri-amycin nephrotic rats was 261±14 mg/d. ACEi induced a significant reduction inproteinuria, whereas proteinuria was unaffected by VEH. PM did not affect proteinuria atany time. In the PM/ACEi group, proteinuria initially decreased, but with a gradual es-cape afterwards, resulting in annihilation of the antiproteinuric response during long termfollow-up. The time-course of the antiproteinuric response is shown in Figure 1B. The ab-

VEH ACEi PM PM/ACEi CON (n=11) (n=11) (n=12) (n=10) (n=6)

Body weight (g) 491 ± 12 513 ± 13 437 ± 18 #†‡ 419 ± 30 #†‡ 516 ± 20

Serum creatinine (μmol/L) 71 ± 4 63 ± 6 81 ± 7 146 ± 39* 55 ± 1

Creatinine clearance (mL/min) 1,24 ± 0,14 # 1,55 ± 0,20 0,87 ± 0,14 #‡ 0,80 ± 0,29 #‡ 1.85 ± 0,14

Serum urea (mmol/L) 14 ± 2 11 ± 1 14 ± 2 33 ± 9* 6 ± 0.2

Serum sodium (mmol/L) 133 ± 1 129 ± 5 135 ± 1 134 ± 1 137 ± 1

Serum potassium (mmol/L) 5.5 ± 0.2 # 5.2 ± 0.2 4.9 ± 0.2 6.9 ± 0.3* 4.5 ± 0.1

Table 1. Clinical parameters at the end of the study. Abbreviations are: VEH, vehicle; ACEi, angiotensin converting enzyme inhibition; PM, pyridox-amine; CON, healthy control. *p<0.05 versus all other groups, #p<0.05 versus CON, †p<0.05 versus VEH, ‡p<0.05 versus ACEi.


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solute values of proteinuria at the end of the experiment are given in Figure 3 (right, darkgray bars), showing nephrotic range proteinuria in all AN groups. A significant reductionin proteinuria was only observed in the group treated by ACEi monotherapy.

Creatinine clearance, Urea and ElectrolytesSerum creatinine, urea and potassium were significantly higher in the PM/ACEi groupcompared to all other groups (Table 1). Creatinine clearance was reduced in both the PM

Figure 1. Timecourse of blood pressure development (A) and antiproteinuric response (B) is shown. Abbreviations are: VEH, vehicle; ACEi, angioten-sin converting enzyme inhibition; PM, pyridoxamine; CON, healthy control. *p<0.05 versus all other groups, +p<0.05 versus VEH, ACEi and CON.

+

*


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and the PM/ACEi groups compared to the CON and ACEi groups (Table 1). No sig-nificant differences in serum sodium concentration were observed between the groups(Table 1).

Renal structural changes, renal pentosidine accumulation and glomerular macrophagesIn all adriamycin nephrotic rats FGS and IF were significantly elevated compared to CON.Compared to VEH, ACEi significantly reduced FGS, but this did not reach statistical sig-nificance for IF. In the PM/ACEi group FGS was significantly elevated compared to VEH,ACEi and CON. IF in this group was significantly elevated compared to all other groups(Figure 2A and B, photographs of morphology are presented in the upper 5 panels of Figure4). Also, in the PM/ACEi group a significant glomerular influx of macrophages was seen(Figure 2C). Pentosidine was found in the brush border and cytoplasm of dilated tubularstructures (Figure 5). In PM/ACEi treated rats with AN, total immunoreactive tubularpentosidine was most pronounced, consistent with severe interstitial renal damage.

Figure 2. Quantitative renal parameters of damage, glomerular macrophages and renal ACE activity. A) Focal glomerulosclerosis, semi-quantitative score (arbitrary units 0 to 400). B) Interstitial fibrosis, semi-quantitative score (arbitrary units 0 to 400). C) Mean glomerular macrophage, mean number per glomerulus of 50 counted glomeruli. D) Renal ACE activity. Abbreviations are: VEH, vehicle; ACEi, angiotensin converting enzyme inhibition; PM, pyridoxamine; CON, healthy control. *p<0.05 versus all othergroups, #p<0.05 versus CON, +p<0.05 versus VEH, ACEi and CON


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Renal ACE activityTo assess the pharmacological efficacy of lisinopril on renal tissue ACE we measured renalACE activity. In the VEH group renal ACE activity was significantly increased comparedto all other groups. ACEi normalized renal ACE activity in both the ACEi group and inthe PM/ACEi group. Remarkably, PM also significantly reduced renal ACE activity com-pared to VEH, albeit not to normal levels (Figure 2D).

