Luciana G. Pereira

ACE-Dependent and Chymase-Dependent Angiotensin II Generation in Normal and Glucose-Stimulated Human Mesangial Cells

High glucose (HG) increases angiotensin II (AngII) generation in mesangial cells (MC). Chymase, an alternative AngII-generating enzyme, is upregulated in the glomeruli of diabetic kidneys. In this study, we examined AngII synthesis by human MC via angiotensin-converting enzyme (ACE)-dependent and chymase-dependent pathways under normal glucose (NG, 5 mM) and HG (30 mM) conditions. NG cells expressed ACE and chymase mRNA. Under NG conditions the chymase inhibitor chymostatin reduced AngII levels in cell lysates and in the culture medium, and the ACE inhibitor captopril had no effect. HG induced a 3-fold increase in chymase mRNA and protein but not in ACE mRNA; however, HG induced a 10-fold increase in intracellular ACE activity. The increase in AngII generation induced by HG was found in the cell lysate but not in the culture medium. The rise in intracellular AngII was not prevented by captopril or by chymostatin. Moreover, captopril inhibited extracellular ACE activity but failed to block intracellular ACE activity; these results suggested that captopril was unable to reach intra-cellular ACE. Losartan did not change the intracellular AngII content in either NG or HG conditions, and this lack of change suggested that the increase in AngII was due to intracellular generation. Together these results suggest that chymase may be active in human MC and that both ACE and chymase are involved in increased AngII generation during the HG stimulus by different mechanisms, including an upregulation of chymase mRNA and a rise in intracellular ACE activity, favoring the generation and accumulation of intracellular AngII.

Mesenchymal stem cells (MSC) prevented the progression of renovascular hypertension, improved renal function and architecture.

Renovascular hypertension induced by 2 Kidney-1 Clip (2K-1C) is a renin-angiotensin-system (RAS)-dependent model, leading to renal vascular rarefaction and renal failure. RAS inhibitors are not able to reduce arterial pressure (AP) and/or preserve the renal function, and thus, alternative therapies are needed. Three weeks after left renal artery occlusion, fluorescently tagged mesenchymal stem cells (MSC) (2×105 cells/animal) were injected weekly into the tail vein in 2K-1C hypertensive rats. Flow cytometry showed labeled MSC in the cortex and medulla of the clipped kidney. MSC prevented a further increase in the AP, significantly reduced proteinuria and decreased sympathetic hyperactivity in 2K-1C rats. Renal function parameters were unchanged, except for an increase in urinary volume observed in 2K-1C rats, which was not corrected by MSC. The treatment improved the morphology and decreased the fibrotic areas in the clipped kidney and also significantly reduced renal vascular rarefaction typical of 2K-1C model. Expression levels of IL-1β, TNF-α angiotensinogen, ACE, and Ang II receptor AT1 were elevated, whereas AT2 levels were decreased in the medulla of the clipped kidney. MSC normalized these expression levels. In conclusion, MSC therapy in the 2K-1C model (i) prevented the progressive increase of AP, (ii) improved renal morphology and microvascular rarefaction, (iii) reduced fibrosis, proteinuria and inflammatory cytokines, (iv) suppressed the intrarenal RAS, iv) decreased sympathetic hyperactivity in anesthetized animals and v) MSC were detected at the CNS suggesting that the cells crossed the blood-brain barrier. This therapy may be a promising strategy to treat renovascular hypertension and its renal consequences in the near future.

Role of chymase in diabetic nephropathy

Chymase is an alternative pathway for angiotensin-converting enzyme in angiotensin II (Ang II) formation, and its expression is increased in human diabetic kidneys and in human mesangial cells (MCs) stimulated with high glucose. In addition, chymase activates transforming growth factor (TGF-β1) via an Ang II-independent pathway. The aim of this study was to evaluate the role of chymase on TGF-β1 activation in diabetic rats and in rat MCs (RMCs) stimulated with high glucose (HG). Diabetes was induced in male Wistar rats by streptozotocin (60 mg/kg, intravenous). After 30 (D30) or 60 (D60) days, chymase activity and the expression of profibrotic markers were evaluated. RMCs were stimulated with HG in the presence or absence of 50 μmol/L chymostatin, a chymase inhibitor, or 100 nmol/L of losartan, an Ang II antagonist. Chymase activity and expression increased in D60 kidneys, with increased expression of fibronectin, type I and III collagen, TGF-β1 and Smad 3 and with no change in Smad 7 expression. RMCs exposed to HG presented increases in chymase activity and expression, together with upregulation in fibrosis markers and in the TGF-β1 signaling pathway. All these effects were reversed by chymostatin and by losartan, but type 1 angiotensin II receptor blockade did not interfere with the Smad 3 and 7 pathway. Similar to HG-stimulated RMCs, control RMCs treated with chymase responded with increased expression of TGF-β1, Smad 3 and fibrosis markers. These effects were reversed by chymostatin but not by losartan. The results indicate an important role for chymase in inducing fibrosis through TGF-β1 activation, parallel with Ang II effects.

