RENAL DISEASE’
IRENE L. NORONHA1, CAMILE ALBA PEREIRA1, HUMBERTO DELLE1, DENISE M. A. MALHEIROS1, SOLOMON MARGOLIN2, IRENE L. NORONHA1
Renal Division, University of Sao Paulo, Brazil1, Marnac, Inc, USA2
Chronic progressive renal disease is characterized by excessive deposition of extracellular matrix leading to the development of fibrosis. The use of angiotensin blockers, such as losartan (LOS), and anti-inflammatory drugs as mycophenolate mofetil (MMF), do not completely block the progression of renal fibrosis. A pos-sible alternative therapeutic approach could be the use of anti-fibrotic drugs, such as pirfenidone. The aim of the present study was to analyze the effect of pir-fenidone as monotherapy and also associated with LOS and MMF, in an exper-(CTGF), Plasminogen activator inhibitor-1 (PAI-1), Tissue inhibitor of
metalloproteinase-1 (TIMP-1) and Type I Collagen (Col I) in kidney tissue were semi-quantitatively determined by RT-PCR and immunohistochemical staining respectively.
Results: Compared with CTR, proteinuria, urinary b2 microgloubumin and Scr were significantly increased in the CAAN group (P < 0.05). Relative area of interstitial fibrosis was also significantly enlarged (P < 0.01). The expressions of TGF-b1, CTGF, PAI-1, TIMP-1 and Col I mRNA and protein were up-regulated (P < 0.01). After intervention with valsartan and bosentan, the up-regulation of the above mentioned parameters were all markedly reduced (P < 0.05). But there was no different between valsartan group and bosentan group.
Conclusion: Valsartan and bosentan might ameliorate renal tubulointerstitial fibrosis, probably through inhibiting the accumulation of extracellular matrix in renal interstitium.
W-PO20057 FUNCTIONAL SIGNIFICANCE OF PI3 KINASE AND MTOR IN THE REGULATION OF RENAL FIBROBLAST FUNCTION
CATHERINE WINBANKS1, TIM HEWITSON1, LAUREN GRIMWOOD1, ANNA GASSER1, IAN DARBY2, GAVIN BECKER1
Royal Melbourne Hospital1, RMIT University2
The recruitment of fibroblasts is an essential part in the pathogenesis of tubu-lointerstitial fibrosis. Despite their significance their regulation remains poorly understood. In this study we investigated the role of specific PI3 kinase/mTOR pathway inhibitors in the regulation of renal fibroblast function.
Rat renal fibroblasts were propagated from kidney tissue post-ureteric obstruc-tion. Specific inhibitors upstream (LY294002) and downstream (rapamycin deriv-ative RAD) of Akt phosphorylation were used to examine the effects on cell kinetics (changes in thymidine incorporation and cell number) and myofibrob-last differentiation (cytochemistry for alpha smooth muscle actin; SMA). A mito-chondrial activity test (MTT dye reduction) and propidium iodide staining were used to substantiate that effects were independent of cell toxicity.
As expected, western blotting confirmed that LY294002 (10 mM) but not RAD prevented phosphorylation of Akt. RAD (10, 100, 200 nM) inhibited serum induced mitogenesis by 53 ± 7%, 50 ± 9% and 42 ± 7% respectively (all p < 0.01 vs basal) with 100, 200 nM RAD decreasing population growth over 5 days by 63 ± 10%, and 65 ± 14% respectively (both p < 0.001 vs basal). Likewise 10 mM LY294002 decreased mitogenesis by 32 ± 12% and population growth by 44 ± 8%
(both p < 0.05 vs basal). Treatment with 100, 200 nM RAD and LY294002 increased the proportion of cells staining for SMA, and therefore myofibroblasts, by 18%, 31% and 20% respectively (all p < 0.05 vs basal, n = 3 each group).
These effects were independent of cell toxicity.
In conclusion, the regulation of fibroblast activity in vitro involves the PI3 kinase pathway and mTOR. Two specific inhibitors have been shown to decrease cell proliferation but interestingly increase myofibroblast differentiation.
