Andrea B. Kramer

Reduction of proteinuria in adriamycin-induced nephropathy is associated with reduction of renal kidney injury molecule (Kim-1) over time

Tubulointerstitial lesions are important in the progression of proteinuric renal disease. Tubular kidney injury molecule-1 (Kim-1) is induced in acute renal injury and reversible as a natural course. Kim-1 is also present in chronic renal damage; however, the 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 renin-angiotensin system blockade. A renal biopsy was taken 6 wk after adriamycin injection to study renal damage and Kim-1 expression. Subsequently, ACE inhibition (ACEi; n = 23), angiotensin II antagonist (AT(1A); n = 23), or vehicle (n = 10) was given for 6 wk; healthy rats served as controls (CON; n = 8). In AN, renal Kim-1 mRNA was induced 26-fold vs. CON at week 6, with further increase in vehicle to week 12 (40-fold) but was reduced by ACEi and AT(1A) to 10- and 12-fold vs. CON (P < 0.05 vs. week 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 weeks 6-12 in vehicle and decreased in ACEi- and AT(1A)-treated groups (P < 0.05). In vehicle, urinary Kim-1 was increased (P < 0.05 vs. CON), with a reduction by ACEi and AT(1A) (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 tubulointerstitial 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 tubulointerstitial processes of damage and repair.

Induction of glomerular heparanase expression in rats with Adriamycin nephropathy is regulated by reactive oxygen species and the renin-angiotensin system

Heparan sulfate (HS) in the glomerular basement membrane (GBM) is important for regulation of the charge-dependent permeability. Heparanase has been implicated in HS degradation in several proteinuric diseases. This study analyzed the role of heparanase in HS degradation in Adriamycin nephropathy (AN), a model of chronic proteinuria-induced renal damage. Expression of heparanase, HS, and the core protein of agrin (to which HS is attached) was determined on kidney sections from rats with AN in different experiments. First, expression was examined in a model of unilateral AN in a time-course study at 6-wk intervals until week 30. Second, rats were treated with the hydroxyl radical scavenger dimethylthiourea (DMTU) during bilateral AN induction. Finally, 6 wk after AN induction, rats were treated with angiotensin II receptor type 1 antagonist (AT1A) or vehicle for 2 wk. Heparanase expression was increased in glomeruli of rats with AN, which correlated with HS reduction at all time points and in all experiments. Treatment with DMTU prevented the increased heparanase expression, the loss of GBM HS, and reduced albuminuria. Finally, treatment of established proteinuria with AT1A significantly reduced heparanase expression and restored glomerular HS. In conclusion, an association between heparanase expression and reduction of glomerular HS in AN was observed. The effects of DMTU suggest a role for reactive oxygen species in upregulation of heparanase. Antiproteinuric treatment by AT1A decreased heparanase expression and restored HS expression. These results suggest involvement of radicals and angiotensin II in the modulation of GBM permeability through HS and heparanase expression.

Regulation of glomerular heparanase expression by aldosterone, angiotensin II and reactive oxygen species

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.

Inter-individual differences in anti-proteinuric response to ACEi in established adriamycin nephrotic rats are predicted by pretreatment renal damage

ACE inhibition (ACEi) reduces proteinuria and provides reno‐protection, but not all subjects benefit from ACEi. Individual differences in the reduction in proteinuria at the onset of treatment and in residual proteinuria during therapy predict differences in renal outcome. The present study investigated whether individual differences in the anti‐proteinuric efficacy of ACEi are explained by differences in the severity of pretreatment renal structural damage and whether differences in the level of residual proteinuria during therapy are explained by the severity of renal structural damage at that time, in adriamycin nephrosis in the rat. Pretreatment renal structural damage was assessed in biopsies 6 weeks after exposure to adriamycin (2 mg/kg iv). Then ACEi (75 mg/l lisinopril, n = 23) or vehicle (n = 10) was administered; renal biopsies were repeated after stabilization of the anti‐proteinuric response (week 8). Early renal damage (interstitial α‐smooth muscle actin expression and macrophage accumulation) and established lesions [focal glomerulosclerosis (FGS) and interstitial fibrosis] were scored. During ACEi, proteinuria fell from 834 (487–851) mg/24 h pretreatment to 153 (66–265) mg/24 h at week 8 (p < 0.05); FGS stabilized from 27 (4–70) arbitrar units (AU) pretreatment to 26 (4–84) at week 12, whereas the vehicle did not affect proteinuria, resulting in progressive FGS: 18 (10–26) AU pretreatment versus 88 (46–130) at week 12 (p < 0.05). All parameters of pretreatment damage significantly predicted the anti‐proteinuric response. Residual proteinuria during ACEi correlated significantly with renal structural damage parameters at that time. Pretreatment renal damage also predicted renal outcome during extended treatment. Thus, in this experimental setting, in rats with the same renal disorder and the same duration of disease, individual differences in pretreatment renal damage, albeit relatively modest, explain individual differences in renal responsiveness to ACEi. This implies that the limits of the efficacy of ACEi are set by prevalent renal damage. Further studies into the mechanisms of individual resistance to the anti‐proteinuric action of ACEi are needed to develop additive intervention strategies. Copyright

Modulation of osteopontin in proteinuria-induced renal interstitial fibrosis

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, α‐smooth muscle actin (α‐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. Copyright

