BACKGROUND: Inhibition of the renin-angiotensin-aldosterone system (RAAS) provides renoprotection in adriamycin nephropathy (AN), along with a decrease in overexpression of glomerular heparanase. Angiotensin II (AngII) and reactive oxygen species (ROS) are known to regulate heparanase expression in vivo. However, it is unknown whether this is also the case for aldosterone. Therefore, we further assessed the role of aldosterone, AngII and ROS in the regulation of glomerular heparanase expression. METHODS: Six weeks after the induction of AN, rats were treated with vehicle (n = 8), lisinopril (75 mg/L, n = 10), spironolactone (3.3 mg/day, n = 12) or the combination of lisinopril and spironolactone (n = 14) for 12 weeks. Age-matched healthy rats served as controls (n = 6). After 18 weeks, renal heparanase and heparan sulfate (HS) expression were examined by immunofluorescence staining. In addition, the effect of aldosterone, AngII and ROS on heparanase expression in cultured podocytes was determined. RESULTS: Treatment with lisinopril, spironolactone or their combination significantly blunted the increased glomerular heparanase expression and restored the decreased HS expression in the GBM. Addition of aldosterone to cultured podocytes resulted in a significantly increased heparanase mRNA and protein expression, which could be inhibited by spironolactone. Heparanase mRNA and protein expression in podocytes were also significantly increased after stimulation with AngII or ROS. CONCLUSIONS: Our in vivo and in vitro results show that not only AngII and ROS, but also aldosterone is involved in the regulation of glomerular heparanase expression.

Tubulointerstitial lesions are important in the progression of proteinuric renal disease. Tubular Kim-1 is induced in acute renal injury and reversible as a natural course. Kim-1 is also present in chronic renal damage; however dynamics of Kim-1 in chronic renal damage and effects of antiproteinuric treatment on Kim-1 are unknown. We studied Kim-1 in adriamycin nephrosis (AN) before and after RAS-blockade. A renal biopsy was taken 6 wks after adriamycin injection to study renal damage and Kim-1 expression. Subsequently, ACE-inhibition (ACEi, n=23), angiotensin II-antagonist (AT1A, n=23) or vehicle (n=10) was given for 6 wks, healthy rats served as controls (CON, n=8). In AN renal Kim-1 mRNA was induced 26-fold vs CON at wk 6, with further increase in vehicle to wk 12 (40-fold), but reduced by ACEi and AT1A to 10- and 12-fold vs CON (p<0.05 vs wk 6). Kim-1 protein was undetectable in CON; in AN it was present in brush border of dilated tubules in areas with adjacent interstitial lesions. Renal Kim-1 protein levels increased from wk 6 to 12 in vehicle and decreased in ACEi and AT1A-treated groups (p<0.05). In vehicle, urinary Kim-1 was increased (p<0.05 vs CON), with a reduction by ACEi and AT1A (p<0.05 vs vehicle). Renal and urinary Kim-1 correlated with proteinuria and interstitial damage cross-sectionally. Reductions in proteinuria and renal Kim-1 correlated, which was not associated by corresponding changes in tubulo-interstitial fibrosis. In conclusion, on longitudinal follow-up during antiproteinuric treatment increased renal Kim-1 expression is reversible in proportion to proteinuria reduction, likely reflecting reversibility of early tubular injury, supporting its potential as a biomarker for tubulo-interstitial processes of damage and repair. Key words: proteinuria, ACE inhibitors, proximal tubule, chronic kidney disease.

BACKGROUND: Minimal change nephrotic syndrome (MCNS) is the most frequent form of nephrotic syndrome in childhood. In the glomerular basement membrane (GBM) of adult patients with MCNS, a reduced expression of a specific heparan sulphate (HS) domain has been reported. In children with MCNS, urinary activity of the HS-degrading enzyme heparanase was increased. It is, therefore, possible that a decreased GBM HS expression is associated with the pathogenesis of proteinuria in patients with MCNS. METHODS: In this study, HS in glomeruli of five adult and six paediatric patients with MCNS were analysed by immunofluorescence staining using four different antibodies, each defining a specific sulphated HS domain. The pediatric patients were subdivided into three groups depending on the presence or absence of podocyte foot process effacement, the level of proteinuria and prednisone administration at the time of the biopsy. In addition, kidneys of rats with adriamycin nephropathy (ADRN), a model for MCNS, were included in the study. RESULTS: Expression of sulphated HS domains was not aberrant in adult or paediatric patients compared with control subjects. Children with and without proteinuria had the same HS content. In contrast, rats with ADRN showed a decreased glomerular expression of sulphated HS domains. CONCLUSIONS: These results suggest that in patients with MCNS proteinuria is not associated with major changes in glomerular expression of sulphated HS domains.