These results highlight the potential significance of these pathways in renal fibrogenesis.
W-PO20058 ALL-TRANS RETINOIC ACID MARKEDLY HALTED RENAL DAMAGE OF 5/6 RENAL ABLATION RATS
MAN LI1, AI PENG1, YONG GU1, SHANYAN LIN1
Nephrology Research Institute and Division of Nephrology, Huashan Hospital, Fudan University, China1
Objective: Glomerulosclerosis is a common pathological event to different kidney diseases. Some evidences showed that all trans retinoic acid (atRA) could regress the mesangial cells proliferation and reduce podocytes damage in acute experimental glomerulonephritis, which clued on a hypothesis that atRA halts the development of glomerulosclerosis.
Methods: 5/6 renal ablation rats divided into atRA treated groups (5 mg/kg/d, 10 mg/kg/d and 20 mg/kg/d, n = 8) and vehicle group (n = 8). Health rats consist of sham-operation group (n = 8). Concentrations of atRA in plasma and renal tissues were measured by Reversed Phase High Performance Liquid Chromatog-raphy (RP-HPLC). Glomerulosclerosis was evaluated by glomerulosclerosis index system. activator protein-1 (AP-1) mRNA expressions were quantitated by real-time PCR. The level of TGFb1 was assayed by renal immunohistochemical staining and Western blot.
Results: From 6 to 10 weeks mean artery pressures of all nephroectomic rats treated by atRA became much lower than those of vehicle group, which
paral-imental model of progressive renal disease induced by chronic inhibition of nitric oxide, using L-NAME.
Male Wistar rats were distributed into six groups: Control (n = 5), rats receiving only high salt diet (HS, 3.2% Na); NAME (n = 8), rats receiving L-NAME (200 mg/L) and HS diet during 30 days to induce progressive renal disease; LOS (n = 5), NAME-rats receiving losartan (50 mg/kg/day) in the water; MMF (n = 8), NAME-rats receiving MMF (10 mg/kg/day); PIRF (n = 10), NAME-rats receiv-ing pirfenidone (chow with 500 mg/kg/day); and PIRF + LOS + MMF (n = 10), NAME-rats receiving triple therapy consisting of pirfenidone + losartan + MMF.
The parameters analyzed after thirty days were: body weight (BW), delta body weight (DBW), tail-cuff pressure (TCP), albuminuria (UalbV), glomerulosclero-sis index (GS), % of collapsed glomeruli (collapsed G), and % of interstitial fibro-sis (Int Fibrofibro-sis).
Results: mean ± SEM; ap < 0.05 vs Control; bp < 0.05 vs NAME; cp < 0.05 vs LOS
Control NAME LOS MMF PIRF PIRF + LOS
+ MMF Initial BW 245 ± 25 226 ± 11 231 ± 25 234 ± 12 254 ± 21 223 ± 13 Final BW 368 ± 31 267 ± 18 330 ± 17 315 ± 11a 339 ± 16 334 ± 9a
D BW (%) 50% 18% 43% 44% 33% 50%
TCP 140 ± 8 179 ± 3a 177 ± 0.7a 177 ± 2a 171 ± 7a 171 ± 4a (mmHg)
UalbV 0.6 ± 0.5 83.2 ± 11.8a 20.0 ± 5.0a,b 34.8 ± 17.3a 32.0 ± 10.0a,b 11.0 ± 3.0a,b (mg/24 h)
GS (%) 0.1 ± 0.1 8.3 ± 2.2a 2.2 ± 0.6a,b 2.6 ± 0.8a,b 2.9 ± 0.9a 2.0 ± 0.4a,b Collapsed G 0.2 ± 0.2 9.6 ± 2.5a 0.7 ± 0.4b 5.7 ± 3.0 1.6 ± 1.0b 0.9 ± 0.3b
(%)
Int Fibrosis 0.1 ± 0.0 2.8 ± 0.7a 0.8 ± 0.1b 0.7 ± 0.2b 0.4 ± 0.1b,c 0.4 ± 0.1b,c (%)
After 30 days, NAME-rats developed hypertension, albuminuria, glomeruloscle-rosis and interstitial fibglomeruloscle-rosis, with consequent lower body weight gain. Monother-apy with pirfenidone significantly diminished albuminuria, collapsed G, and interstitial fibrosis, similar to LOS and MMF monotherapies. However, the best renoprotection was achieved with triple therapy (PIRF + LOS + MMF), which significantly decreased albuminuria, glomerulosclerosis, collapsed G and intersti-tial fibrosis, and was associated with a normal weight gain.