Serum and renal lipidsIn all AN groups cholesterol was significantly higher than in healthy control rats. Re-markably, in PM and in PM/ACEi treated rats serum cholesterol was significantly hig-her than in VEH and ACEi treated rats, indicating that in these groups the increase inserum cholesterol was stronger than expected from proteinuria alone (Figure 3). Oil red O staining of kidney sections showed massive glomerular accumulation of lipidpositive cells in PM and even more in PM/ACEi treated rats. In addition, focal accu-mulation of lipid-positive cells in proximal tubules was observed, but this was not nearlyas dramatic as the glomerular accumulation. Less accumulation of Oil red O stainable li-pids was observed in glomeruli of VEH treated rats, compared to PM and PM/ACEitreated rats. In ACEi treated rats and CON rats renal lipid deposition was not observed(Figure 4, lower 5 panels). Double staining formacrophages revealed no co-localization ofthese cells with lipid-positive cells (Figure 6).

Figure 3. Cholesterol (left bars, gray, left y axis) and proteinu-ria (right bars, dark gray, right y-axis) values at the end of thestudy. In PM and in PM/ACEi treated rats serum cholesterol wassignificantly elevated versus CON, VEH, ACEi treated rats. The inc-rease in serum cholesterol was stronger than expected from pro-teinuria alone. Abbreviations are: VEH, vehicle; ACEi, angiotensinconverting enzyme inhibition; PM, pyridoxamine; CON, healthycontrol. *p<0.05 versus all other groups (proteinuria), +p<0.05versus VEH, ACEi and CON (cholesterol).

Figure 6. Representative photograph of a kidney section double stained for ma-crophages and Oil red O stainable lipids. No colocalization of macrophages withlipid-positive cells was observed. Section shown is from a rat treated with pyri-doxamine.


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Figure 4. Magnifications are 200x. Upper 5 panels: re-presentative figures of histology. Lower 5 pa-nels: representative figures of Oil red O stainedkidney sections. Renal morphology. The firstof the 5 upper panels is a photograph of a PASstained section from a kidney of a rat with adri-amycin nephrosis treated with vehicle (VEH).Glomerular and interstitial damage was obser-ved, as evidenced by focal glomerulosclerosisand marked tubular basement membrane thic-kening. The photograph of the section from akidney of a proteinuric rat treated with lisinopril(ACEi) shows that the overall extent of tubularand glomerular damage is less severe than invehicle treated rats. In the representative exam-ple of a section from a proteinuric rat treatedwith pyridoxamine (PM) severe glomeruloscle-rosis and expansion of the interstitial space, withthe presence of atrophied and dilated tubulesand thickened tubular basement membranes ispresent. The photograph of a section from a ratwith adriamycin nephrosis treated with the com-bination of pyridoxamine and lisinopril(PM/ACEi) shows that in this group more andseverer renal structural damage in glomeruli aswell as interstitium was observed compared toall other groups. For comparison of renal mor-phology, a photograph of a section from a he-althy control kidney (CON) is presented as well.Glomerular lipid deposition. The first of the fivelower panels is a photograph of a section froma healthy control kidney (CON) in which no renallipid deposition was observed. The photographof an Oil red O stained section from a kidney ofa rat with adriamycin nephrosis treated with ve-hicle (VEH) shows that some accumulation ofOil red O stainable lipids was observed in glo-meruli. In the photograph of section from a kid-ney of a proteinuric rat treated with lisinopril(ACEi) no renal lipid deposition was observed.The representative photograph of a Oil red Ostained kidney section from a rat treated withpyridoxamine (PM) shows that massive glome-rular accumulation of lipid positive cells was pre-sent. In addition, focal accumulation oflipid-positive cells in proximal tubules was ob-served. Also, in rats treated with the combinationpyridoxamine and lisinopril (PM/ACEi) massiveglomerular accumulation of lipid positive cellswas observed. This was even more pronouncedthan in rats treated with pyridoxamine alone asis shown in the lower right panel.


