The dipeptidyl peptidase inhibitor linagliptin and the angiotensin II receptor blocker telmisartan show renal benefit by different pathways in rats with 5/6 nephrectomy.

Dipeptidyl peptidase (DPP)-4 inhibitors delay chronic kidney disease (CKD) progression in experimental diabetic nephropathy in a glucose-independent manner. Here we compared the effects of the DPP-4 inhibitor linagliptin versus telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. Animals were allocated to 1 of 4 groups: sham operated plus placebo; 5/6 nephrectomy plus placebo; 5/6 nephrectomy plus linagliptin; and 5/6 nephrectomy plus telmisartan. Interstitial fibrosis was significantly decreased by 48% with linagliptin but a non-significant 24% with telmisartan versus placebo. The urine albumin-to-creatinine ratio was significantly decreased by 66% with linagliptin and 92% with telmisartan versus placebo. Blood pressure was significantly lowered by telmisartan, but it was not affected by linagliptin. As shown by mass spectrometry, the number of altered peptide signals for linagliptin in plasma was 552 and 320 in the kidney. For telmisartan, there were 108 peptide changes in plasma and 363 in the kidney versus placebo. Linagliptin up-regulated peptides derived from collagen type I, apolipoprotein C1, and heterogeneous nuclear ribonucleoproteins A2/B1, a potential downstream target of atrial natriuretic peptide, whereas telmisartan up-regulated angiotensin II. A second study was conducted to confirm these findings in 5/6 nephrectomy wild-type and genetically deficient DPP-4 rats treated with linagliptin or placebo. Linagliptin therapy in wild-type rats was as effective as DPP-4 genetic deficiency in terms of albuminuria reduction. Thus, linagliptin showed comparable efficacy to telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. However, the underlying pathways seem to be different.

Renal and cardiac effects of DPP4 inhibitors--from preclinical development to clinical research.

Inhibitors of type 4 dipeptidyl peptidase (DDP-4) were developed and approved for the oral treatment of type 2 diabetes. Its mode of action is to inhibit the degradation of incretins, such as type 1 glucagon like peptide (GLP-1), and GIP. GLP-1 stimulates glucose-dependent insulin secretion from pancreatic beta-cells and suppresses glucagon release from alpha-cells, thereby improving glucose control. Besides its action on the pancreas type 1 glucagon like peptide has direct effects on the heart, vessels and kidney mainly via the type 1 glucagon like peptide receptor (GLP-1R). Moreover, there are substrates of DPP-4 beyond incretins that have proven renal and cardiovascular effects such as BNP/ANP, NPY, PYY or SDF-1 alpha. Preclinical evidence suggests that DPP-4 inhibitors may be effective in acute and chronic renal failure as well as in cardiac diseases like myocardial infarction and heart failure. Interestingly, large cardiovascular meta-analyses of combined phase II/III clinical trials with DPP-4 inhibitors point all in the same direction: a potential reduction of cardiovascular events in patients treated with these agents. A pooled analysis of pivotal phase III, placebo-controlled, registration studies of linagliptin further showed a significant reduction of urinary albumin excretion after 24 weeks of treatment. The observation suggests direct renoprotective effects of DPP-4 inhibition that may go beyond its glucose-lowering potential. Type 4 dipeptidyl peptidase inhibitors have been shown to be very well tolerated in general, but for those excreted via the kidney dose adjustments according to renal function are needed to avoid side effects. In conclusion, the direct cardiac and renal effects seen in preclinical studies as well as meta-analysis of clinical trials may offer additional potentials - beyond improvement of glycemic control - for this newer class of drugs, such as acute kidney failure, chronic kidney failure as well as acute myocardial infarction and heart failure.

DPP-4 inhibition on top of angiotensin receptor blockade offers a new therapeutic approach for diabetic nephropathy.

