Tubulointerstitial fibrosis in chronic renal disease is strongly associated with progressive loss of renal function. We studied the potential involvement of lysophosphatidic acid (LPA), a growth factor-like phospholipid, and its receptors LPA 1-4 in the development of tubulointerstitial fibrosis (TIF). Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) for up to 8 d, and kidney explants were prepared from the distal poles to measure LPA release into conditioned media. After obstruction, the extracellular release of LPA increased approximately 3-fold. Real-time reverse transcription PCR (RT-PCR) analysis demonstrated significant upregulation in the expression of the LPA 1 receptor subtype, downregulation of LPA 3 , and no change of LPA 2 or LPA 4 . TIF was significantly attenuated in LPA 1 (Ϫ/Ϫ) mice compared to wild-type littermates, as measured by expression of collagen III, ␣-smooth muscle actin (␣-SMA), and F4/80. Furthermore, treatment of wild-type mice with the LPA 1 antagonist Ki16425 similarly reduced fibrosis and significantly attenuated renal expression of the profibrotic cytokines connective tissue growth factor (CTGF) and transforming growth factor ␤ (TGF␤). In vitro, LPA induced a rapid, dose-dependent increase in CTGF expression that was inhibited by Ki16425. In conclusion, LPA, likely acting through LPA 1 , is involved in obstruction-induced TIF. Therefore, the LPA 1 receptor might be a pharmaceutical target to treat renal fibrosis.

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Background-The physiological effects of ACE inhibitors may act in part through a kinin-dependent mechanism. We investigated the effect of chronic ACE-inhibitor treatment on functional kinin B 1 -and B 2 -receptor expression, which are the molecular entities responsible for the biological effects of kinins. Methods and Results-Rats were subjected to different 6-week treatments using various mixtures of the following agents:ACE inhibitor, angiotensin AT 1 -receptor antagonist, and B 1 -and B 2 -receptor antagonists. Chronic ACE inhibition induced both renal and vascular B 1 -receptor expression, whereas B 2 -receptor expression was not modified. Furthermore, with B 1 -receptor antagonists, it was shown that B 1 -receptor induction was involved in the hypotensive effect of ACE inhibition. Using microdissection, we prepared 10 different nephron segments and found ACE-inhibitor-induced expression of functional B 1 -receptors in all segments. ACE-inhibitor-induced B 1 -receptor induction involved homologous upregulation, because it was prevented by B 1 -receptor antagonist treatment. Finally, using B 2 -receptor knockout mice, we showed that ACE-inhibitor-induced B 1 -receptor expression was B 2 -receptor independent. Conclusions-This study provides the first evidence that chronic ACE-inhibitor administration is associated with functional vascular and renal B 1 -receptor induction, which is involved in ACE-inhibitor-induced hypotension. The observed B 1 -receptor induction in the kidney might participate in the known renoprotective effects of ACE inhibition.

Renal fibrosis is the common histological feature of advanced glomerular and tubulointersti‐tial disease leading to end‐stage renal disease (ESRD). However' specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persis‐tent inflammation is a key event in the development of fibrosis' we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that' in the unilateral ureteral obstruction model of renal fibrosis' the B1R is overexpressed and that delayed treatment with an orally active non‐peptide B1R antagonist blocks macrophage infiltration' leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.—Klein, J., Gonzalez, J., Duchene, J., Esposito, L., Pradere, J. P., Neau, E., Delage, C., Calise, D., Ahluwalia, A., Carayon, P., Pesquero, J. B., Bader, M., Schanstra, J. P., Bas‐cands, J. L. Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy. FASEBJ. 23′ 134‐142 (2009)