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Discussion

In contrast to previous findings in experimental chronic allograft nephropathy from ourown laboratory7 and in rat models of diabetic nephropathy4 and obese Zucker rats by oth-ers,8 PM in a dose similar to these prior studies was not renoprotective in adriamcyin-in-duced proteinuria, neither as a monotherapy, nor in combination with ACEi. By contrast,

Figure 5. Immunohistochemical analysis of pentosidine. Pentosidine wasfound in the brush border and cytoplasm of dilated tubular structures. Pre-sumably, this reflects impaired clearance of filtered and reabsorbed AGEsby the dysfunctional tubular cells. We did not observe a clear-cut inhibitoryeffect of PM on tubular AGE accumulation. In contrast, in PM/ACEi-trea-ted rats, total immunoreactive tubular pentosidine was most pronounced,consistent with the presence of severe interstitial renal damage.


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renal damage was markedly aggravated when PM was used in combination with ACEi.

PM is generally believed to be a non-toxic, safe and well tolerated vitamin from the B6family, which inhibits the glycoxidative breakdown of Amadori products to AGEs, a keystep in the formation of AGEs.6;8;19 Unexpectedly, we found aggravated renal glomerularand interstitial damage along with renal function decline in our adriamycin nephrotic ratswhen the AGE formation inhibitor PM was given in combination with standard an-tiproteinuric treatment with ACEi. In contrast, previous studies found renoprotective,and sometimes added effects of combined intervention in the RAAS and in advancedglycation, two different pathways of renal damage. In non-hypertensive diabetic nephropa-thy in B6 db/db mice, the progression of albuminuria and glomerular lesions was reducedby the combination of PM and the ACEi enalapril. In diabetic spontaneously hyperten-sive rats the combination of the AGE-inhibitor aminoguanidine and the ACEi perindo-pril attenuated the development of albuminuria more effectively than eithermonotherapy.20 In normotensive streptozocin-diabetic rats no additional renoprotectiveeffect of the addition of the AGE cross-link breaker alagebrium to ACEi with ramiprilwas observed.21 However, adverse renal effects of combined intervention as observed in ourstudy have not been described previously.

In our non-diabetic nephrotic rats PM monotherapy did not reduce proteinuria nor ame-liorated renal morphological abnormalities. In previous studies, PM prevented the rise inproteinuria or, in line with our current data, was without effect.22 We observed a gradualincrease in blood pressure during PM monotherapy, which is at variance with data in pre-vious studies, in which PM either prevented the rise in blood pressure8 or was without ef-fect.6;22 Renal damage in our PM groups was accompanied by an increase in serumcholesterol which was stronger than expected from proteinuria alone, and by pronouncedglomerular lipid deposition. These effects, which were observed in both PM treatedgroups, have not been described previously. Remarkably, renal ACE activity in PM treatedrats was lower than in untreated adriamycin nephrosis. The reduction in ACE activity byPM has been observed previously,22 but in our study this was apparently not associatedwith a renoprotective effect. Body weight was significantly lower in both PM treatedgroups compared to the other groups, in line with adverse effects of PM in these rats. An-other explanation of the lower body weight in the PM/ACEi group could be volume de-pletion, as this group had not only an elevated serum creatinine, but also a disproportionallyhigh urea, consistent with a component of pre-renal failure. However, this does not ex-


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plain the lower body weight of the PM-treated group, that did not have elevated serumurea, creatinine or potassium levels. In our previous study in rats with CAN, PM treatedFisher-Fisher isograft controls gained less weight than the vehicle treated controls.7 Oth-ers have observed a decrease in body weight in PM treated diabetic rats,4 but in most stud-ies, PM had no effect on body weight.6;8;23;24

Unexpectedly, combined therapy of PM and ACEi abrogated the renoprotective effectsof ACEi. After an initial decrease in proteinuria and blood pressure, these parameters re-turned to their baseline values during long term therapy. Unexpectedly, the combinationof PM/ACEi was associated with a marked increase in glomerular and interstitial renaldamage, with a pronounced glomerular influx of macrophages. This was associated withrenal function impairment as well. In AN, renal AGEs accumulated in dilated tubularstructures, which is in accordance with our prior findings in AN and in CAN.7;10 Pre-sumably, this reflects impaired clearance of filtered and reabsorbed AGEs by the dys-functional tubular cells. We did not observe a clear-cut inhibitory effect of PM on tubularAGE accumulation. In contrast, in PM/ACEi-treated rats, total immunoreactive tubularpentosidine was most pronounced, consistent with the presence of severe interstitial renaldamage. To test whether the abrogation of the renoprotective effects of ACEi might bedue to a pharmacological interaction, we measured renal ACE activity. This was similarlydecreased in the ACEi and the PM/ACEi group, so apparently PM did not interfere withthe primary pharmacological effect of the ACEi. Also, model-related factors should betaken into account, as under certain circumstances the adriamycin model is resistant totherapy.25 However, ACEi monotherapy reduced proteinuria and focal glomerulosclero-sis, supporting the responsiveness to ACEi intervention in our experimental animals.