These results confirmed that pirfenidone has anti-fibrotic effects. Pirfenidone associated with LOS and MMF induced the best renoprotective effect in this model, and may be considered a promising alternative for the treatment of chronic progressive nephropathy.
W-PO20061 EXPRESISON OF CTGF MRNA AND TGF-BETA 1 MRNA IN TUBULOINTERSTITIAL AREA ASSOCIATED WITH URINARY PROTEIN IN IGA NEPHROPATHY
SACHIKO TAKAHASHI1, TAKAYUKI FUJITA1, YUKI WADA1, KEN ITO1, YOSHINOBU FUKE1, ATSUSHI SATOMURA1, KOICHI MATSUMOTO1
Division of Nephrology and Endocrinology Department of Internal medicine, Nihon University School of medicine1
Background: Tubulointerstitial fibrosis may become the prognostic indicator in IgA nephropathy (IgA-N). Many factors are known to be key mediators of tubulointerstitial fibrosis. Recently, connective tissue growth factor (CTGF) was observed to be strongly upregulated in human proliferative and fibrogenic diseases. In IgA-N, CTGF may play an important role in the development and progression of tubulointerstitial fibrosis. We examined the expression and local-ization of CTGF mRNA and TGF-beta 1 mRNA in the tubulointerstitial area using rapid in situ hybridization and its association with urinary protein.
Methods: Renal biopsy specimens were obtained from 10 patients with IgA-N.
None of the patients had been treated with steroid or immunosuppressive drugs before the renal biopsy. We examined the expression of CTGF mRNA and TGF-beta 1 mRNA in renal tissue using a rapid in situ hybridization, and assessed the relationship between urinary proteins. The clinical parameters were examined at the time of renal biopsy.
Results: Our rapid in situ hybridization method demonstrated the presence of positive staining for CTGF mRNA and TGF-beta 1 mRNA in tubular epithelial cells in all renal tissue specimens. The positive cells for CTGF mRNA and TGF-beta 1 mRNA were higher in patients with heavy proteinuria than the patients with mild proteinuria. Very faint positive signals were observed in the fibrotic area.
Conclusion: These findings suggest that expression of CTGF mRNA and TGF-beta 1 mRNA were upregulated in the tubulointerstitial area and these expressions are associated with urinary protein in IgA-N.
W-PO20062 LOCALISATION OF PROTEOLYTIC ACTIVITY, MATRIX METALLOPROTEINASES (MMP) AND THEIR INHIBITORS (TIMPS) IN EXPERIMENTAL KIDNEY SCARRING
AIMUN K. H. AHMED1, E. KHEDR1, A. M. EL NAHAS1, T. S. JOHNSON1
Sheffield Kidney Institute (University of Sheffield), Northern General Hospital, Sheffield, United Kingdom1
Background: Reduced MMP activity contributes to the ECM accumulation characterising kidney scarring. Homogenate studies show reduced proteolysis occurs predominantly due to overexpression of TIMPs. However homogenate studies fail to take into account in-vivo compartmentalisation and separation and may therefore be artefactual. To address this we have localised both ECM prote-olysis and specific components of the MMP and TIMP families within kidney sections.
Methods: Wistar Rats were subjected to 5/6thsubtotal nephrectomy (SNx) to induce kidney scarring. Groups of 4–6 rats had the remnant kidney recovered between 7 & 120 days following surgery. Overall changes in ECM proteolysis in homogenates was measured. Localisation of proteolysis was performed using in-situ Zymography. Immunhistochemistry was used to localize MMPs 1,2 3 &9 and TIMPs 2&3 within the scarring kidney and TIMP1 by western blotting.