Aldosterone has pro-fibrotic properties and is a potential target for additional intervention in patients with chronic renal disease showing resistance to therapy during treatment with angiotensin-converting enzyme inhibitors (ACEi). Combining ACEi and aldosterone receptor blockade (aldoRB) in proteinuric renal disease reduces proteinuria, but effects on proteinuria-induced renal damage are unknown. We studied the effect of ACEi/aldoRB in adriamycin nephrosis (AN). Six weeks after injection of adriamycin in Wistar rats, randomized treatment with vehicle (VEH, n=8), aldoRB (n=12), ACEi (n=10), or a combination of ACEi/aldoRB (n=14) was given for 12 weeks. Healthy rats served as controls (n=6). Renal damage was quantified by markers of tubular injury (osteopontin (OPN) and kidney injury molecule-1 (Kim-1)), pre-fibrotic lesions (alpha-smooth muscle actin (SMA)), interstitial fibrosis (IF), and focal glomerulosclerosis (FGS). In AN animals, proteinuria was increased compared with controls. ACEi and ACEi/aldoRB significantly reduced proteinuria compared with VEH, whereas aldoRB monotherapy was without effect. Blood pressure was reduced in ACEi and ACEi/aldoRB compared with VEH and aldoRB. OPN and Kim-1 were increased in AN animals, but significantly reduced by ACEi/aldoRB. Treatment with ACEi and ACEi/aldoRB prevented an increase of SMA, IF, and FGS. In conclusion, ACEi/aldoRB effectively reduced proteinuria and markers of tubular injury and prevented renal damage in this rat model of chronic proteinuria-induced renal damage.Kidney International advance online publication, 10 January 2007; doi:10.1038/sj.ki.5002075.

Kidney injury molecule-1 (Kim-1) is associated with ischemic and proteinuric tubular injury, however whether dysregulation of the renin-angiotensin system (RAS) can also induce Kim-1 is unknown. We studied Kim-1 expression in homozygous Ren2 rats, characterized by renal damage through excessive RAS activation. Furthermore, we investigated whether antifibrotic treatment (RAS blockade or p38 MAP kinase inhibition) would affect Kim-1 expression. Seven-wk old homozygous Ren2 rats received a non-hypotensive dose of candesartan (0.05 mg/kg/day s.c.) or the p38 inhibitor SB239063 (15 mg/kg/day s.c.) for 4 wks; untreated Ren2 and Sprague Dawley (SD) rats served as controls. Kim-1 mRNA (qPCR) and protein (IHC) expression were determined and related to markers of prefibrotic renal damage. Urinary Kim-1 was measured in 8-wk old Ren2 and SD rats with and without ACE inhibition (ramipril 1 mg/kg/day in drinking water for 4 wks). Untreated Ren2 rats showed >20-fold increased renal Kim-1 mRNA (ratio Kim-1/GAPDH 75.5+/-43.6 vs 3.1+/-1.0 in SD, p<0.01). Both candesartan (3.1+/-1.5, p<0.01) and SB239063 (9.8+/-4.2, p<0.05) strongly reduced Kim-1 mRNA. Kim-1 protein expression in damaged tubules paralleled mRNA expression. Kim-1 expression correlated with renal osteopontin, alpha-smooth muscle actin and collagen III expression and with tubulointerstitial fibrosis. Damaged tubular segments expressing activated p38 also expressed Kim-1. Urinary Kim-1 was increased in Ren2 (458+/-70 pg/ml) vs SD (27+/-2, p<0.01) and abolished in Ren2 animals treated with ramipril (33+/-5, p<0.01). Kim-1 is associated with the development of RAS-mediated renal damage. Antifibrotic treatment through RAS blockade or p38 MAP kinase inhibition reduces Kim-1 in the homozygous Ren2 model. Key words: Kidney injury molecule-1, Homozygous TGR(mRen2)27, Angiotensin II type 1 receptor, p38 MAP kinase.

