A method for accurate measurement of GFR in conscious, spontaneously voiding rats

A method for accurate measurement of GFR in conscious, spontaneously voiding rats. Renal function measurement by clearance methods relies on accurately timed urine collection. In small experimental animals, renal function measurement is usually performed under anesthesia and/or with the application of bladder catheters to ensure accurate urine collection. To avoid both anesthesia and the need for bladder catheters we developed a method to measure glomerular filtration rate (GFR) in spontaneously voiding conscious rats. GFR was measured as the urinary clearance of constantly infused 125I-iothalamate. To correct for incomplete bladder emptying, urinary clearance of 125I-iothalamate was multiplied by the ratio of plasma and urinary clearance of simultaneously infused 131I-hippuran, a correction method that has been previously validated in humans. Reproducibility of the technique was evaluated by analysis of the results of four consecutive clearance periods during the day (intra-assay variation) in a group of 17 rats and of two consecutive clearance periods on two or three separate days in a group of 20 rats (inter-assay variation), all with normal renal function. Application of the correction method reduced the intra-assay coefficient of variation (mean ± SD) from 37.4 ± 14.3 to 5.4 ± 2.3% (P < 0.05). The mean inter-assay coefficient of variation fell slightly from 23.4 ± 10.3 to 11.0 ± 7.2% (P < 0.10). In rats with moderately impaired renal function (N = 8) the intra-assay variation fell from 27.9 ± 20.7 to 2.7 ± 1.6% (P < 0.05). Our data show that this correction method is a useful technique to assess renal function in conscious, spontaneously voiding rats

Author index for volume 286

Susceptibility to renal injury varies among individuals. Previously, we found that individual endothelial function of healthy renal arteries in vitro predicted severity of renal damage after 5/6 nephrectomy. Here we hypothesized that individual differences in endothelial function in vitro and renal perfusion in vivo predict the severity of renal damage in a model of adriamycin-induced nephropathy. In three separate studies, the following baseline parameters were measured in healthy male Wistar rats: (1) acetylcholine (ACh)-induced relaxation in small renal arteries in vitro (n = 16) and the contribution of prostaglandins, nitric oxide (NO) and endothelium-dependent hyperpolarizing factor (EDHF) to the relaxation; (2) glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) in spontaneously voiding rats in vivo (n = 16) and (3) the acute effect of the NO-synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME, n = 12) on renal blood flow (RBF) as compared to vehicle infusion (n = 9). Following these measurements, adriamycin (1.75 mg/kg i.v.) was injected and subsequent renal damage after 6 weeks was related to the baseline parameters. Total ACh-induced (r = 0.51, P <0.05) and EDHF-mediated relaxation (r = 0.68, P <0.05), as well as ERPF (r = 0.66, P <0.01), positively correlated with the severity of proteinuria 6 weeks after injection. In contrast, pronounced baseline NO-mediated dilation was associated with lower proteinuria (r = 0.71, P <0.01). Nevertheless, an acute L-NAME infusion, strongly reducing RBF by 22 +/- 8%, during adriamycin administration provided protection against the development of proteinuria. Individual animals with pronounced baseline endothelial dilatory ability measured in vitro and high ERPF in vivo are vulnerable to renal damage after the adriamycin injection. Acute inhibition of NO during adriamycin administration, resulting in a decrease of RBF, protects against renal injury, probably by limiting the delivery of the drug to the kidney. Therefore, interindividual variability in renal haemodynamics might be crucially involved in susceptibility to nephrotoxic renal damag

Canagliflozin and Renal Outcomes in Type 2 Diabetes and Nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to 300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m 2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Genetic association study of childhood aggression across raters, instruments, and age

Childhood aggressive behavior (AGG) has a substantial heritability of around 50%. Here we present a genome-wide association metaanalysis (GWAMA) of childhood AGG, in which all phenotype measures across childhood ages from multiple assessors were included. We analyzed phenotype assessments for a total of 328 935 observations from 87 485 children aged between 1.5 and 18 years, while accounting for sample overlap. We also meta-analyzed within subsets of the data, i.e., within rater, instrument and age. SNP-heritability for the overall meta-analysis AGGoverall was 3.31% (SE= 0.0038). We found no genome-wide significant SNPs for AGGoverall. The gene-based analysis returned three significant genes: ST3GAL3 (P= 1.6E-06), PCDH7 (P= 2.0E-06), and IPO13 (P= 2.5E-06). All three genes have previously been associated with educational traits. Polygenic scores based on our GWAMA significantly predicted aggression in a holdout sample of children (variance explained = 0.44%) and in retrospectively assessed childhood aggression (variance explained = 0.20%). Genetic correlations rg among rater-specific assessment of AGG ranged from rg= 0.46 between self- and teacher-assessment to rg= 0.81 between mother- and teacher-assessment. We obtained moderate-to-strong rgs with selected phenotypes from multiple domains, but hardly with any of the classical biomarkers thought to be associated with AGG. Significant genetic correlations were observed with most psychiatric and psychological traits (range |rg|: 0.19-1.00), except for obsessive-compulsive disorder. Aggression had a negative genetic correlation (rg=∼-0.5) with cognitive traits and age at first birth. Aggression was strongly genetically correlated with smoking phenotypes (range |rg| : 0.46-0.60). The genetic correlations between aggression and psychiatric disorders were weaker for teacher-reported AGG than for mother- and self-reported AGG. The current GWAMA of childhood aggression provides a powerful tool to interrogate the rater-specific genetic etiology of AGG.</p