BACKGROUND: The need for an improved treatment for diabetic nephropathy is greatest in patients who do not adequately respond to angiotensin II receptor blockers (ARBs). This study investigated the effect of the novel dipeptidyl peptidase-4 inhibitor linagliptin alone and in combination with the ARB telmisartan on the progression of diabetic nephropathy in diabetic endothelial nitric oxide synthase (eNOS) knockout mice. METHODS: Sixty male eNOS knockout C57BL/6J mice were divided into four groups after receiving intraperitoneal highdose streptozotocin: telmisartan (1 mg/kg), linagliptin (3 mg/kg), linagliptin + telmisartan (3 mg/kg + 1 mg/kg) and vehicle. Fourteen mice were used as non-diabetic controls. RESULTS: After 12 weeks, urine and blood were obtained and blood pressure measured. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan alone reduced systolic blood pressure by 5.9 mmHg versus diabetic controls (111.2 ± 2.3 mmHg vs 117.1 ± 2.2 mmHg; mean ± SEM; P=0.071). Combined treatment significantly reduced albuminuria compared with diabetic controls (71.7 ± 15.3 µg/24 h vs. 170.8 ± 34.2 µg/24 h; P=0.017), whereas the effects of single treatment with either telmisartan (97.8 ± 26.4 µg/24 h) or linagliptin (120.8 ± 37.7 µg/24 h) were not statistically significant. DPP-4 inhibition, alone and in combination, led to significantly lower plasma osteopontin levels compared with telmisartan alone. Histological analysis revealed reduced glomerulosclerosis after Linagliptin alone and in combination with telmisartan in comparison to non treated diabetic animals (p<0.01 and p<0.05). Kidney malonaldehyde immune-reactivity, a marker of oxidative stress, was significantly lower in animals treated with linagliptin. CONCLUSIONS: DPP-4 inhibition on top of ARB treatment significantly reduced urinary albumin excretion and oxidative stress in diabetic eNOS knockout mice. Linagliptin on top of an angiotensin II receptor blocker may offer a new therapeutic approach for patients with diabetic nephropathy.

Early urinary and plasma biomarkers for experimental diabetic nephropathy.

BACKGROUND: As the prevalence of diabetes rises, its complications such as diabetic nephropathy affect an increaseing number of patients. Consequently, the need for biomarkers in rodent models which reflect the stage and course of diabetic nephropathy is high. This article focuses on Heart-type fatty acid binding protein (H-FABP), osteopontin (OPN), nephrin, and Neutrophil gelatinase-associated lipocalin (NGAL) in urine, and kidney injury molecule (KIM)-1, clusterin, and tissue inhibitior of metalloproteinases (TIMP) 1 in plasma in uni-nephrectomized rats with streptocotozin-induced type 1 diabetes mellitus, a common animal model to explore renal impairment in the setting of diabetes mellitus. METHODS: 23 male Wistar rats were uni-nephrectomized and subsequently divided into two study groups. The diabetic group received streptozotocin (STZ) via tail-vein injection, the non-diabetic group received citrate buffer without STZ. Subsequently, blood glucose, body weight, and blood pressure were checked regularly. After 18 weeks, animals were placed in metabolic cages, blood and urine obtained and subsequently organs were harvested after sacrifice. RESULTS: Blood glucose levels were highly increased in diabetic animals throughout the experiment, whereas systolic blood pressure did not differ between the study groups. At study end, classical biomarkers such as urinary albumin and protein and plasma cystatin c were only slightly but not significantly different between groups indicating a very early disease state. In contrast, urinary excretion of H-FABP, OPN, nephrin, and NGAL were highly increased in diabetic animals with a highly significant p-value (p < 0.01 each) compared to non-diabetic animals. In plasma, differences were found for calbindin, KIM-1, clusterin, TIMP-1, and OPN. These findings were confirmed by means of the area under the receiver operating characteristic curve (ROC-AUC) analysis. CONCLUSIONS: In summary, our study revealed elevated levels of new plasma and urinary biomarkers (urinary osteopontin, urinary nephrin, urinary NGAL, urinary H-FABP, plasma KIM-1, plasma TIMP-1) in uni-nephrectomized diabetic rats, an established rat model of diabetic nephropathy. These biomarkers appeared even before the classical biomarkers of diabetic nephropathy such as albuminuria and urinary protein excretion. The new biomarkers might offer an advantage to urinary albumin and plasma cystatin c with respect to early detection.

Renoprotective effects of combined endothelin-converting enzyme/neutral endopeptidase inhibitor SLV338 in acute and chronic experimental renal damage.

BACKGROUND: Acute kidney injury (AKI) as well as chronic renal failure are associated with a huge mortality/morbidity. However, so far no drugs have been approved for the treatment of acute kidney failure and only a few for the treatment of chronic kidney disease (CKD). We analysed the effect of SLV338, a neutral endopeptidase (NEP)/endothelin converting enzyme (ECE)-inhibitor in animal models of acute kidney failure as well as chronic renal failure. METHODS: Acute renal failure was induced in male Wistar rats by uninephrectomy and clamping of the remaining kidney for 55 minutes. SLV338 (total dose: 4.9 mg/kg) or vehicle was continuously infused for 2 hours (starting 20 minutes prior to clamping). Sham operated animals served as controls. Plasma creatinine was measured at baseline and day 2 and 8 after renal ischemia-reperfusion. Hypertensive renal damage was induced in male Sprague Dawley rats by nitric oxide deficiency using L-NAME (50 mg/kg per day, added to drinking water for 4 weeks). One group was treated over the same time period with SLV338 (30 mg/kg per day, mixed with food). Systolic blood pressure was monitored weekly. At study end, urine and blood samples were collected and kidneys were harvested. RESULTS: Acute renal ischemia-reperfusion caused a 5-fold plasma creatinine elevation (day 2), which was significantly attenuated by more than 50% in animals treated with SLV338 (p < 0.05). Renal failure was accompanied by a 67% mortality in vehicle-treated rats, but only 20% after SLV338 treatment (p = 0.03 compared to sham controls). Chronic L-NAME administration caused hypertension, urinary albumin excretion, glomerulosclerosis, renal arterial remodelling, and renal interstitial fibrosis. Treatment with SLV338 did not significantly affect blood pressure, but abolished renal tissue damage (interstitial fibrosis, glomerulosclerosis, renal arterial remodelling (p < 0.05 versus L-NAME group in each case). CONCLUSIONS: The dual ECE/NEP inhibitor SLV338 preserves kidney function and reduces mortality in severe acute ischemic renal failure. Moreover, combined ECE/NEP inhibition prevents hypertensive renal tissue damage in a blood pressure independent manner in L-NAME-treated rats.