Background: Antiproteinuric treatment by ACE inhibition (ACEi) provides renoprotection. However, resistance to antiproteinuric intervention occurs frequently, resulting in progressive renal damage. The extent of renal damage prior to treatment with ACEi reversely correlates with the antiproteinuric effects of ACEi in established adriamycin nephrosis. Sodium restriction enhances the response to ACEi, but whether it can overcome the negative predictive value of preceding renal damage on the therapeutic response is unknown. We studied the impact of preceding renal damage on the efficacy of ACEi in adriamycin nephrosis on different oral sodium loads. Methods: Male Wistar rats were randomly assigned to a low (LS), normal (NS) or high (HS) sodium diet, initiated 1 week before adriamycin induction. At week 6, proteinuria was stabilized (195 +/- 172 mg/24 h), a renal biopsy was performed for analysis of preceding damage and rats were instituted on lisinopril for 6 weeks until sacrifice at week 12. Results: ACEi reduced proteinuria in LS and NS animals. On univariate analysis, the antiproteinuric response was significantly predicted by preceding renal damage (focal glomerulosclerosis, interstitial macrophages and interstitial alpha-smooth muscle cell actin expression). On multivariate analysis, both sodium intake and preceding renal damage independently predicted residual proteinuria during ACEi (R(2) model: 80% and 75% for data after 3 and 6 weeks of therapy, respectively). Conclusion: Our data confirm the predictive value of pretreatment renal damage for the antiproteinuric response to ACEi, despite the fact that the renal damage prior to the ACEi was very mild. The impact of pretreatment damage on the therapeutic response, however, was overcome by low sodium. Thus, the impact of pretreatment damage does not warrant therapeutic nihilism, but rather optimization of therapy response by dietary sodium restriction. Further studies are needed to elucidate whether this also applies to more severe damage, and whether combining ACEi with low sodium diet can improve long-term renal outcome in human. Copyright (c) 2006 S. Karger AG, Basel.

Proteinuria is associated with macrophage-dependent interstitial fibrosis (IF). Osteopontin (OPN), a macrophage chemoattractant, may be involved in the transition of proteinuria to IF but protective properties have also been reported. To elucidate whether OPN may be involved in the proteinuria-induced cascade of tubulointerstitial damage, renal expression of OPN was studied during the development of proteinuria-induced renal damage and during anti-proteinuric intervention with ACE inhibition (ACEi). First, the temporal relationships between proteinuria, interstitial OPN induction, and IF in adriamycin nephrosis (AN), a model of chronic proteinuria-induced renal damage, were studied. Second, the effect of anti-proteinuric treatment on OPN expression was investigated. The time course of OPN induction and markers of renal damage was studied in rats with unilateral AN at 6-week intervals until week 30. In a second study, a renal biopsy was taken 6 weeks after induction of bilateral AN; subsequently, rats were treated with ACEi until termination (week 12). In unilateral AN, proteinuria developed gradually and stabilized at week 10. In proteinuric kidneys, OPN expression was induced from week 12 onwards. Simultaneously, a progressive increase in interstitial macrophages, alpha-smooth muscle actin (alpha-SMA), collagen type III, and focal glomerulosclerosis (FGS) was observed. In bilateral AN, ACEi reduced proteinuria and OPN protein and stabilized fibrosis. In untreated animals, OPN mRNA increased, with stable OPN protein and fibrosis and increased FGS. Thus, in AN, development of proteinuria is followed by up-regulation of OPN along with markers of renal damage. The up-regulation of OPN is reversible by anti-proteinuric treatment without a corresponding reduction in fibrosis. Whereas these data are consistent with a role for OPN in the cascade of transition from proteinuria to fibrosis, intervention with ACEi showed that reduction of OPN does not attenuate established fibrosis.

