Markus Lassila

Imatinib Attenuates Diabetic Nephropathy in Apolipoprotein E-Knockout Mice

In the diabetic kidney, clinical as well as experimental observations have shown an upregulation of growth factors such as PDGF. These studies, however, were not designed to address whether upregulation of PDGF is merely a manifestation of diabetic renal injury or whether PDGF plays an active role in the pathophysiology of diabetic nephropathy. The objectives of this study were first to assess whether PDGF-dependent pathways are involved in the development of diabetic nephropathy and second to determine the effects of PDGF receptor antagonism on this disorder and associated molecular and cellular processes. This study used the diabetic apolipoprotein E-knockout (apoE-KO) mouse, a recently described model of accelerated diabetic nephropathy. Diabetes was induced by injection of streptozotocin in 6-wk-old apoE-KO mice. Diabetic animals received treatment with a tyrosine kinase inhibitor that inhibits PDGF action, imatinib (STI-571, 10 mg/kg per d orally) or no treatment for 20 wk. Nondiabetic apoE-KO mice served as controls. This model of accelerated renal disease with albuminuria as well as glomerular and tubulointerstitial injury was associated with increased renal expression of PDGF-B, proliferating cells, and alpha-smooth muscle actin-positive cells. Furthermore, there was increased accumulation of type I and type IV collagen as well as macrophage infiltration. Imatinib treatment ameliorated both renal functional and structural parameters of diabetes as well as overexpression of a number of growth factors, collagens, proliferating cells, alpha-smooth muscle actin-positive cells, and macrophage infiltration within the kidney. Tyrosine kinase inhibition with imatinib seems to retard the development of experimental diabetic nephropathy.

The MIF Receptor CD74 in Diabetic Podocyte Injury

Although metabolic derangement plays a central role in diabetic nephropathy, a better understanding of secondary mediators of injury may lead to new therapeutic strategies. Expression of macrophage migration inhibitory factor (MIF) is increased in experimental diabetic nephropathy, and increased tubulointerstitial mRNA expression of its receptor, CD74, has been observed in human diabetic nephropathy. Whether CD74 transduces MIF signals in podocytes, however, is unknown. Here, we found glomerular and tubulointerstitial CD74 mRNA expression to be increased in Pima Indians with type 2 diabetes and diabetic nephropathy. Immunohistochemistry confirmed the increased glomerular and tubular expression of CD74 in clinical and experimental diabetic nephropathy and localized glomerular CD74 to podocytes. In cultured human podocytes, CD74 was expressed at the cell surface, was upregulated by high concentrations of glucose and TNF-alpha, and was activated by MIF, leading to phosphorylation of extracellular signal-regulated kinase 1/2 and p38. High glucose also induced CD74 expression in a human proximal tubule cell line (HK2). In addition, MIF induced the expression of the inflammatory mediators TRAIL and monocyte chemoattractant protein 1 in podocytes and HK2 cells in a p38-dependent manner. These data suggest that CD74 acts as a receptor for MIF in podocytes and may play a role in the pathogenesis of diabetic nephropathy.

β-Catenin mediates adriamycin-induced albuminuria and podocyte injury in adult mouse kidneys

BACKGROUND Glomerular slit diaphragm (SD) represents a modified adherens junction composed of molecules belonging to both immunoglobulin and cadherin superfamilies. Cadherins associate with the cytosolic scaffolding protein beta-catenin, but the precise role of beta-catenin in mature or injured podocytes is not known. METHODS The conditional podocyte-specific beta-catenin-deficient mouse line was generated using the doxycycline-inducible Cre-loxP system. Expression of the beta-catenin-deficient gene was turned off at the age of 8 weeks by doxycycline treatment and the kidney phenotype was analysed. In addition, beta-catenin-deficient and control mice were treated with adriamycin (ADR) and analysed for albuminuria and morphological alterations. RESULTS Deletion of beta-catenin in mature podocytes did not change the morphology of podocytes nor did it lead to albuminuria. However, lack of beta-catenin attenuated albuminuria after ADR treatment. Electron microscopic examination showed increased podocyte foot process effacement associated with SD abnormalities in ADR-treated control mice compared to beta-catenin-deficient mice. CONCLUSIONS These results show that beta-catenin in podocytes is dispensable for adult mice, but appears to be important in modulating the SD during ADR-induced perturbation of the filtration barrier.

