| Abstract |
Type 1 diabetes mellitus is an autoimmune disease, one of the most common in childhood worldwide. Pancreatic beta cells are destroyed and the resulting hyperglycemia is the trigger to a wide array of complications that affect target- organs,
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including the kidney. Renal cells do not depend on insulin for glucose uptake, but instead glucose diffuses across the cell membrane down its concentration gradient. In diabetes, renal intracellular glucose levels rise in proportion to the degree of hyperglycemia. Metabolic changes and hemodynamic stress induce the release of cytokines and growth factors that ultimately mediate renal lesions in terms of renal cell hypertrophy, extracellular matrix deposition and podocyte abnormalities. As the disease progresses, the 24h urinary albumin excretion increases.
In diabetic nephropathy, despite the suppression of systemic renin angiotensin system (RAS), the renal renin angiotensin system is activated, causing detrimental effects in the kidney. Data from animal studies and human kidney biopsies provide sufficient evidence. In glomeruli from male Sprague- Dawley rats made diabetic by streptozotocin, there were detected increased levels of angiotensinogen and angiotensin II, as compared to their respective controls. In addition, exogenous administration of angiotensin I to the glomeruli resulted in an increase in angiotensin II production in diabetic glomeruli. In renal biopsies from diabetic patients with nephropathy, immunohistochemical studies showed enhanced angiotensin-converting enzyme (ACE) staining in glomeruli from diabetic patients, as opposed to their healthy controls, implicating increased ACE as one of the factors responsible for the activation of renal renin angiotensin system in diabetic nephropathy.
Furthermore, the glomerulus and the podocytes have a central role in the pathophysiology of diabetic nephropathy.
Evidence from cell culture systems link angiotensin II and increased mTOR activity. PI3K- AKT- mTOR pathway is activated and involved in the pathogenesis of diabetic nephropathy, mediating pathologic changes. Both AKT and mTOR are activated in diabetic nephropathy, mediating diabetic kidney lesions. mTOR, a serine- threonine kinase, is a component of 2 multiprotein signaling complexes, mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2). PI3K receives input from amino acids and growth factors and phosphorylates PIP2 to PIP3 that activates PDK1. PDK1 activates AKT, through phosphorylation at Thr308 (pAKT308). AKT is a key regulator
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of cell proliferation and survival. Activated AKT leads to mTORC1 activation through reversion of a series of inhibitory signals that involve the tuberous sclerosis system –TSC complex. AKT can also activate directly mTORC1 without the involvement of TSC complex by phosphorylation at Ser2448 (pTOR2448). AMPK is a cellular energy sensor, that is activated at low energy states and inhibits mTOR, ensuring that in conditions of cellular energy depletion, energy- consuming reactions are inhibited. AKT is also phosphorylated at Ser473 by mTORC2 (pAKT473).
No data, currently exists as to the effects of losartan on the PI3K-AKT-mTOR pathway in type 1 diabetic nephropathy in vivo.
Therefore, the purpose of the present thesis, was to investigate, in an experimental model of type 1 diabetic nephropathy:
(a) Τhe glomerular content of total AKT (tAKT), phosphorylated (p)AKT Thr308, pAKT Ser473, mTOR, and activated mTOR phosphorylated at residue Ser2448 (pTOR2448),
(b) Τhe podocytic content of the aforementioned molecules, employing co-localization analysis with the podocyte-specific marker nephrin,
(c) Τhe degree of mesangial matrix expansion and glomerular basement membrane thickening,
(d) Clinical and biochemical parameters of the animals, such as systolic blood pressure and 24h urinary albumin excretion.
