Edgar Maquigussa

Effect of Long-Term Type 1 Diabetes on Renal Sodium and Water Transporters in Rats

The effects of long-term diabetes in the presence of established nephropathy on tubular function remains poorly understood. We evaluated the levels of the main sodium and water transport proteins expressed in the kidney after long-term (8 weeks) of streptozotocin (STZ)-induced type 1 diabetes mellitus (DM) in untreated (D) and insulin (4 U/s.c./day)-treated (D+I) rats. D animals presented upregulation (∼4.5-fold) of Na/glucose cotransporter (SGLT1), whereas the α-subunit of the epithelial sodium channel (α-ENaC) and aquaporin 1 (AQP1) were downregulated (∼20 and 30% respectively) with no change in the Na/H exchanger (NHE3), Na/Cl cotransporter (TSC) and AQP2. Insulin replacement partially prevented these alterations and caused increases in the expression of α-ENaC and AQP2. These effects suggest an action of insulin in the tubular transport properties. The upregulation of SGLT1 may constitute a mechanism to prevent greater glucose losses in the urine but it may result in glucotoxicity to the proximal epithelial cells contributing to the diabetic nephropathy. The decrease of α-ENaC in D animals may compensate for the increased sodium reabsorption via SGLT1 resulting in discrete natriuresis. DM-induced polyuria was not due to changes in AQP2 expression.

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.

Bradykinin receptor 1 activation exacerbates experimental focal and segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and α-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling.

Soluble uric acid increases intracellular calcium through an angiotensin II-dependent mechanism in immortalized human mesangial cells

Hyperuricemia is associated with increases in cardiovascular risk and renal disease. Mesangial cells regulate glomerular filtration rates through the release of hormones and vasoactive substances. This study evaluates the signaling pathway of uric acid (UA) in immortalized human mesangial cells (ihMCs). To evaluate cell proliferation, ihMCs were exposed to UA (6–10 mg/dL) for 24–144 h. In further experiments, ihMCs were treated with UA (6–10 mg/dL) for 12 and 24 h simultaneously with losartan (10−7 mmol/L). Angiotensin II (AII) and endothelin-1 (ET-1) were assessed using the enzyme-linked immunosorbent assay (ELISA) technique. Pre-pro-ET mRNA was evaluated by the real-time PCR technique. It was observed that soluble UA (8 and 10 mg/dL) stimulated cellular proliferation. UA (10 mg/dL) for 12 h significantly increased AII protein synthesis and ET-1 expression and protein production was increased after 24 h. Furthermore, UA increased [Ca2+]i, and this effect was significantly blocked when ihMCs were preincubated with losartan. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. In addition, UA can potentially affect glomerular function due to UA-induced proliferation and contraction of mesangial cells. The latter mechanism could be related to the long-term effects of UA on renal function and chronic kidney disease.

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.

Regulation of Glucose Uptake in Mesangial Cells Stimulated by High Glucose: Role of Angiotensin II and Insulin

Mesangial cells (MCs) play a central role in the pathogenesis of diabetic nephropathy (DN). MC dysfunction arises from excessive glucose uptake through insulin-independent glucose transporter (GLUT1). The role of the insulin-dependent transporter (GLUT4) remains unknown. This study evaluated the effect of high glucose on GLUT1, GLUT4, and fibronectin expression levels. Glucose uptake was determined in the absence and presence of insulin. Angiotensin II has been implicated as a mediator of MC abnormalities in DN, and its effects on the GLUTs expression were evaluated in the presence of losartan. MCs were exposed to normal (NG, 10 mM) or high (HG, 30 mM) glucose for 1, 4, 12, 24, and 72 hrs. Glucose uptake was elevated from 1 hr up to 24 hrs of HG, but returned to NG levels after 72 hrs. HG induced an early (1-, 4-, and 12-hrs) rise in GLUT1 expression, returning to NG levels after 72 hrs, whereas GLUT4 was overexpressed at later timepoints (24 and 72 hrs). HG during 4 hrs induced a 40% rise in glucose uptake, which was unaffected by insulin. In contrast, after 72 hrs, glucose uptake was increased by 50%, only under insulin stimulus. Losartan blunted the effects of HG on GLUT1, GLUT4, and fibronectin expression and on glucose uptake. Results suggest that MCs can be highly susceptible to the HG environment since they uptake glucose in both an insulin-independent and insulin-dependent manner. The beneficial effects of angiotensin II inhibition in DN may also involve a decrease in the rate of glucose uptake by MCs.

(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.

Escherichia coli lipopolysaccharide impairs the calcium signaling pathway in mesangial cells: role of angiotensin II receptors.

Sepsis causes impaired vascular reactivity, hypotension and acute renal failure. The ability of the Escherichia coli endotoxin (lipopolysaccharide [LPS]) to impair agonist-induced contractility in mesangial cells, which contributes to LPS-induced renal dysfunction, was evaluated. Agonist-induced intracellular calcium ([Ca2+ ]i) mobilization was analyzed using angiotensin II (AngII). The effect of LPS on the levels of the renin–angiotensin system (RAS) components and the roles of vasodilatation-inducing molecules including AT2 receptor (AT2R) and nitric oxide (NO) in the cell reactivity were also evaluated. Confluent human mesangial cells (HMCs) were stimulated with LPS (0111-B4, 100 μg/mL). AngII-induced [Ca2+ ]i mobilization was measured by fluorometric analysis using Fura-2AM in the absence and presence of an AT2R antagonist (PD123319). The mRNA and protein levels for angiotensinogen, renin, angiotensin-converting enzyme, AT1R and AT2R were analyzed by realtime reverse transcriptase-polymerase chain reaction and Western blot, respectively. NO production was measured by the chemiluminescence method in the culture media after 24, 48 and 72 h of LPS incubation. After 24 h, LPS-stimulated HMCs displayed lower basal [Ca2+ ]i and an impaired response to AngII-induced rise in [Ca2+ ]i. LPS significantly increased AT2R levels, but did not cause significant alterations of RAS components. PD123319 restored both basal and AngII-induced [Ca2+ ]i peak, suggesting an involvement of AT2R in these responses. The expected increase in NO production was significant only after 72 h of LPS incubation and it was unaffected by PD123319. Results showed that LPS reduced the reactivity of HMCs to AngII and suggest that the vasodilatation induced by AT2R is a potential mediator of this response through a pathway independent of NO.

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.