Advanced Glycation End Products in Diabetes- Associated Atherosclerosis and Renal Disease Interventional Studies

Abstract: There is increasing evidence that advanced glycation end products (AGEs) and their interactions with various receptors (in particular, the receptor RAGE) play a pivotal role in the development and progression of diabetic macro‐ and microvascular complications. Several approaches have been used to inhibit tissue accumulation of AGEs in diabetes, including inhibitors of AGE formation such as aminoguanidine, ALT 946, and pyridoxamine—or putative cross‐link breakers such as ALT 711. Alternative interventions have also included the administration of a soluble receptor for RAGE, sRAGE, thus capturing circulating AGEs and preventing them from binding to the cell‐bound full‐length receptor RAGE, thereby inhibiting the proinflammatory and profibrotic response following AGE‐RAGE binding. In this review we summarize the evidence for such antiglycation therapies in retarding or delaying the development and progression of diabetes‐associated atherosclerosis and renal disease while focusing on interventional strategies inhibiting AGE accumulation. In summary, all approaches have been shown to confer some degree of antiatherosclerotic and renoprotective effects, albeit to different degrees and by different mechanisms.

Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet

Background. The introduction of cyclosporine (CsA) has led to an improvement in the prognosis of solid organ transplantation. However, drug-induced hypertension and nephrotoxicity, associated with the development of atherosclerosis and coronary heart disease, still worsen the long-term outcome of CsA-treated patients. Whether the CsA-induced myocardial changes are associated with the induction of connective tissue growth factor (CTGF), a recently found polypeptide implicated in extracellular matrix synthesis, is not known. Methods. Spontaneously hypertensive rats (8–9 weeks old) were treated with CsA (5 mg·kg−1·d−1 subcutaneously) for 6 weeks. The influence of angiotensin-converting enzyme inhibition (enalapril 30 mg·kg−1·d−1 orally) and angiotensin-1 receptor blockade (valsartan 3 and 30 mg·kg−1·d−1 orally) on CsA toxicity was also investigated. Myocardial morphology was examined, and vascular lesions were scored. Localization and the quantitative expression of CTGF, as well as collagen I and collagen III, mRNA were evaluated by in situ hybridization and Northern blot. Results. CsA-induced hypertension and nephrotoxicity were associated with myocardial infarcts and vasculopathy of the coronary arteries. CsA increased myocardial CTGF, collagen I, and collagen III mRNA expressions by 91%, 198%, and 151%, respectively. CTGF mRNA expression colocalized with the myocardial lesions. Blockade of the renin-angiotensin system prevented vascular damage and the CsA-induced CTGF, collagen I, and collagen III mRNA overexpressions in the heart. Conclusions. CsA increases CTGF, collagen I, and collagen III mRNA expressions in the heart. The induction of CTGF gene is mediated, at least in part, by angiotensin II.

Comparison of enalapril and valsartan in cyclosporine A‐induced hypertension and nephrotoxicity in spontaneously hypertensive rats on high‐sodium diet