Results: Gelatinases activity was reduced from day 7 post SNx onwards by 49%
(p < 0.01), collagenases activity decreased from day 60 (79% reduction, p < 0.05) onwards.
In-situ Zymography (Collagen IV substrate) showed proteolytic activity in normal kidney to be predominantly cytoplasmic within the tubules. This was decreased by 59 and 81% at 90 and 120 days post SNx respectively. In contrast, glomeru-lar proteolytic activity increased in the SNx, 27 fold by 120 days.
Immunohistochemistry for MMPs 1, 2, 3 & 9 and TIMPs 2&3 confirmed the in-situ zymography location within the tubules. By day 120, MMP1 immunostain-ing increased from 0.1 to 7%, and MMP2 by from 0.27 to 9.36%. In contrast MMP3 decreased by 55% and TIMP2 increased by 31%, Western blot analysis of TIMP1 showed increase by 320.
Conclusion: ECM proteolytic activity is reduced in homogenates from scarred kidneys with changes in MMPs and TIMPs occurring in the same cellular com-partment. However the vast bulk of this proteolytic potential is cytoplasmic and that contradicts to the interstitial location of ECM accumulation in scarring.
W-PO20063 ANTI-FIBROTIC VERSUS PRO-FIBROTIC: DUAL POTENTIAL OF ALL-TRANS RETINOIC ACID IN MESANGIAL CELLS AND RENAL FIBROBLASTS
QIHE XU1, AYDA YEGANEH1, YING HONG2, XIONGZHONG RUAN1, JAVIER LUCIO-CAZANA3, JILL T NORMAN1
Department of Medicine, Royal Free and University College Medical School, London, United Kingdom; Department of Nephrology, General Hospital of Chinese PLA, Beijing, China1, Wolfson Institute for Biomedical Research, Royal Free and University College Medical School, London, United Kingdom2, Departamento de Fisiologia, Facultad de Medicina, Universidad de Alcala, Alcala de Henares, Madrid, Spain3
All-trans retinoic acid (tRA) suppresses both inflammatory and fibrotic changes in experimental nephritides, however, whether tRA has inflammation-independent anti-fibrotic effects remains controversial. We hypothesise that tRA may play an anti-fibrotic role by targeting the major extracellular matrix (ECM)-producing cells, renal mesangial cells and fibroblasts. To test this hypothesis, we examined the effect of tRA (1–5 mM) on the expression of fibrogenic genes in primary cultures of human mesangial cells (HMC) and a rat kidney fibroblast cell line (NRK-49F).
Results: (1) tRA suppressed transforming growth factor-b1 (TGF-b1)-induced fibronectin, collagen III and connective tissue growth factor (CTGF) mRNA expression in both HMC and NRK-49F cells. (2) tRA suppressed TGF-b1-induced collagen I mRNA expression in HMC but not in NRK-49F cells. (3) In HMC, 10 mU/ml glucose oxidase induced expression of fibronectin and colla-gens I, III and IV at the mRNA level, which was blocked by catalase, a H2O2
scavenger, and also by tRA. In contrast, tRA slightly, but reproducibly, enhanced
Forty three untreated patients (35 M, 8 F, and age 37.2 ± 10.5) with biopsy proven GN participated in the study. In the kidney biopsy specimens, the per cent of the sclerotized glomeruli and the intensity of interstitial fibrosis were assessed. In the first freshly voided morning urine sample the concentration of collagen type IV (ELISA method) and creatinine were measured and urinary excretion of the collagen type IV (coll.t.IV) was expressed as coll.t.IV/creatinine ratio. The results obtained in healthy subjects matched for sex and age, served as control (4.7 ± 2.1 ng/gCr).