BACKGROUND: Advanced glycation end-products (AGEs) contribute to the pathogenesis of diabetic glomerulopathy. The role of AGEs in non-diabetic renal damage is not well characterized. First, we studied whether renal AGE accumulation occurs in non-diabetic proteinuria-induced renal damage and whether this is ameliorated by renoprotective treatment. Secondly, we investigated whether renal AGE accumulation was due to intrarenal effects of local protein trafficking. METHODS: Pentosidine was measured (by high-performance liquid chromatography) in rats with chronic bilateral adriamycin nephropathy (AN), untreated and treated with lisinopril. Age-matched healthy rats served as negative controls. Secondly, we compared renal pentosidine in mild proteinuric and non-proteinuric kidneys of unilateral AN and in age-matched controls at 12 and 30 weeks. Intrarenal localization of pentosidine was studied by immunohistochemistry. RESULTS: Renal pentosidine was elevated in untreated AN (0.14+/-0.04 micromol/mol valine) vs healthy controls (0.04+/-0.01 micromol/mol valine, P<0.01). In lisinopril-treated AN, pentosidine was lower (0.09+/-0.02 micromol/mol valine) than in untreated AN (P<0.05). In unilateral proteinuria, pentosidine was similar in non-proteinuric and proteinuric kidneys. After 30 weeks of unilateral proteinuria, pentosidine was increased in both kidneys (0.26+/-0.10 micromol/mol valine) compared with controls (0.18+/-0.06 micromol/mol valine, P<0.05). Pentosidine (AN, week 30) was also increased compared with AN at week 12 (0.16+/-0.06 micromol/mol valine, P<0.01). In control and diseased kidneys, pentosidine was present in the collecting ducts. In proteinuric kidneys, in addition, pentosidine was present in the brush border and cytoplasm of dilated tubular structures, i.e. at sites of proteinuria-induced tubular damage. CONCLUSION: Pentosidine accumulates in non-diabetic proteinuric kidneys in damaged tubules, and renoprotective treatment by angiotensin-converting enzyme (ACE) inhibitors inhibits AGE accumulation, supporting a relationship between abnormal renal protein trafficking, proteinuria-induced tubular damage and tubular pentosidine accumulation. Future studies, applying specific AGE inhibitors, should be conducted to provide insight into the pathophysiological significance of renal AGEs in non-diabetic renal disease.

BACKGROUND: In man, differences in angiotensin-converting enzyme (ACE) levels, related to ACE (I/D) genotype, are associated with renal prognosis. This raises the hypothesis that individual differences in renal ACE activity are involved in renal susceptibility to inflicted damage. Therefore, we studied the predictive effect of renal ACE activity for the severity of renal damage induced by a single injection of adriamycin in rats. METHODS: Renal ACE activity (Hip-His-Leu cleavage by cortical homogenates) was determined by renal biopsy in 27 adult male Wistar rats. After 1 week of recovery, proteinuria was induced by adriamycin [1.5 mg/kg intravenously (i.v.) n = 18; controls, saline i.v. n = 9]. Proteinuria was measured every 2 weeks. After 12 weeks, rats were sacrificed and their kidneys harvested. RESULTS: As anticipated, adriamycin elicited nephrotic range proteinuria, renal interstitial damage and mild focal glomerulosclerosis. Baseline renal ACE positively correlated with the relative rise in proteinuria after adriamycin (r = 0.62, P<0.01), renal interstitial alpha-smooth muscle actin (r = 0.49, P<0.05), interstitial macrophage influx (r = 0.56, P<0.05), interstitial collagen III (r = 0.53, P<0.05), glomerular alpha-smooth muscle actin (r = 0.74, P<0.01) and glomerular desmin (r = 0.48, P<0.05). Baseline renal ACE did not correlate with focal glomerulosclerosis (r = 0.22, NS). In controls, no predictive values for renal parameters were observed. CONCLUSION: Individual differences in renal ACE activity predict the severity of adriamycin-induced renal damage in this outbred rat strain. This supports the assumption that differences in renal ACE activity predispose to a less favourable course of renal damage.