Approaches and methods in gene therapy for kidney disease

Renal gene therapy may offer new strategies to treat diseases of native and transplanted kidneys. Several experimental techniques have been developed and employed using nonviral, viral, and cellular vectors. The most efficient vector for in vivo transfection appears to be adenovirus. Glomeruli, blood vessels, interstitial cells, and pyelum can be transfected with high efficiency. In addition, electroporation and microbubbles with ultrasound, both being enhanced naked plasmid techniques, offer good opportunities. Trapping of mesangial cells into the glomeruli as well as natural targeting of monocytes or macrophages to inflamed kidneys are elegant methods for site-specific delivery of genes. For gene therapy in kidney transplantation, hemagglutinating virus of Japan liposomes are efficient vectors for tubular transfection, whereas enhanced naked plasmid techniques are suitable for glomerular transfection. However, adenovirus offers the best opportunities in a renal transplantation setup because varying parameters of graft perfusion allows targeting of different cell types. In renal grafts, lymphocytes can be used for selective targeting to sites of inflammation. In conclusion, for both in vivo and ex vivo renal transfection, enhanced naked plasmids and adenovirus offer the best perspectives for effective clinical application. Moreover, the development of safer, nonimmunogenic vectors and the large-scale production could make clinical renal gene therapy a realistic possibility for the near future

Early, but not late therapy with a vasopressin V1a-antagonist ameliorates the development of renal damage after 5/6 nephrectomy

INTRODUCTION: Vasopressin, mainly through the V1a-receptor, is thought to be a major player in the maintenance of hyperfiltration. Its inhibition could therefore lead to a decrease in progression of chronic renal failure. To this end, the effect of the vasopressin V1a-receptor-selective antagonist, YM218, was studied on proteinuria and focal glomerulosclerosis in early and late intervention after 5/6 nephrectomy in rats, and compared with an angiotensin-converting enzyme inhibitor (ACE-I). MATERIALS AND METHODS: After 5/6 nephrectomy, early intervention was performed between week 2 and 10 thereafter with the V1a-receptor-selective antagonist (VRA, 10 mg/kg/day, n=10), enalapril (ACE-I, 10 mg/kg/day, n=9), or vehicle (n=8). Late intervention was performed in another group between week 6 and 12 with VRA (10 mg/kg/day, n=7), lisinopril (ACE-I, 5 mg/kg/day, n=7), or vehicle (n=7). RESULTS: In early intervention, proteinuria and focal glomerulosclerosis were significantly decreased by VRA compared to vehicle (44+7% and 59+8% respectively). ACE-I significantly decreased proteinuria (67+7%) and a trend towards a decrease in focal glomerulosclerosis was observed (30+18%). In late intervention, VRA did not decrease proteinuria and focal glomerulosclerosis compared to vehicle (21+20% and 0%, respectively), ACE-I significantly lowered proteinuria (92+2%) and a focal glomerulosclerosis (69+1%) lowering trend was observed. CONCLUSION: These results indicate that VRA may protect against early progression of renal injury after 5/6 nephrectomy, whereas its effectiveness seems limited in established renal damage

Myocardial infarction does not further impair renal damage in 5/6 nephrectomized rats

Background. Recent observational studies show that reduced renal function is an independent risk factor for the development of cardiovascular disease. Previously, we reported that myocardial infarction (MI) indeed enhanced mild renal function decline in rats after unilateral nephrectomy (NX) and that RAAS intervention inhibited this decline. The effects of an MI on pre-existing sever renal function loss and the effects of RAAS intervention interrupting this hypothesized cardiorenal interaction are however unknown and clinically even more relevant. Methods. Male Wistar rats underwent MI, sham MI, 5/6NX, or 5/6NX and MI. Six weeks later, the NX rats were treated with an angiotensin-converting enzyme inhibitor (ACEi) or vehicle for 6 weeks. Results. An MI did not significantly induce more proteinuria (303 +/- 46 versus 265 +/- 24 mg/24 h) and glomerulosclerosis (40 +/- 11 versus 28 +/- 4 arbitrary units) in 5/6NX+MI compared to 5/6NX, and ACEi therapy was equally effective in reducing renal damage in these groups. In the 5/6NX+MI group, decreased renal blood flow and creatinine clearance were observed compared to 5/6NX (2.2 +/- 0.6 versus 3.6 +/- 0.4 ml/min/kg and 2.1 +/- 0.3 versus 2.9 +/- 0.3 ml/min/kg), which both increased after ACEi to levels comparable found in the group that underwent 5/6NX alone. Conclusions. MI does not further deteriorate structural renal damage induced by 5/6NX compared with 5/6NX alone. Furthermore, renal haemodynamic impairment occurs after MI, which can be improved applying ACEi therapy. Therefore, we conclude that treatment with ACEi should be optimized in patients with chronic kidney disease after MI to improve renal function