Biophysical characterization of KCNQ1 P320 mutations linked to long QT syndrome 1.

Hereditary long QT syndrome (LQTS) is a cardiovascular disorder characterized by prolongation of the QT interval on the surface ECG and a high risk for arrhythmia-related sudden death. Mutations in a cardiac voltage-gated potassium channel, KCNQ1, account for the most common form of LQTS, LQTS1. The objective of this study was the characterization of a novel KCNQ1 mutation linked to LQTS. Electrophysiological properties and clinical features were determined and compared to characteristics of a different mutation at the same position. Single-strand conformation polymorphism analysis followed by direct sequencing was performed to screen LQTS genes for mutations. A novel missense mutation in the KCNQ1 gene, KCNQ1 P320H, was identified in the index patient presenting with recurrent syncope and aborted sudden death triggered by physical stress and swimming. Electrophysiological analyses of KCNQ1 P320H and the previously reported KCNQ1 P320A mutation indicate that both channels are non-functional and suppress wild type I(Ks) in a dominant-negative fashion. Based on homology modeling of the KCNQ1 channel pore region, we speculate that the proline residue at position 320 limits flexibility of the outer pore and is required to maintain the functional architecture of the selectivity filter/pore helix arrangement. Our observations on the KCNQ1 P320H mutation are consistent with previous studies indicating that pore mutations in potassium channel alpha-subunits are associated with more severe electrophysiological and clinical phenotypes than mutations in other regions of these proteins. This study emphasizes the significance of mutation screening for diagnosis, risk-assessment, and mutation-site specific management in LQTS patients.

Maternal and fetal PROGINS progesterone receptor polymorphism reduces the risk for transient tachypnea of the newborn.

BACKGROUND: Transient tachypnea of the newborn (TTN) is the most common perinatal respiratory disorder. It was suggested that the pathogenesis of TTN might involve altered activity of female sex hormones. This study analyzed whether the PROGINS progesterone receptor polymorphism, which is less responsive to progesterone, is associated with TTN. METHODS: A cohort of 2352 infants born to Caucasian women at the Obstetrics Department of the Charite was investigated prospectively. The collected information included the occurrence of respiratory disorders, birth weight, gestational age at delivery, mode of delivery, and maternal morbidity. Mothers and newborns were genotyped for the PROGINS progesterone receptor polymorphism. Statistical analyses considered correction for confounding factors. RESULTS: The presence of the mutated T2-allele either in mothers or in infants was associated with a reduction of the incidence of TTN in a gene dose-dependent manner (mothers T1/T1: 6.6%, T1/T2: 4.3% T2/T2: 2.3%, p < 0.01; infants T1/T1: 6.5%, T1/T2: 4.7%, T2/T2: 0.0%, p = 0.02 in a multivariable regression model). The total number of mutated T2-alleles present in a mother/child pair was associated with a reduction of TTN (4 T2-alleles: 6.4%, n=95; 3: 5.9%, n=30; 2: 3.1%, n=9; 1: 1.4%, n=1; 0:0%, n=0; p < 0.01 in a multivariable regression model). CONCLUSIONS: Both the maternal and fetal mutated alleles of the PROGINS progesterone receptor polymorphism seem to protect from TTN. The same phenotype occurs regardless of whether the mutation is localized in the mother or in the infant. Fetal as well as maternal T2-alleles synergistically reduce the risk for TTN in a gene dose-dependent manner.

ADMA predicts major adverse renal events in patients with mild renal impairment and/or diabetes mellitus undergoing coronary angiography.

Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of the nitric oxide (NO)-synthase and a biomarker of endothelial dysfunction (ED). ED plays an important role in the pathogenesis of contrast-induced nephropathy (CIN). The aim of our study was to evaluate serum ADMA concentration as a biomarker of an acute renal damage during the follow-up of 90 days after contrast medium (CM) application.Blood samples were obtained from 330 consecutive patients with diabetes mellitus or mild renal impairment immediately before, 24 and 48 hours after the CM application for coronary angiography. The patients were followed for 90 days. The composite endpoints were major adverse renal events (MARE) defined as occurrence of death, initiation of dialysis, or a doubling of serum creatinine concentration.Overall, ADMA concentration in plasma increased after CM application, although, there was no differences between ADMA levels in patients with and without CIN. ADMA concentration 24 hours after the CM application was predictive for dialysis with a specificity of 0.889 and sensitivity of 0.653 at values higher than 0.71 µmol/L (area under the curve: 0.854, 95% confidential interval: 0.767-0.941, P < 0.001). This association remained significant in multivariate Cox regression models adjusted for relevant factors of long-term renal outcome. 24 hours after the CM application, ADMA concentration in plasma was predictive for MARE with a specificity of 0.833 and sensitivity of 0.636 at a value of more than 0.70 µmol/L (area under the curve: 0.750, 95% confidence interval: 0.602-0.897, P = 0.004). Multivariate logistic regression analysis confirmed that ADMA and anemia were significant predictors of MARE. Further analysis revealed that increased ADMA concentration in plasma was highly significant predictor of MARE in patients with CIN. Moreover, patients with CIN and MARE had the highest plasma ADMA levels 24 hours after CM exposure in our study cohort. The impact of ADMA on MARE was independent of such known CIN risk factors as anemia, pre-existing renal failure, pre-existing heart failure, and diabetes.ADMA concentration in plasma is a promising novel biomarker of major contrast-induced nephropathy-associated events 90 days after contrast media exposure.

Atypical tetracyclic antidepressant maprotiline is an antagonist at cardiac hERG potassium channels.

Maprotiline is an antidepressant compound with an atypical tetracyclic structure that is widely used in elderly patients due to its favourable side-effect profile. However, there have been reports of proarrhythmia associated with maprotiline and in vitro studies of its electrophysiological properties have been lacking. Therefore, we characterised the effects of maprotiline on cardiac hERG channels. hERG channels were expressed in HEK cells and in the Xenopus oocyte expression system. Currents were measured using a whole-cell patch clamp and a two-microelectrode voltage-clamp. Maprotiline inhibited hERG currents with an IC(50) of 8.2 micromol/l in HEK cells and 29.2 micromol/l in Xenopus oocytes. Onset of the effect was rather slow and took several minutes. No wash-out of effect was observed. Maprotiline blocked hERG channels in the open and inactivated states, but not in the closed states. In mutant hERG channels Y652A and F656A, the effect was markedly attenuated (hERG-F656A) or completely abolished (hERG-Y652A). Voltage dependence of hERG current activation and inactivation was not affected by maprotiline. hERG inactivation was accelerated at positive potentials. The effect of maprotiline on hERG currents was voltage-dependent with a marked reduction at a more positive potential. hERG blockade by maprotiline was not frequency-dependent. Maprotiline is an antagonist of cardiac hERG potassium channels that preferably accesses the putative pore binding site Y652/F656. Although the affinity of maprotiline to hERG channels is low, its use in patients with risk factors for acquired long QT syndrome should be monitored appropriately.

In vitro modulation of HERG channels by organochlorine solvent trichlormethane as potential explanation for proarrhythmic effects of chloroform.

Acute chloroform intoxication can cause depression of the central nervous system and may lead to death from lethal arrhythmias or respiratory arrest. Thus, the organic solvent is no longer in clinical use as an anaesthetic, but still plays a role in cases of suicide, homicide or inhalation for psychotropic effects. Several cases of lethal arrhythmia after intoxication with chloroform have been described. Pharmacological inhibition of cardiac "human ether-à-go-go-related gene" (HERG) potassium channels is linked to proarrhythmic effects of cardiac and noncardiac drugs. To further investigate the electrophysiological basis of the arrhythmogenic potential of chloroform, we analysed inhibitory effects of chloroform on cloned HERG potassium channels, heterologously expressed in Xenopus oocytes and in Human Embryonic Kidney (HEK 293) cells using the double-electrode voltage-clamp technique and the whole-cell patch-clamp technique, respectively. In HEK cells, chloroform blocked HERG tail currents with an IC(50) of 4.97mM. Biophysical properties were further investigated in the Xenopus oocyte expression system. Onset and wash-out of block was fast and inhibition was completely reversible. Chloroform did not alter channel activation, however, direct channel inactivation was accelerated significantly. Steady-state-inactivation of HERG was not affected. Chloroform dependent block of HERG channels was voltage dependent with a decrease of inhibition at more positive membrane potentials. No frequency-dependence of block could be observed. In summary, chloroform blocked HERG potassium channels probably in a toxicologically relevant concentration. These findings contribute to the pathophysiology of proarrhythmic effects in acute chloroform intoxication.