Severe inflammation characterizes rapidly progressive glomerulonephritides, and expression of the kinin B1 receptor (B1R) associates with inflammation. Delayed B1R blockade reduces renal inflammation in a model of unilateral ureteral obstruction, but whether B1R modulates the pathophysiology of glomerulonephritides is unknown. Here, we observed an association of B1R protein expression and inflammation, in both glomeruli and the renal interstitium, in biopsies of patients with glomerulonephritides, HenochSchö nlein purpura nephropathy, and ANCA-associated vasculitis. In the nephrotoxic serum-induced glomerulonephritis model, we observed upregulation of the B1R receptor; treatment with a B1R antagonist beginning 2 weeks after the onset of disease reduced both glomerular and tubular lesions and improved renal function. B1R blockade reduced renal chemokine expression and macrophage accumulation. Collectively, our data demonstrate that blockade of the kinin B1R has significant potential for the treatment of glomerulonephritis. Rapidly progressive glomerulonephritides constitute a group of kidney diseases sharing common pathologic features. These nephropathies are characterized by severe glomerular inflammation, mainly composed of infiltrating macrophages and T cells. This leads to the formation of glomerular crescents composed of immune and proliferating epithelial cells from the Bowman's capsule. After the primary glomerular insult, inflammation and lesions extend to the tubulointerstitial compartment and induce tubular damage and progressive interstitial fibrosis, leading to the loss of renal function; therefore, renal inflammation is a key player in the initiation and the progression of these pathologies. It has been suggested that any strategy or agent able to limit or attenuate renal inflammation should significantly slow the progression of glomerulonephritides to ESRD. 1 Endogenous kinins (bradykinin-related peptides)are produced through cleavage of kininogens by kallikreins. These peptides mediate their biologic effects by activation of two G protein-coupled receptors: a constitutively expressed B2 receptor (B2R) and an inducible B1 receptor (B1R). 2 Bradykinin and kallidin are the natural agonists of the B2R, whereas their metabolites, generated by the enzyme kininase I, desArg9-BK and Lys-des-Arg9-BK, respectively, are spe-

Angiotensin-converting enzyme (ACE) inhibitors reduce the progression of various fibrotic renal diseases both in humans and in animal models. Unilateral ureteral obstruction (UUO) is an animal model of accelerated renal tubulointerstitial fibrosis that is attenuated by ACE inhibition. Although ACE inhibitors increase bradykinin concentrations in addition to their effect on angiotensin II formation, the role of bradykinin in renal fibrosis has not been studied. We show here that genetic ablation (B2(-/-) mice) or pharmacological blockade of the bradykinin B2 receptor increases UUO-induced interstitial fibrosis in mice, whereas transgenic rats expressing increased endogenous bradykinin show reduced UUO-induced interstitial fibrosis. The increased interstitial fibrosis in B2(-/-) mice was accompanied by a decreased activity of plasminogen activators (PAs) and metalloproteinase-2 (MMP-2), enzymes involved in ECM degradation, suggesting that the protective effects of bradykinin involve activation of a B2 receptor/PA/MMP-2 cascade. This ability of bradykinin to increase PA activity was confirmed in primary culture proximal tubular cells. Thus, in both mice and rats, bradykinin B2 receptor activation reduces renal tubulointerstitial fibrosis in vivo, most likely by increasing ECM degradation.

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We investigated the effects of a 3-week treatment with various combinations of angiotensin-converting enzyme inhibitor (ACEI) and B1 and B2 bradykinin receptor (B1R and B2R) antagonists (B1A and B2A) and AT1 receptor antagonist on ERK 1 and 2 phosphorylation in isolated glomeruli from streptozotocin-treated diabetic rats (STZ rats). Body weight, glycemia, and blood pressure were monitored. The rats were divided into nine groups: (1) control; and groups 2-9 were STZ treated with (3) insulin, (4) ACEI, (5) ACEI + B1A, (6) ACEI + B2A, (7) B2A, (8) B1A, (9) AT1 antagonist. ERK 1 and 2 phosphorylation and expression of B1R and B2R were assessed by Western blot analysis. ERK 1 and 2 phosphorylation was higher in STZ rats; this activation was normalized by insulin and reduced by ACEI but not by AT1 antagonist. The reduction of ERK 1 and 2 phosphorylation by the ACEI was reversed by B1A and B2A. The induction of B1R was confirmed by increased expression of mRNA and B1 receptor protein. Since ERK 1 and 2 phosphorylation is an early event in the induction of matrix secretion and hyperproliferation associated with diabetic nephropathy, activation of B1R and B2R appears to be a useful pharmacological target in the management of this pathology.