Diabetes mellitus was induced to male Sprague-Dawley rats by intraperitoneal injection of streptozotocin. Three days after the induction of diabetes, blood glucose was measured by a glucometer and animals with values >350 mg/dl were included in the study. The diabetic rats received, subcutaneously, insulin glargine, every other day, individually adjusted, to ensure blood glucose levels between 350-500 mg/dl. Monthly, the animals were put in specific metabolic cages for 24h urine collection. During the collection, the rats had ad libitum access to water and standard rat feed. The collected urine was intended for measurement of albumin excretion. Five months after the induction of diabetes, the rats were divided in four study groups and the per os
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administration of losartan commenced (Dm diabetic animals, DmRx diabetic animals on losartan potassium daily, Ctrl healthy control animals, CtrlRx and healthy control animals on losartan potassium daily). After 2 months of losartan administration, the animals were housed in specific metabolic cages for 24h urine collection and measurement of albumin excretion. Furthermore, measurements of the animals’ blood pressure took place, after the rats had been habituated in specific warming chambers of the blood pressure system. Afterwards, the animals were injected with sodium pentobarbital and killed by exsaguination. The kidneys were excised and weighed and parts of the tissue were processed differently for diverse techniques. Part of the kidneys was used for glomeruli isolation, employing sequential sieving through stainless steel sieves of different pore size. The kidney cortex was dissected and it was forced, using a spatula, through the screen of 150μm. The tissue that remained on the 150μm sieve was mainly tubular and vascular elements and it was discarded. The material collected onto the next screen, i.e. the 106 μm one, was extensively washed with normal saline. After the washes, the tissue remaining on the middle sieve, mostly tubular elements, was discarded and the glomeruli had already passed and collected on the last screen of 75μm. The glomeruli were, also, washed with normal saline so that small tubular parts had passed through. Purity of the glomerular isolate was estimated >95%. Isolated glomeruli were put in lysis buffer containing protease inhibitors for Western Blot analysis. The samples were electrophorized and transferred to nitrocellulose membranes that were blotted with the following primary antibodies: tAKT, pAKT308, pAKT473, mTOR, pTOR2448 and nephrin. Appropriate secondary antibodies were applied. Furthermore, for indirect immunofluorescence studies, tissue was snap frozen in liquid nitrogen and stored at −80C till further analysis. The following primary antibodies were used: tAKT, pAKT308, pAKT473, mTOR, pTOR2448 and nephrin. Tissue was also processed appropriately for optical microscope studies (estimation of mesangial matrix expansion) and transmission electron microscope observation (measurement of the glomerular basement membrane width).
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As far as the clinical and biochemical parameters of the animals are concerned, results are as following: From diabetes induction to the fifth month of experimentation, 24h albumin excretion increased in the urine of diabetic subjects. Losartan decreased urinary albumin excretion in diabetic animals. Diabetes mellitus caused an increase in systolic blood pressure. However, losartan did not cause any effect on blood pressure of diabetic animals, implying that the beneficial role of losartan on diabetic kidney, may not be, exclusively, due to its effects on glomerular hemodynamics.
As far as the effect of losartan on glomerular basement membrane thickening is concerned, in diabetic animals, the glomerular basement membrane width was restored to normal.
Diabetes mellitus caused an increase in the mesangium. Concerning the influence of losartan on mesangial matrix expansion, there was a tendency towards reduction that did not reach statistical significance.
Regarding the immunofluorescence experiments, the sections were incubated with the
following primary antibodies: total AKT, pAKT308, pAKT473, mTOR, pTOR2448 and nephrin. Diabetes increased glomerular pAKT308, mTOR, and pTOR2448 protein content, findings that are in accordance with those from previous studies. In losartan treated animals, glomeruli showed decreased mTOR, pTOR2448, pAKT308 and pAKT473 but increased AKT protein levels. These data demonstrate that losartan reversed the signaling events that led to AKT and mTOR activation.
The podocyte- specific marker nephrin was used for colocalization experiments with the molecules under study. In podocytes, diabetes mellitus caused an increase in total AKT, pAKT308, and pTOR2448 protein levels, indicating activation of the PI3K-AKT-mTOR pathway, despite the decrease in mTOR and pAKT473 content. In diabetic animals, losartan administration, despite the increase in podocytic mTOR, decreased its activation as it is shown by the decreased of pTOR2448. In losartan- treated healthy animals the observed effect was reversed: podocytic pTOR2448 levels were increased as opposed to the respective levels in healthy control animals.
Immunofluorescence results were further confirmed by Western blot.
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Conclusively, losartan decreased albuminuria in diabetic animals and reversed the effect of diabetes on the glomerular basement membrane width. However, it had no effect on the blood pressure of diabetic animals. Furthermore, there was a differential pattern of in situ activation of the PI3K-AKT-mTOR pathway in glomeruli and podocytes as a result of diabetes and losartan. The PI3K-AKT-mTOR pathway is involved in the pathogenesis of diabetic nephropathy through cell hypertrophy and extracellular matrix production. Losartan influences its components in a beneficial way, implying a crosstalk between the Renin Angiotensin System and the PI3K-AKT-mTOR pathway.
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