We compared the effects of the angiotensin converting enzyme (ACE) inhibitor enalapril and the angiotensin AT1 receptor antagonist valsartan in cyclosporine A (CsA)‐induced hypertension and nephrotoxicity in spontaneously hypertensive rats (SHR). SHR (8–9 weeks old) on high‐sodium diet were given CsA (5 mg kg−1d −1 s.c.) for 6 weeks. The rats were treated concomitantly either with enalapril (30 mg kg−1d −1 p.o.) or valsartan (3 or 30 mg kg−1 d −1 p.o.). To evaluate the role of bradykinin in the action of enalapril, some rats received a bradykinin B2 receptor antagonist icatibant (HOE 140, 500 μg kg−1 d −1 s.c.) during the last 2 weeks of enalapril treatment. Blood pressure was recorded every second week by tail cuff method. Renal function was measured by serum creatinine, creatinine clearance and urinary excretion of proteins at the end of the experiment. The activity of the renal kallikrein‐kinin system was estimated by urinary kallikrein excretion. CsA caused hypertension, impaired renal function and induced morphological nephrotoxicity with glomerular damage and interstitial fibrosis. Enalapril and the lower dose of valsartan attenuated the CsA‐induced hypertension to the same extent, while the higher dose of valsartan totally abolished it. Icatibant did not reduce the antihypertensive effect of enalapril. Urinary kallikrein excretion was similar in all groups. Enalapril and valsartan equally prevented the CsA‐induced deterioration of kidney function and morphology. The renin‐angiotensin but not the kallikrein‐kinin system plays a crucial role in CsA‐toxicity during high intake of sodium in SHR.

Anti-atherosclerotic and renoprotective effects of combined angiotensin-converting enzyme and neutral endopeptidase inhibition in diabetic apolipoprotein E-knockout mice.

Objective To investigate the effects of the combined angiotensin-converting enzyme (ACE)/neutral endopeptidase (NEP) inhibitor omapatrilat on atherosclerosis and renal injury in a model of diabetes-associated accelerated atherosclerosis and renal injury. Design The study was performed using diabetic apolipoprotein E-knockout (apo E-KO) mice, a model combining hyperlipidemia and hyperglycemia, which leads to accelerated atherosclerosis and renal injury. Methods Diabetes was induced by the injection of streptozotocin in 6-week old apo E-KO mice. Diabetic animals received no treatment (n = 12) or treatment with the ACE/NEP inhibitor omapatrilat (30 mg/kg per day, via gavage, n = 12) or quinapril (10 mg/kg per day, in drinking water, n = 12) for 20 weeks. Non-diabetic apo E-KO mice (n = 12) served as controls. Results Omapatrilat reduced atherosclerosis and protected the mice from renal structural injury and albuminuria. The protective effects were associated with tissue inhibition of aortic and renal ACE and NEP as well as a significant reduction in blood pressure. Omapatrilat had similar anti-atherosclerotic effects compared with the ACE inhibitor quinapril in association with an almost complete inhibition of aortic ACE activity by both drugs. Omapatrilat conferred superior renoprotection in the diabetic apo E-KO mouse compared with quinapril in the context of greater renal ACE inhibition by omapatrilat than seen with quinapril, additional renal NEP inhibition and a modestly enhanced antihypertensive response. Conclusions These studies demonstrate the anti-atherosclerotic and renoprotective effects of omapatrilat in diabetic apo E-KO mice, a model of accelerated atherosclerosis and renal injury. These effects were observed in association with the local inhibition of ACE and NEP at the tissue level in the aorta and kidney. These results suggest that the anti-atherosclerotic effect conferred by omapatrilat treatment in the diabetic apo E-KO mouse is predominantly mediated by its capacity to inhibit local vascular ACE. By contrast, in the kidney, local renal ACE and NEP inhibition and the superior antihypertensive effect of omapatrilat all contribute to the renoprotective effect conferred by omapatrilat treatment in the diabetic apo E-KO mouse.

Genistein treatment reduces arterial contractions by inhibiting tyrosine kinases in ovariectomized hypertensive rats

The aim of the present study was to evaluate the vascular effects of genistein in a short-term study. The ovariectomized spontaneously hypertensive rats (SHR) were divided into four groups (n = 8 in each), which received the following subcutaneous treatments either for 2 days or for 2 weeks: (1) solvent control (96% dimethylsulphoxide (DMSO) 1 ml/kg), (2) estradiol-17beta (25 microg/kg), (3) genistein (2.5 mg/kg; low-dose), and (4) genistein (25 mg/kg; high-dose). The renal arterial rings were studied using organ bath system. The renal artery contractions were attenuated by the 2-day low-dose genistein treatment as follows: angiotensin II (46%), noradrenaline (42%) KCl (36%), and endothelin-1 (34%). Only the angiotensin II-induced contractions were reduced by the 2-week treatment with estradiol-17beta (38%) and with the low-dose of genistein (31%). The 2-day genistein treatment reduced tyrosine phosphorylation, while the other treatments or treatment times had no effect. The 2-day low-dose genistein treatment had no estrogenic effect on the uterine morphology. The mechanism for attenuated contractility in the renal arteries after the 2-day low-dose genistein treatment is independent of the estrogenic effect of genistein, but is due to the tyrosine kinase inhibitory property of genistein.