(Pro)renin receptor: another member of the system controlled by angiotensin II?

The prorenin receptor [(P)RR] is upregulated in the diabetic kidney and has been implicated in the high glucose (HG)-induced overproduction of profibrotic molecules by mesangial cells (MCs), which is mediated by ERK1/2 phosphorylation. The regulation of (P)RR gene transcription and the mechanisms by which HG increases (P)RR gene expression are not fully understood. Because intracellular levels of angiotensin II (AngII) are increased in MCs stimulated with HG, we used this in vitro system to evaluate the possible role of AngII in (P)RR gene expression and function by comparing the effects of AT1 receptor blockers (losartan or candesartan) and (P)RR mRNA silencing (siRNA) in human MCs (HMCs) stimulated with HG. HG induced an increase in (P)RR and fibronectin expression and in ERK1/2 phosphorylation. These effects were completely reversed by (P)RR siRNA and losartan but not by candesartan (an angiotensin receptor blocker that, in contrast to losartan, blocks AT1 receptor internalization). These results suggest that (P)RR gene activity may be controlled by intracellular AngII and that HG-induced ERK1/2 phosphorylation and fibronectin overproduction are primarily induced by (P)RR activation. This relationship between AngII and (P)RR may constitute an additional pathway of MC dysfunction in response to HG stimulation.

Calcitriol ameliorates renal damage in a pre‑established proteinuria model

Proteinuria is critical in the tubulointerstitial changes that ultimately lead to renal insufficiency. Increased protein filtration has direct toxic effects on tubular epithelial cells, leading to epithelial mesenchymal transition (EMT) to a myofibroblast phenotype. Angiotensin II and transforming growth factor (TGF)-β1 are the main mediators of EMT. Calcitriol may exert a potential renoprotective effect by reducing the activity of the renin angiotensin system by suppressing renin gene expression and also by inhibiting the proinflammatory nuclear factor-κB pathway. The present study investigated the benefits of calcitriol treatment in a puromycin-induced protein-uric nephropathy model. Uninephrectomized adult male Wistar rats received intraperitoneal administration of a single dose of puromycin (100 mg/kg) or vehicle. After eight weeks, the animals were divided into two groups and received vehicle or calcitriol (0.5 μg/kg) for four weeks. The vehicle-treated, proteinuric rats developed progressive proteinuria and tubulointerstitial fibrosis after 12 weeks. Increased collagen deposition and fibrosis were significantly ameliorated by calcitriol treatment. Calcitriol was effective in preventing an increase in the EMT markers, α-smooth muscle actin and fibroblast-specific protein 1, reducing macrophage infiltration as evidenced by levels of ED-1. In addition, calcitriol increased the anti-inflammatory cytokine interleukin-10 and reduced the pro-oxidant p47 phox enzyme. These effects were paralleled by a reduction in TGF-β/Smad3 expression. Calcitriol may have therapeutic potential in the proteinuric nephropathy model used in the present study by inhibiting the TGF-β1 axis.

Inhibition of cellular transdifferentiation by losartan minimizes but does not reverse type 2 diabetes-induced renal fibrosis

Hypothesis/Introduction: Transformer Growth Factor (TGF-β1) and angiotensin II (AngII) induce epithelial mesenchymal transition (EMT) and myofibroblastic transdifferentiation (MFT) contributing to renal fibrosis. The present study evaluated the capacity of an AT1 receptor blocker (losartan) to induce the regression of pre-existing fibrosis via interference with MFT and EMT in a rat model of type 2 diabetes, and in cultured mesangial cells (MCs) stimulated with high glucose and AngII. Materials and methods: After 12 weeks of diabetes induction (D12 group), animals showing evidence of nephropathy, were divided in groups untreated for additional 8 weeks (D20 group) and treated for additional 8 weeks with losartan (D20+los group). Results: D12 animals presented hyperglycemia, insulin resistance, hypertension, proteinuria, increased levels of TGF-β1 and MFT/EMT markers. Losartan stabilized all of these parameters and hindered the progression of fibrosis, but it did not reverse the pre-existing fibrotic manifestations. Losartan reduced TGF-β1 in the tubules, but not in the glomeruli. Stimulated MC exhibited myofibroblast phenotype and capacity for migration, which were completely reversed by losartan. Conclusions: Cellular transition may play a role in diabetes-inducing renal fibrogenesis in both AngII-TGF-β1 axis-dependent and independent manners. Losartan was efficient in preventing cells from undergoing further transdifferentiation, but this strategy was not sufficient to induce regression of the pre-existing tissue fibrosis.

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT1 and AT2), defined as intracrine response. The aim of this study was to examine the presence of AT1 and AT2 receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT1 through an intracrine mechanism. Subcellular distribution of AT1 and AT2 was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.