Dominant-negative I(Ks) suppression by KCNQ1-deltaF339 potassium channels linked to Romano-Ward syndrome.

OBJECTIVE: Hereditary long QT syndrome (LQTS) is a genetically heterogeneous disease characterized by prolonged QT intervals and an increased risk for ventricular arrhythmias and sudden cardiac death. Mutations in the voltage-gated potassium channel subunit KCNQ1 induce the most common form of LQTS. KCNQ1 is associated with two different entities of LQTS, the autosomal-dominant Romano-Ward syndrome (RWS), and the autosomal-recessive Jervell and Lange-Nielsen syndrome (JLNS) characterized by bilateral deafness in addition to cardiac arrhythmias. In this study, we investigate and discuss dominant-negative I(Ks) current reduction by a KCNQ1 deletion mutation identified in a RWS family. METHODS: Single-strand conformation polymorphism analysis and direct sequencing were used to screen LQTS genes for mutations. Mutant KCNQ1 channels were heterologously expressed in Xenopus oocytes, and potassium currents were recorded using the two-microelectrode voltage clamp technique. RESULTS: A heterozygous deletion of three nucleotides (CTT) identified in the KCNQ1 gene caused the loss of a single phenylalanine residue at position 339 (KCNQ1-deltaF339). Electrophysiological measurements in the presence and absence of the regulatory beta-subunit KCNE1 revealed that mutant and wild type forms of an N-terminal truncated KCNQ1 subunit (isoform 2) caused much stronger dominant-negative current reduction than the mutant form of the full-length KCNQ1 subunit (isoform 1). CONCLUSION: This study highlights the functional relevance of the truncated KCNQ1 splice variant (isoform 2) in establishment and mode of inheritance in long QT syndrome. In the RWS family presented here, the autosomal-dominant trait is caused by multiple dominant-negative effects provoked by heteromultimeric channels formed by wild type and mutant KCNQ1-isoforms in combination with KCNE1.

Urinary Vitamin D Binding Protein and KIM-1 Are Potent New Biomarkers of Major Adverse Renal Events in Patients Undergoing Coronary Angiography.

BACKGROUND: Vitamin-D-binding protein (VDBP) is a low molecular weight protein that is filtered through the glomerulus as a 25-(OH) vitamin D 3/VDBP complex. In the normal kidney VDBP is reabsorbed and catabolized by proximal tubule epithelial cells reducing the urinary excretion to trace amounts. Acute tubular injury is expected to result in urinary VDBP loss. The purpose of our study was to explore the potential role of urinary VDBP as a biomarker of an acute renal damage. METHOD: We included 314 patients with diabetes mellitus or mild renal impairment undergoing coronary angiography and collected blood and urine before and 24 hours after the CM application. Patients were followed for 90 days for the composite endpoint major adverse renal events (MARE: need for dialysis, doubling of serum creatinine after 90 days, unplanned emergency rehospitalization or death). RESULTS: Increased urine VDBP concentration 24 hours after contrast media exposure was predictive for dialysis need (no dialysis: 113.06 ± 299.61 ng/ml, n = 303; need for dialysis: 613.07 ± 700.45 ng/ml, n = 11, Mean ± SD, p<0.001), death (no death during follow-up: 121.41 ± 324.45 ng/ml, n = 306; death during follow-up: 522.01 ± 521.86 ng/ml, n = 8; Mean ± SD, p<0.003) and MARE (no MARE: 112.08 ± 302.00 ng/ml, n = 298; MARE: 506.16 ± 624.61 ng/ml, n = 16, Mean ± SD, p<0.001) during the follow-up of 90 days after contrast media exposure. Correction of urine VDBP concentrations for creatinine excretion confirmed its predictive value and was consistent with increased levels of urinary Kidney Injury Molecule-1 (KIM-1) and baseline plasma creatinine in patients with above mentioned complications. The impact of urinary VDBP and KIM-1 on MARE was independent of known CIN risk factors such as anemia, preexisting renal failure, preexisting heart failure, and diabetes. CONCLUSIONS: Urinary VDBP is a promising novel biomarker of major contrast induced nephropathy-associated events 90 days after contrast media exposure.

Urinary ET-1 excretion after exposure to radio-contrast media in diabetic patients and patients with preexisting mild impaired renal function.