Our study was not designed to unravel the mechanisms underlying the detrimental renaleffects of combined therapy of PM with ACEi, but several inferences can be made. It islikely that hypertension, hypercholesterolaemia and intrarenal fat deposition, as observedin both PM groups, contributed to the renal damage. The role of hypertension as a pro-moter of renal damage is well-established.26 Lipid deposition could be another mecha-nism of renal damage observed here, as marked glomerular lipid deposition was observed,in particular in the PM/ACEi rats. Lipid and lipoprotein abnormalities secondary tonephrotic syndrome can contribute to the vicious circle of proteinuria-associated pro-gressive renal damage.27;28 Lipid deposition in the kidney occurs in several animal modelsof renal damage such as experimental diabetes, and obese Zucker rats29;30 as well as in man.


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In humans, abnormal lipid accumulation has been found in non-fibrotic glomeruli of pa-tients with glomerulopathy.31 A recent study showed co-localization of renal lipid depo-sition and increased TGF-β mRNA expression in rats,32 suggesting that renal lipiddeposition can trigger a profibrotic response in the kidney, and thus may play a patho-genetic role in renal damage. It should be noted that prior studies with PM in rat modelsof renal disease were generally conducted in models where proteinuria was onlymild,4;6-8;21-23 whereas the AN in our study was characterized by overt nephrotic range pro-teinuria. Therefore, it cannot be excluded that the severity of the proteinuria in our modelwas a predisposing factor for the adverse renal effects of the PM/ACEi regimen. Furtherstudies are obviously needed to elucidate the mechanisms underlying the adverse renal ef-fects of the combination of PM and ACEi.

Adverse effects of PM have been suggested by reports showing that pyridoxine, anothervitamin from the B6 family, causes dose and duration dependent sensory neuropathy.33;34

This vitamin B6 associated neuropathy is enhanced by uremia35 and a protein-deficientdiet.36 Unfortunately, these pyridoxine toxicity studies did not investigate renal morphol-ogy and the uremia study was done in an anephric rat model. Furthermore, recent phase IIclinical trials in type 1 and type 2 diabetic patients with overt nephropathy (PYR-206 andPYR-205/207) show that there were slightly more deaths and serious adverse events in thePM compared to the placebo treated group.37 In these studies PM was given for six monthsin addition to standard treatment with ACEi and/or angiotensin II type 1 receptor block-ade.37 Although these were randomized trials, the authors ascribed the adverse effects tothe existence of medical conditions at baseline predisposing patients in the PM group tothe adverse events.

PM is emerging as a promising therapeutic agent, now on the FDA “fast track” to phase IIIclinical trials for treatment of diabetic nephropathy.38 However, in a dose that was previouslyrenoprotective in animal models of diabetic nephropathy,4;6 Zucker obese rats,8 and ex-perimental chronic allograft nephropathy,7 PM was not renoprotective in AN. By con-trast, renal damage was aggravated when PM was used in combination with ACEi. Theserenal effects need further analysis. Despite the fact that is no current evidence that thesefindings apply to the drug as used in diabetic nephropathy in humans, we caution that in fu-ture studies with PM in renal patients close monitoring of blood pressure, lipids and pos-sible direct toxic effects is needed, especially when adding PM to ACEi.


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Acknowledgements

This study was supported by the Dutch Kidney Foundation (C03.2055). The compoundpyridoxamine was a kind gift from Thorsten Degenhardt (BioStratum Incorporated,Durham, NC 27703). These data were presented at the 39th Annual Meeting of theAmerican Society of Nephrology, San Diego, CA, November 14 through 19, 2006 ( JAm Soc Nephrol 17: 433A). We would like to thank Pieter Klok, Ingrid van Veen andMarian Bulthuis for their skilled (bio)technical assistance.None of the authors have involvements that might raise the question of bias in the workreported nor in the conclusions, implications or opinions stated.


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2. Tanji N, Markowitz GS, Fu C et al. Expression of advanced glycation end products and their cellular receptorRAGE in diabetic nephropathy and nondiabetic renal disease. J Am Soc Nephrol 2000; 11: 1656-1666

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