Increased protein aggregation in Zucker diabetic fatty rat brain: identification of key mechanistic targets and the therapeutic application of hydrogen sulfide.

BACKGROUND: Diabetes and particularly high blood glucose levels are implicated in neurodegeneration. One of the hallmarks of neurodegeneration is protein aggregation. We investigated the presence of protein aggregation in the frontal brain of Zucker diabetic fatty (ZDF) rats, an animal model for diabetes. Further, the effect of NaHS in suppressing protein aggregation in cultured brain slices from ZDF was assessed. RESULTS: The levels of protein synthesis, protein/gene expression, autophagy and anti-oxidant defense were evaluated in ZDF and control (Lean) brains.Compared to Lean, ZDF brains displayed a significant increase in protein aggregates, p-tau, fibronectin expression and protein glycosylation. Increased phosphorylation of mTOR and S6 ribosomal protein in ZDF indicated higher protein synthesis, while the increase in ubiquitinated proteins and LC3-I in ZDF brains accompanied by lower LC3-II expression and LC3-II/LC3-I levels indicated the blockage of proteolytic pathways. CBS (cystathionine beta synthase) protein and mRNA expression and thiol group levels in ZDF brains were lower compared to Lean. ZDF brains show a higher level of reactive oxygen species. In vitro NaHS treatment normalized proteostasis while counteracting oxidative stress. CONCLUSION: Our data demonstrate increased protein synthesis and aggregation in the diabetic ZDF rat brain, which was reversible by NaHS treatment.This is the first report on the potential use of NaHS as a novel strategy against protein aggregation in diabetic brain.

Vildagliptin restores renal myogenic function and attenuates renal sclerosis independently of effects on blood glucose or proteinuria in zucker diabetic fatty rat.

Type 2 diabetes mellitus (T2DM) is associated with risk for chronic kidney disease (CKD), which is associated with a decrease in renal myogenic tone - part of renal autoregulatory mechanisms. Novel class of drugs used for the treatment of T2DM, dipeptidyl peptidase-4 (DPP-4) inhibitors, have protective effects on the cardiovascular system. A Zucker Diabetic Fatty (ZDF) rat is an animal model of T2DM that displays progressive nephropathy in which inflammation leads to initiation of renal fibrosis and CKD. We hypothesized that CKD in the ZDF rat is related to decrease in myogenic constriction (MC) of intrarenal arteries and that treatment with the DPP-4 inhibitor, vildagliptin, prevents such changes. Renal arteries isolated from 25 weeks old lean, ZDF and ZDF treated with vildagliptin (n=7 in each group) were transferred to an arteriograph to assess agonist and pressure induced contractile responses. Furthermore, blood glucose, proteinuria, focal glomerulosclerosis (FGS) and p22phox mRNA expression of renal tissue were measured. Compared to lean controls, ZDF had significantly increased plasma glucose and cholesterol levels, focal glomerulosclerosis and interstitial α-SMA expression, and urinary protein excretion. ZDF rats also had impaired MC of renal arteries and increased renal p22phox expression. Vildagliptin did not affect plasma glucose levels or proteinuria, but effectively decreased glomerulosclerosis and restored MC and p22phox expression to the levels found in lean rats. Based on these data, it can be suggested that vildagliptin treatment protects diabetic rats from the loss of renal vascular reactivity and the development of glomerulosclerosis perhaps secondary to a reduction in oxidative stress.

Renal myogenic constriction protects the kidney from age-related hypertensive renal damage in the Fawn-Hooded rat.

INTRODUCTION: Intact myogenic constriction plays a role in renal blood flow autoregulation and protection against pressure-related (renal) injury. However, to what extent alterations in renal artery myogenic constriction are involved in development of renal damage during aging is unknown. Therefore, we studied two strains of fawn-hooded rats, which differ in expression of hypertension and chronic renal failure. METHODS: Ten-week-old fawn-hooded hypertensive (FHH) and fawn-hooded low blood pressure (FHL) rats were followed for SBP and proteinuria for 1 year. At 52 weeks of age, the kidney was removed and studied for focal glomerulosclerosis (FGS) and glomerular cross-sectional area, and myogenic constriction of isolated small renal arteries in a vessel perfusion set up. Renal myogenic constriction and FGS were additionally determined in 10-week-old fawn-hooded rats. RESULTS: At young age, fawn-hooded rats did not differ in SBP, FGS, and urinary protein excretion, but renal artery myogenic constriction already was significantly smaller (∼50%) in FHH compared with FHL rats. Aging in fawn-hooded rats was associated with increase in SBP and urinary protein excretion and loss of renal artery myogenic constriction. These changes occurred in both fawn-hooded strains, although that in FHH rats the onset of hypertension occurred earlier and the increase in proteinuria by far exceeded (>4 times) that observed in FHL rats, and came along with 5.5 times increase in FGS and 1.3 times increase in glomerular cross-sectional area and significantly less preserved renal artery myogenic constriction in FHH rats. CONCLUSION: Better preservation of renal myogenic constriction protects the kidney from age-related hypertensive renal damage in the fawn-hooded rat.

Epidermal growth factor receptor inhibitor PKI-166 governs cardiovascular protection without beneficial effects on the kidney in hypertensive 5/6 nephrectomized rats.