AIMS: Contrast media-induced nephropathy (CIN) is associated with increased morbidity and mortality. The renal endothelin system has been associated with disease progression of various acute and chronic renal diseases. However, robust data coming from adequately powered prospective clinical studies analyzing the short and long-term impacts of the renal ET system in patients with CIN are missing so far. We thus performed a prospective study addressing this topic. MAIN METHODS: We included 327 patients with diabetes or renal impairment undergoing coronary angiography. Blood and spot urine were collected before and 24 h after contrast media (CM) application. Patients were followed for 90 days for major clinical events like need for dialysis, unplanned rehospitalization or death. KEY FINDINGS: The concentration of ET-1 and the urinary ET-1/creatinine ratio decreased in spot urine after CM application (ET-1 concentration: 0.91±1.23 pg/ml versus 0.63±1.03 pg/ml, p<0.001; ET-1/creatinine ratio: 0.14±0.23 versus 0.09±0.19, p<0.001). The urinary ET-1 concentrations in patients with CIN decreased significantly more than in patients without CIN (-0.26±1.42 pg/ml vs. -0.79±1.69 pg/ml, p=0.041), whereas the decrease of the urinary ET-1/creatinine ratio was not significantly different (non-CIN patients: -0.05±0.30; CIN patients: -0.11±0.21, p=0.223). Urinary ET-1 concentrations as well as the urinary ET-1/creatinine ratio were not associated with clinical events (need for dialysis, rehospitalization or death) during the 90 day follow-up after contrast media exposure. However, the urinary ET-1 concentration and the urinary ET-1/creatinine ratio after CM application were higher in those patients who had a decrease of GFR of at least 25% after 90 days of follow-up. SIGNIFICANCE: In general the ET-1 system in the kidney seems to be down-regulated after contrast media application in patients with moderate CIN risk. Major long-term complications of CIN (need for dialysis, rehospitalization or death) are not associated with the renal ET system.

Blood pressure and glucose independent renoprotective effects of dipeptidyl peptidase-4 inhibition in a mouse model of type-2 diabetic nephropathy.

BACKGROUND: Despite the beneficial effects of type 4 dipeptidyl peptidase (DPP-4) inhibitors on glucose levels, its effects on diabetic nephropathy remain unclear. METHOD: This study examined the long-term renoprotective effects of DPP-4 inhibitor linagliptin in db/db mice, a model of type 2 diabetes. Results were compared with the known beneficial effects of renin-angiotensin system blockade by enalapril. Ten-week-old male diabetic db/db mice were treated for 3 months with either vehicle (n = 10), 3 mg linagliptin/kg per day (n = 8), or 20 mg enalapril/kg per day (n = 10). Heterozygous db/m mice treated with vehicle served as healthy controls (n = 8). RESULTS: Neither linagliptin nor enalapril had significant effects on the parameters of glucose metabolism or blood pressure in diabetic db/db mice. However, linagliptin treatment reduced albuminuria and attenuated kidney injury. In addition, expression of podocyte marker podocalyxin was normalized. We also analysed DPP-4 expression by immunofluorescence in human kidney biopsies and detected upregulation of DPP-4 in the glomeruli of patients with diabetic nephropathy, suggesting that our findings might be of relevance for human kidney disease as well. CONCLUSION: Treatment with DPP-4 inhibitor linagliptin delays the progression of diabetic nephropathy damage in a glucose-independent and blood-pressure-independent manner. The observed effects may be because of the attenuation of podocyte injury and inhibition of myofibroblast transformation.

Effects of telmisartan and linagliptin when used in combination on blood pressure and oxidative stress in rats with 2-kidney-1-clip hypertension.

OBJECTIVE: To investigate the effects of linagliptin alone and in combination with the angiotensin II receptor blocker (ARB), telmisartan on blood pressure (BP), kidney function, heart morphology and oxidative stress in rats with renovascular hypertension. METHODS: Fifty-seven male Wistar rats underwent unilateral surgical stenosis of the renal artery [2-kidney-1-clip (2k1c) method]. Animals were randomly divided into four treatment groups (n = 14-18 per group) receiving: telmisartan (10 mg/kg per day in drinking water), linagliptin (89 ppm in chow), combination (linagliptin 89 ppm + telmisartan 10 mg/kg per day) or placebo. An additional group of 12 rats underwent sham surgery. BP was measured one week after surgery. Hypertensive animals entered a 16-week dosing period. BP was measured 2, 4, 8, 12 and 16 weeks after the initiation of treatment. Blood and urine were tested for assessment of kidney function and oxidative stress 6, 10, 14 and 18 weeks after surgery. Blood and urine sampling and organ harvesting were finally performed. RESULTS: Renal stenosis caused an increase in mean ±â€ŠSD systolic BP as compared with the sham group (157.7 ±â€Š29.3 vs. 106.2 ±â€Š20.5 mmHg, respectively; P < 0.001). Telmisartan alone and in combination with linagliptin, normalized SBP (111.1 ±â€Š24.3 mmHg and 100.4 ±â€Š13.9 mmHg, respectively; P < 0.001 vs. placebo). Telmisartan alone and in combination with linagliptin significantly prevented cardiac hypertrophy, measured by heart weight and myocyte diameter. Renal function measured by cystatin C was not affected by 2k1c surgery. Telmisartan significantly increased plasma concentration of cystatin C. 2k1c surgery initiated fibrosis in both kidneys. Telmisartan promoted further fibrotic changes in the clipped kidney, as measured by protein expression of Col1a1 and histology for interstitial fibrosis and glomerulosclerosis. In non-clipped kidneys, telmisartan demonstrated antifibrotic properties, reducing Col1a1 protein expression. Plasma levels of oxidized low-density lipoprotein were higher in the placebo-treated 2k1c rats as compared to sham-operated animals. The increase was abolished by linagliptin alone (P = 0.03 vs. placebo) and in combination with telmisartan (P = 0.02 vs. placebo). Combination therapy also significantly reduced plasma concentration of carbonyl proteins (P = 0.04 vs. placebo). CONCLUSION: Inhibition of type 4 dipeptidyl peptidase with linagliptin did not counter BP-lowering effects of ARB in 2k1c rats. Linagliptin reduced lipid and protein oxidation in 2k1c rats, and this effect was BP-independent.