Effects of ACE inhibition on cyclosporine A-induced hypertension and nephrotoxicity in spontaneously hypertensive rats on a high-sodium diet.

Cyclosporine A (CsA)-induced hypertension has been shown to be dependent on the level of dietary salt. The present study assessed the role of the renin-angiotensin system in the development of CsA-induced hypertension and nephrotoxicity in spontaneously hypertensive rats (SHR) on a high-sodium diet. In addition, we examined whether ACE inhibition prevents the detrimental effects of CsA on blood pressure, kidney function and vascular morphology in SHR on high sodium intake. Eight-week-old SHR were divided into three different groups (n = 8 in each group): (i) SHR control group receiving a high-sodium diet (Na 2.6% of the dry weight of the chow), (ii) CsA group (5 mg/kg s.c.) on a high-sodium diet and (iii) CsA + enalapril group (30 mg/kg p.o.) on a high-sodium diet. At the end of the six-week experimental period, systolic blood pressure in the CsA group was significantly higher compared to the control group (245+/-6 vs 208+/-9 mmHg, respectively, p < 0.05). Plasma renin activity was increased 20-fold by CsA treatment (p < 0.05 compared to controls). CsA increased serum creatinine by 22%, the 24-h urinary protein excretion by 190% and the 24-h urinary excretions of calcium, phosphorus and magnesium by 150%, 25% and 140%, respectively (p < 0.05 compared to controls). Histologically, the kidneys of CsA-treated SHR showed severe thickening of the media of the afferent arteriole and fibrinoid necrosis of the arteriolar wall. Interestingly, CsA induced vascular injury also in the small myocardial arteries. Enalapril treatment prevented CsA-induced hypertension and deterioration of kidney function as well as CsA-induced vascular injuries in the kidneys and myocardium. Enalapril also decreased left ventricular weight-to body weight ratio and prevented CsA-induced increases in urinary calcium and phosphorus excretions. Our findings indicate that CsA has a detrimental effect on blood pressure, kidney function and vascular morphology in SHR on high sodium intake. ACE inhibition prevents the CsA-induced hypertension, nephrotoxicity and vascular injuries. Our findings thus suggest that increased activity of the renin-angiotensin system is involved in the pathogenesis of CsA-induced hypertension and nephrotoxicity in SHR on a high-sodium diet.

Podocyte Cell–Specific Expression of Doxycycline Inducible Cre Recombinase in Mice

Conventional silencing of many podocyte-specific genes in mice is associated with embryonic or perinatal lethality. Therefore, it would be of great importance to generate mouse models that allow the modification of genes that are expressed in podocytes at later stages of age. Herein is described a transgenic mouse with doxycycline-inducible podocyte-specific expression of Cre recombinase. For the generation of this binary system, a single transgenic construct that contained two separate genes was used: One encoding the optimized M2 version of the doxycycline-dependent transcription transactivator reverse tetracycline-controlled transcriptional activator (rtTA) under control of the human podocin (NPHS2) promoter and the other encoding the recombinase Cre under control of the rtTA/doxycycline-responsive minimal cytomegalovirus (CMV) Tet operator sequence 7 promotor. Microinjection of the JRC-CRE construct in fertilized oocytes from FVB/N mice resulted in 16 transgenic founders. Double-transgenic offspring from breeding of a selected founder with the Z/AP reporter mouse showed alkaline phosphatase staining only upon doxycycline administration and exclusively in podocytes. These data indicate that this new inducible Cre recombinase mouse line is an excellent tool in conditional, kidney glomerular podocyte-specific gene deletion in adult mice.