Transactivation of epidermal growth factor receptor (EGFR) signaling by G protein-coupled receptors has been implicated in several cardiovascular (CV) conditions, including hypertension, heart failure, and cardiac and vascular hypertrophy. However, the therapeutic potential of EGFR inhibition in these conditions is currently unknown. The main objective of the present study was to investigate cardiac, vascular, and renal effects of EGFR inhibition by 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]phenol (PKI-166) in the hypertensive chronic kidney disease model. Rats underwent 5/6 nephrectomy (5/6Nx) and were treated with PKI-166, lisinopril or vehicle from week 6 after disease induction until week 12. Sham animals received either PKI-166 or vehicle. Treatment with PKI-166 did not affect the development of the characteristic renal features in 5/6Nx, including proteinuria, diminished creatinine clearance, and increased glomerulosclerosis, whereas these were attenuated by lisinopril. Despite absence of effects on progressive renal damage, PKI-166 attenuated the progression of hypertension and maintained cardiac function (left ventricle end-diastolic pressure) to a similar extent as lisinopril. Also, PKI-166 attenuated the increase in phosphorylated EGFR in the heart as induced by 5/6Nx. Moreover, PKI-166 and lisinopril restored the impaired contraction of isolated thoracic aortic rings to phenylephrine and angiotensin II and impaired myogenic constriction of small mesenteric arteries in 5/6Nx rats. Blockade of the EGFR displays a CV benefit independent of limiting the progression of renal injury. Our findings extend the evidence on EGFR signaling as a target in CV disorders.

Growth differentiation factor 15 impairs aortic contractile and relaxing function through altered caveolar signaling of the endothelium.

Growth differentiation factor 15 (GDF15) is an independent predictor of cardiovascular disease, and increased GDF15 levels have been associated with endothelial dysfunction in selected patients. We therefore investigated whether GDF15 modulates endothelial function in aortas of wild-type (WT) and GDF15 knockout (KO) mice. Vascular contractions to phenylephrine and relaxation to ACh were assessed in aortas obtained from healthy WT and GDF15 KO mice. The effects of GDF15 pretreatment and the involvement of ROS or caveolae were determined. Phenylephrine-induced contractions and ACh-mediated relaxations were similar in WT and GDF15 KO mice. Pretreatment with GDF15 inhibited contraction and relaxation in both groups. Inhibition of contraction by GDF15 was absent in denuded vessels or after blockade of nitric oxide (NO) synthase. Relaxation in WT mice was mediated mainly through NO and an unidentified endothelium-derived hyperpolarizin factor (EDHF), whereas GDF15 KO mice mainly used prostaglandins and EDHF. Pretreatment with GDF15 impaired relaxation in WT mice by decreasing NO; in GDF15 KO mice, this was mediated by decreased action of prostaglandins. Disruption of caveolae resulted in a similar inhibition of vascular responses as GDF15. ROS inhibition did not affect vascular function. In cultured endothelial cells, GDF15 pretreatment caused a dissociation between caveolin-1 and endothelial NO synthase. In conclusion, GDF15 impairs aortic contractile and relaxing function through an endothelium-dependent mechanism involving altered caveolar endothelial NO synthase signaling.

Attenuation of renovascular damage in Zucker diabetic fatty rat by NWT-03, an egg protein hydrolysate with ACE- and DPP4-inhibitory Activity.

BACKGROUND: Dipeptidyl peptidase 4 (DPP4) and angiotensin-converting enzyme (ACE) are important target enzymes in glycemic control and renovascular protection. Here, we studied the effect of NWT-03, an egg protein hydrolysate with DPP4- and ACE-inhibitory activity, on renovascular damage in Zucker diabetic fatty (ZDF) rats. Comparisons were made to rats treated with vildagliptin (VIL), included as a positive control for the effect of DPP4 inhibition. METHODS: ZDF rats received NWT-03 (1 g/kg/day) or VIL (3 mg/kg/day) from 10 to 25 weeks of age. Metabolic and renal functions were assessed; the kidney was removed for histological analysis of glomerulosclerosis and expression of pro-inflammatory/fibrotic markers (RT-PCR and Western blotting); and the aorta was removed for studies of endothelium-dependent relaxation (EDR). FINDINGS: Hyperinsulinemic ZDF rats typically developed signs of type-2 diabetes and renovascular damage, as evidenced by albuminuria, glomerulosclerosis, and impaired EDR. Neither NWT-03 nor VIL improved metabolic parameters; for VIL, this was despite a 5-fold increase in glucagon-like peptide (GLP)-1 levels. NWT-03 and VIL both reduced renal interleukin (Il)-1ß/Il-13 mRNA expression and glomerulosclerosis. However, only NWT-03 additionally decreased renal tumor necrosis factor (TNF)-α mRNA and P22(phox) protein expression, reduced albuminuria, and restored aortic EDR. Indomethacin added to the organ bath instantly improved aortic EDR, indicating a role for cyclooxygenase (COX)-derived contractile prostanoids in opposing relaxation in ZDF rats. This indomethacin effect was reduced by NWT-03, but not by VIL, and coincided with decreased renal COX-1/2 protein expression. CONCLUSION AND INTERPRETATION: Long-term supplementation with the egg protein hydrolysate NWT-03 attenuated renovascular damage in this preclinical rat model of type 2 diabetes. A comparison to the DPP4-inhibitor VIL suggests that the effects of NWT-03 were related to both ACE- and DPP4-inhibitory properties. The development of protein hydrolysates with a multiple-targeting strategy may be of benefit to functional food formulations.