Effects of stimulation of soluble guanylate cyclase on diabetic nephropathy in diabetic eNOS knockout mice on top of angiotensin II receptor blockade.

The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN), is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB) treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC) as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy. Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d), riociguat (3 mg/kg/d), riociguat+telmisartan (3+1 mg/kg/d), and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071). Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg). Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002) reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h), whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h) or riociguat (97.1±15.7 µg/24 h) was not statistically significant. The combination treatment led to a significant (p<0.01) decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress. In conclusion, stimulation of sGC significantly reduced urinary albumin excretion in diabetic eNOS knockout mice treated already with ARB. Thus, this new drug class on top of standard ARBs administration may offer a new therapeutic approach for patients resistant to ARB treatment.

Effects of DPP-4 inhibitors on the heart in a rat model of uremic cardiomyopathy.

BACKGROUND: Uremic cardiomyopathy contributes substantially to mortality in chronic kidney disease (CKD) patients. Glucagon-like peptide-1 (GLP-1) may improve cardiac function, but is mainly degraded by dipeptidyl peptidase-4 (DPP-4). METHODOLOGY/PRINCIPAL FINDINGS: In a rat model of chronic renal failure, 5/6-nephrectomized [5/6N] rats were treated orally with DPP-4 inhibitors (linagliptin, sitagliptin, alogliptin) or placebo once daily for 4 days from 8 weeks after surgery, to identify the most appropriate treatment for cardiac dysfunction associated with CKD. Linagliptin showed no significant change in blood level AUC(0-∞) in 5/6N rats, but sitagliptin and alogliptin had significantly higher AUC(0-∞) values; 41% and 28% (p = 0.0001 and p = 0.0324), respectively. No correlation of markers of renal tubular and glomerular function with AUC was observed for linagliptin, which required no dose adjustment in uremic rats. Linagliptin 7 µmol/kg caused a 2-fold increase in GLP-1 (AUC 201.0 ng/l*h) in 5/6N rats compared with sham-treated rats (AUC 108.6 ng/l*h) (p = 0.01). The mRNA levels of heart tissue fibrosis markers were all significantly increased in 5/6N vs control rats and reduced/normalized by linagliptin. CONCLUSIONS/SIGNIFICANCE: DPP-4 inhibition increases plasma GLP-1 levels, particularly in uremia, and reduces expression of cardiac mRNA levels of matrix proteins and B-type natriuretic peptides (BNP). Linagliptin may offer a unique approach for treating uremic cardiomyopathy in CKD patients, with no need for dose-adjustment.

Endothelin-converting enzyme/neutral endopeptidase inhibitor SLV338 prevents hypertensive cardiac remodeling in a blood pressure-independent manner.