Significantly elevated urinary coll t.IV excretion (20.0 ± 17.1 ng/gCr) was found in 19 patients with more than 10% of sclerotized glomeruli and pronounced inter-stitial fibrosis compared to 24 patients with less marked morphological changes (7.9 ± 5.7 ng/gCr; p < 0.01). Increased urinary coll t.IV excretion was also found in 12 patients with elevated serum creatinine level (Scr 1.6 ± 0.2 mg/dl) when compared with 31 patients with normal Scr (coll.t.IV 25.1 ± 19.3 ng/gCr vs 8.6 ± 6.6 ng/gCr; p < 0.0005). A correlation between protein and coll.t.IV excretion was found (p < 0.03), but there was no significant difference in coll.t.IV excretion between patients with and without nephritic syndrome, or with and without hypertension. Immunosuppresive therapy was introduced in 30 patients and in 13 patients conservative treatment was recommended. The remis-sion or marked reduction of proteinuria with stabilization of kidney function was reached in 29 patients with urinary coll.t.IV excretion of 5.2 ± 3.0 ng/gCr before treatment. Fourteen patients did not respond to treatment and their urinary coll.t.IV excretion before therapy was markedly higher (29.8 ± 18.4 ng/gCr;
p < 0.000001).
These results suggest that the determination of urinary coll.t.IV excretion before initiation of treatment in patients with GN may serve as the indirect indicator of the severity of kidney injury and as prognostic marker of the response to therapy.
W-PO20066 THE EFFECTS OF ROSIGLITAZONE ON THE EXPERIMENTAL RENAL TUBULOINTERSTITIAL FIBROSIS OF RATS
HUI GAO1, ZI LI1, WEI QIN1, BAIHAI SU1, JING LI1, JUNMING FAN1 Division of Nephrology, Department of Medicine, West China Hospital of Sichuan University, Chengdu, Sichuan, China 6100411
To illuminate whether rosiglitazone exerted a role to block TEMT in vivo and its anti-fibrotic mechanism.
Method: Sixty male wistar rats were randomly assigned into unilateral ureteral obstruction (UUO), rosiglitazone large dose (PLD), rosiglitazone small dose (PSD), sham-operated (SOR) and ACEI groups. From the first day of initial UUO, PLD, PSD and ACEI groups were administrated intragastrically with rosiglitazone 8 mg/kg, 4 mg/kg and fosinopril 10 mg/kg daily. Identical voluminal normal saline was administrated to UUO and SOR groups. At 7, 14, 21, 28 days after UUO, 3 randomly selected rats of each group were sacrificed. Serum crea-tinine, blood cell differential counts, blood-fasting sugar (BFS), glutamate-pyruvate transaminase (GLT), 24-hour urine protein levels were measured. In HE and masson staining pathological slides, tubulointerstitial damage degree was scored. Expression of CTGF, BMP-7, a-SMA, CD68, fibronection (FN), TIMP1, MMP3 were detected by immunohistochemitry. Localization and expression of CTGF and BMP-7 protein were detected by hybridization in situ.
Results: Serum creatinine was significantly higher in UUO group than other groups at day 7, 14, 21, 28 (p < 0.05), but not in 24-hour urine protein, BFS and GLT. Neutrophil count, categorization and expression of CTGF, TIMP1, colIII and FN were increased conspicuously in UUO group (p < 0.01). a-SMA-positive interstitial cells number increased at day 14–21 of UUO. Renal BMP-7 mRNA and protein levels decreased progressively during the progression of obstructive nephropathy. Compared with the UUO group, levels of BMP-7 mRNA and protein increased significantly in PLD, PSD and ACEI groups. Levels of BFS and GLT in PLD and PSD were not increased markedly. Expression of CTGF, a-SMA, FN, ATIMP1, AMMP-3 in PLD and PSD groups were parallel to AECI group.
Conclusion: Rosiglitazone may suppress fibrosis, via down-regulating CTGF and up-regulating BMP-7 expression in affected kidney. It can also extenuate renal inflammation and decrease accumulation of extracellular matrix.