Urinary Vitamin D Binding Protein: A Potential Novel Marker of Renal Interstitial Inflammation and Fibrosis

Non-invasive tubulointerstitial damage markers may allow better titration and monitoring of renoprotective therapy. We investigated the value of urinary vitamin D binding protein excretion (uVDBP) as a tubulointerstitial inflammation and fibrosis marker in adriamycin rats, and tested whether uVDBP parallels renal damage and responds to therapy intensification in humans. In adriamycin (ADR) rats, uVDBP was strongly elevated vs controls (CON) already 6 wks after nephrosis induction (ADR: 727±674 [mean±SD] vs CON: 9±12 µg/d, p<0.01), i.e. before onset of pre-fibrotic and inflammatory tubulointerstitial damage, and at all following 6-wk time points until end of follow up at 30 wks (ADR: 1403±1026 vs CON: 206±132 µg/d, p<0.01). In multivariate regression analysis, uVDBP was associated with tubulointerstitial macrophage accumulation (standardized beta = 0.47, p = 0.01) and collagen III expression (standardized beta = 0.44, p = 0.02) independently of albuminuria. In humans, uVDBP was increased in 100 microalbuminuric subjects (44±93 µg/d) and in 47 CKD patients with overt proteinuria (9.2±13.0 mg/d) compared to 100 normoalbuminuric subjects (12±12 µg/d, p<0.001). In CKD patients, uVDBP responded to intensification of renoprotective therapy (ACEi+liberal sodium: 9.2±13.0 mg/d vs dual RAAS blockade+low sodium: 2747±4013, p<0.001), but remained still >100-fold increased during maximal therapy vs normoalbuminurics (p<0.001), consistent with persisting tubulointerstitial damage. UVDBP was associated with tubular and inflammatory damage markers KIM-1 (standardized beta = 0.52, p<0.001), beta-2-microglobuline (st.beta = 0.45, p<0.001), cystatin C (st.beta = 0.40, p<0.001), MCP-1 (st.beta = 0.31, p<0.001) and NGAL (st.beta = 0.20, p = 0.005), independently of albuminuria. UVDBP may be a novel urinary biomarker of tubulointerstitial damage. Prospectively designed studies are required to validate our findings and confirm its relevance in the clinical setting.

Sodium intake modifies the negative prognostic value of renal damage prior to treatment with ACE inhibitors on proteinuria induced by adriamycin

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 α-smooth muscle cell actin expression). On multivariate analysis, both sodium intake and preceding renal damage independently predicted residual proteinuria during ACEi (R2 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.

Proteinuria Triggers Renal Lymphangiogenesis Prior to the Development of Interstitial Fibrosis

Proteinuria is an important cause of progressive tubulo-interstitial damage. Whether proteinuria could trigger a renal lymphangiogenic response has not been established. Moreover, the temporal relationship between development of fibrosis, inflammation and lymphangiogenesis in chronic progressive kidney disease is not clear yet. Therefore, we evaluated the time course of lymph vessel (LV) formation in relation to proteinuria and interstitial damage in a rat model of chronic unilateral adriamycin nephrosis. Proteinuria and kidneys were evaluated up to 30 weeks after induction of nephrosis. LVs were identified by podoplanin/VEGFR3 double staining. After 6 weeks proteinuria was well-established, without influx of interstitial macrophages and myofibroblasts, collagen deposition, osteopontin expression (tubular activation) or LV formation. At 12 weeks, a ∼3-fold increase in cortical LV density was found (p<0.001), gradually increasing over time. This corresponded with a significant increase in tubular osteopontin expression (p<0.01) and interstitial myofibroblast numbers (p<0.05), whereas collagen deposition and macrophage numbers were not yet increased. VEGF-C was mostly expressed by tubular cells rather than interstitial cells. Cultured tubular cells stimulated with FCS showed a dose-dependent increase in mRNA and protein expression of VEGF-C which was not observed by human albumin stimulation. We conclude that chronic proteinuria provoked lymphangiogenesis in temporal conjunction with tubular osteopontin expression and influx of myofibroblasts, that preceded interstitial fibrosis.

Budesonide and risk of pneumonia

www.thelancet.com Vol 374 December 19/26, 2009 2051 local immunity. This explanation seems unlikely because budesonide is esterifi ed, leading to sustained retention in the airways and indeed this process may explain the effi cacy of once-daily inhaled budesonide. An alternative explanation may be the eff ect of inhaled steroids on the oropharyngeal fl ora. There is evidence that the use of inhaled steroids is associated with increased colonisation of the mouth with gram-negative bacilli. It is believed that bacterial pneumonia is the result of aspiration of oropharyngeal fl ora into the lower respiratory tract. All of the studies reported in the meta-analysis by Sin and colleagues used budesonide delivered from a turbuhaler. The use of a turbuhaler leads to lower oropharyngeal deposition of budesonide than the same medicine delivered from a metereddose inhaler. It is possible that budesonide delivered from a turbuhaler is associated with less pneumonia in patients with COPD because reduced oropharyngeal deposition of the inhaled steroid results in less change in the oral fl ora. If this hypothesis is correct, the use of a spacer with inhaled steroids delivered from a metered-dose inhaler may also reduce the risk of pneumonia because spacers are associated with a reduction in oropharyngeal deposition of inhaled steroids. Further studies are necessary to explore this possibility.