Hypertensive heart disease is a major contributor to cardiovascular mortality. Endothelin is a potent vasoconstrictive and profibrotic mediator produced by the endothelin-converting enzyme (ECE), whereas natriuretic peptides, degraded by the neutral endopeptidase (NEP), have diuretic, vasodilatory, and antifibrotic properties. Thus, combined ECE/NEP inhibition may halt hypertensive cardiac remodeling. This study examined effects of SLV338, a novel ECE/NEP inhibitor, on cardiac protection in experimental renovascular hypertension (2-kidney, 1-clip [2K1C]). Male rats were allocated to 5 groups: sham-operated rats, untreated animals with 2K1C, 2K1C animals treated with oral SLV338 (30 and 100 mg/kg per day), and 2K1C animals treated with oral losartan (20 mg/kg per day). Treatment duration was 12 weeks. Blood pressure was assessed every 4 weeks. At study end, hearts were taken for histology/computer-aided histomorphometry/immunohistochemistry. Pharmacological properties of SLV338 are described. SLV338 is a dual ECE/NEP inhibitor, as demonstrated both in vitro and in vivo. In the 2K1C study, losartan lowered blood pressure by ≤46 mm Hg, whereas both dosages of SLV338 had no effect. However, SLV338 (both dosages) completely normalized cardiac interstitial fibrosis, perivascular fibrosis, myocyte diameter, and media:lumen ratio of cardiac arteries, as did losartan. Cardiac transforming growth factor-ß1 expression was significantly enhanced in untreated 2K1C rats versus controls, whereas treatment with SLV338 and losartan prevented this effect. Taken together, dual ECE/NEP inhibitor SLV338 prevents cardiac remodeling to the same extent as losartan, but in a blood pressure-independent manner, in a rat model of renovascular hypertension. This effect is at least partially mediated via suppression of cardiac transforming growth factor-ß1 expression.

Renal effects of the novel selective adenosine A1 receptor blocker SLV329 in experimental liver cirrhosis in rats.

Liver cirrhosis is often complicated by an impaired renal excretion of water and sodium. Diuretics tend to further deteriorate renal function. It is unknown whether chronic selective adenosine A(1) receptor blockade, via inhibition of the hepatorenal reflex and the tubuloglomerular feedback, might exert diuretic and natriuretic effects without a reduction of the glomerular filtration rate. In healthy animals intravenous treatment with the novel A(1) receptor antagonist SLV329 resulted in a strong dose-dependent diuretic (up to 3.4-fold) and natriuretic (up to 13.5-fold) effect without affecting creatinine clearance. Male Wistar rats with thioacetamide-induced liver cirrhosis received SLV329, vehicle or furosemide for 12 weeks. The creatinine clearance of cirrhotic animals decreased significantly (-36.5%, p<0.05), especially in those receiving furosemide (-41.9%, p<0.01). SLV329 was able to prevent this decline of creatinine clearance. Mortality was significantly lower in cirrhotic animals treated with SLV329 in comparison to animals treated with furosemide (17% vs. 54%, p<0.05). SLV329 did not relevantly influence the degree of liver fibrosis, kidney histology or expression of hepatic or renal adenosine receptors. In conclusion, chronic treatment with SLV329 prevented the decrease of creatinine clearance in a rat model of liver cirrhosis. Further studies will have to establish whether adenosine A(1) receptor antagonists are clinically beneficial at different stages of liver cirrhosis.

Endothelin-1 overexpression restores diastolic function in eNOS knockout mice.

BACKGROUND: The cardiac nitric oxide and endothelin-1 (ET-1) systems are closely linked and play a critical role in cardiac physiology. The balance between both systems is often disturbed in cardiovascular diseases. To define the cardiac effect of excessive ET-1 in a status of nitric oxide deficiency, we compared left ventricular function and morphology in wild-type mice, ET-1 transgenic (ET(+/+)) mice, endothelial nitric oxide synthase knockout (eNOS(-/-)) mice, and ET(+/+)eNOS(-/-) mice. METHODS AND RESULTS: eNOS(-/-) and ET(+/+)eNOS(-/-) mice developed high blood pressure compared with wild-type and ET(+/+) mice. Left ventricular catheterization showed that eNOS(-/-) mice, but not ET(+/+)eNOS(-/-) , developed diastolic dysfunction characterized by increased end-diastolic pressure and relaxation constant tau. To elucidate the causal molecular mechanisms driving the rescue of diastolic function in ET(+/+)eNOS(-/-) mice, the cardiac proteome was analyzed. Two-dimensional gel electrophoresis coupled to mass spectrometry offers an appropriate hypothesis-free approach. ET-1 overexpression on an eNOS(-/-) background led to an elevated abundance and change in posttranslational state of antioxidant enzymes (e.g., peroxiredoxin-6, glutathione S-transferase mu 2, and heat shock protein beta 7). In contrast to ET(+/+)eNOS(-/-) mice, eNOS(-/-) mice showed an elevated abundance of proteins responsible for sarcomere disassembly (e.g., cofilin-1 and cofilin-2). In ET(+/+)eNOS(-/-) mice, glycolysis was favored at the expense of fatty acid oxidation. CONCLUSION: eNOS(-/-) mice developed diastolic dysfunction; this was rescued by ET-1 transgenic overexpression. This study furthermore suggests that cardiac ET-1 overexpression in case of eNOS deficiency causes specifically the regulation of proteins playing a role in oxidative stress, myocytes contractility, and energy metabolism.