TGF-b1-induced collagen IV and TGF-b1 mRNAs. (4) In HMC, lower con-centrations of H2O2 (glucose oxidase, 1–5 mU/ml) did not induce expression of fibrogenic genes, however, both tRA and catalase enhanced the expression of fibronectin and collagen mRNAs in the presence or absence of 1–5 mU/ml glucose oxidase. (5) In HMC, hypoxia (1% O2), high glucose (30 mM), angiotensin II (1–5 mM), endothelin-1 (5 nM) and CTGF (100 ng/ml) had small and variable effects on ECM mRNA expression but both the basal and inducible expression of fibronectin, collagen III and CTGF mRNAs was suppressed by tRA.
Conclusion: tRA has both anti-fibrotic and pro-fibrotic potential in a cell type-and redox-dependent manner; it exerts its effects by actions on TGFb1-dependent and -inTGFb1-dependent changes. Further investigations are required to evaluate the efficacy of this emerging agent in the treatment and prevention of renal fibrosis.
W-PO20064 GLYCATION OF ALBUMIN MARKEDLY INCREASES ITS NEPHROTOXICITY
MARIE-LUISE PAMELA GROSS1, ANGELIKA BIERHAUS2, MERYEM YÜKSELEM1, KATERINA NEUMANN1, IRINA BERGER1, NADJA KOLEGANOVA1, EBERHARD RITZ2
Institute of Pathology, University of Heidelberg1, Dept. of Internal medicine, University of Heidelberg2
Increasing evidence has been adduced that proteins in tubular fluid are nephro-toxic. After uptake into proximal tubular epithelial cells, an inflammatory phenotype is induced, agonists, cytokines and chemokines are secreted in a pref-erentially abluminal direction and interstitial fibrosis is provoked. In vivo it is difficult to study protein loading of tubular epithelial cells in isolation, i.e.
without glomerular damage, renal hemodynamic changes etc. And there is an ongoing debate whether albumin per se is nephrotoxic.
Recently an amphibian model (Axolotl) has been described which took advan-tage of the unique anatomy of the kidney which is composed of closed as well as
‘open’ nephrons. The latter are connected with the peritoneal cavity by an ori-ficium so that injection of protein i.p. selectively causes protein storage and per-itubular fibrosis.
Endotoxin free preparations of glycated albumin and other forms of metabolized albumin were injected i.p. into Axolotl and the kidneys were obtained after 10 days and studied by light microscopy and immunehistochemistry (TGFb, colla-gen I and IV, PDGF).
Glycated albumin was strikingly more nephrotoxic as indicated by protein storage in tubular epithelial cells and peritubular fibrosis compared to unmodified albumin. Lipid oxidation and carbamylation were only moderately more nephro-toxic as indicated by higher scores.
The data indicate that glycation dramatically increases the nephrotoxicity of albumin.
Table: Renal damage score with different albumin preparations [Score 0– 4]
Protein storage peritubular TGF-b expression fibrosis of tubules
NaCl 1.23 ± 0.5 0.4 ± 0.05 0.45 ± 0.1
Glycated albumin 3.7 ± 0.4* 3.3 ± 0.6* 0.62 ± 0.1
Delipdated 2.4 ± 0.5* 2.45 ± 0.7* 1.0 ± 0.4*
albumin
Albumin lipid 2.6 ± 0.6* 2.3 ± 0.6* 1.16 ± 0.4*
oxidation
Carbamylated 2.8 ± 0.3* 2.67 ± 0.7* 1.49 ± 0.6*
albumin
Albumin 2.74 ± 0.4* 2.87 ± 0.7* 1.0 ± 0.4*
ANOVA p < 0.05 p < 0.05 p < 0.05
* p < 0.05 vs. NaCl injection.
W-PO20065 URINARY EXCRETION OF THE COLLAGEN TYPE IV AS A PROGNOSTIC MARKER IN PATIENTS WITH PRIMARY GLOMERULONEPHRITIS
ILONA IDASIAK-PIECHOCKA1, ANDRZEJ OKO2, ELZBIETA PAWLICZAK2, KRZYSZTOF PAWLACZYK2, STANISLAW CZEKALSKI2
Department of Nephrology, University School of Medical Sciences, Poland1 The aim of this study was an evaluation of urinary excretion of the collagen type IV in patients with newly diagnosed primary glomerulonephritis (GN).