Podocyte Density and Albuminuria in Aging Diabetic Ins2± Mice with or Without Adenosine A1 Receptor Signaling.

Aim Of Study: To investigate podocyte density in aging diabetic Ins2± and Ins2±, A1AR-/- mouse models in C57Bl/6 background.

Methods: Ins2± mice and especially Ins2±, adenosine A1 receptor knockout mice (Ins2±, A1AR-/-) are mouse models with a phenotype of diabetic nephropathy. Aged mice (at ~40 weeks) were assessed for glomerular filtration barrier function by measuring albuminuria, glomerular filtration, glomerular damage by electron microscopy, and podocyte numbers by Wilms Tumor protein (WT-1) staining.

Blood pressure, heart rate and tubuloglomerular feedback in A1AR-deficient mice with different genetic backgrounds.

Aim: Differences in genetic background between control mice and mice with targeted gene mutations have been recognized as a potential cause for phenotypic differences. In this study, we have used A1AR-deficient mice in a C57Bl/6 and SWR/J congenic background to assess the influence of background on the effect of A1AR-deficiency on cardiovascular and renal functional parameters.

Methods: In A1AR+/+ and A1AR-/- mice in C57Bl/6 and SWR/J congenic backgrounds, we assessed blood pressure and heart rate using radio-telemetry, plasma renin concentrations and tubuloglomerular feedback.

Fluid reabsorption in proximal convoluted tubules of mice with gene deletions of claudin-2 and/or aquaporin1.

Deletions of claudin-2 (Cldn2) and aquaporin1 (AQP1) reduce proximal fluid reabsorption (PFR) by about 30% and 50%, respectively. Experiments were done to replicate these observations and to determine in AQP1/claudin-2 double knockout mice (DKO) if the effects of deletions of these established water pores are additive. PFR was determined in inactin/ketamine-anesthetized mice by free-flow micropuncture using single-nephron I(125)-iothalamate (io) clearance.

Tubuloglomerular feedback and renal function in mice with targeted deletion of the type 1 equilibrative nucleoside transporter.

A(1) adenosine receptors (A1AR) are required for the modulation of afferent arteriolar tone by changes in luminal NaCl concentration implying that extracellular adenosine concentrations need to change in synchrony with NaCl. The present experiments were performed in mice with a null mutation in the gene for the major equilibrative nucleoside transporter ENT1 to test whether interference with adenosine disposition by cellular uptake of adenosine may modify TGF characteristics. Responses of stop flow pressure (P(SF)) to maximum flow stimulation were measured in mice with either C57Bl/6 or SWR/J genetic backgrounds.

Renal afferent arteriolar and tubuloglomerular feedback reactivity in mice with conditional deletions of adenosine 1 receptors.

Adenosine 1 receptors (A1AR) have been shown in previous experiments to play a major role in the tubuloglomerular feedback (TGF) constrictor response of afferent arterioles (AA) to increased loop of Henle flow. Overexpression studies have pointed to a critical role of vascular A1AR, but it has remained unclear whether selective deletion of A1AR from smooth muscle cells is sufficient to abolish TGF responsiveness. To address this question, we have determined TGF response magnitude in mice in which vascular A1AR deletion was achieved using the loxP recombination approach with cre recombinase being controlled by a smooth muscle actin promoter (SmCre/A1ARff).

Impaired glucose tolerance in the absence of adenosine A1 receptor signaling.

Objective: The role of adenosine (ADO) in the regulation of glucose homeostasis is not clear. In the current study, we used A1-ADO receptor (A1AR)-deficient mice to investigate the role of ADO/A1AR signaling for glucose homeostasis.

Research Design And Methods: After weaning, A1AR(-/-) and wild-type mice received either a standard diet (12 kcal% fat) or high-fat diet (HFD; 45 kcal% fat).

Angiotensin II overcomes strain-dependent resistance of rapid CKD progression in a new remnant kidney mouse model.

The remnant kidney model in C57BL/6 mice does not develop progressive chronic kidney disease (CKD). In this study we modified the model to mimic features of human CKD and to define accelerants of disease progression using three strains of mice. Following the procedure, there was a progressive increase in albuminuria, progressive loss in renal function, severe glomerulosclerosis and interstitial fibrosis, hypertension, cardiac fibrosis, and anemia by 4 weeks in CD-1 mice and by 12 weeks in 129S3 mice.

Renal failure in mice with Gsalpha deletion in juxtaglomerular cells.

Background: Mice with deletion of Gsalpha in renin-producing cells (RC/FF mice) have been shown to have greatly reduced renin production and lack of responsiveness of renin secretion to acute stimuli. In addition, young RC/FF mice are hypotensive and have a vasopressin-resistant concentrating defect. In the present study we have determined the long-term effect on renal function, blood pressure, and renal pathology in this low renin and diuretic mouse model.

Stimulation of renin secretion by angiotensin II blockade is Gsalpha-dependent.

Angiotensin II converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB) presumably stimulate renin secretion by interrupting angiotensin II feedback inhibition. The increase in cytosolic calcium caused by activation of Gq-coupled AT1 receptors may mediate the renin-inhibitory effect of angiotensin II at the cellular level, implying that ACEI and ARB may work by reducing intracellular calcium. Here, we investigated whether angiotensin II blockade acts predominantly through Gs-mediated stimulation of adenylyl cyclase (AC) by testing the effect of ACEI and ARB in mice with juxtaglomerular cell-specific deficiency of the AC-stimulatory Gsalpha.

Major contribution of tubular secretion to creatinine clearance in mice.

This study was performed to quantify the fraction of excreted creatinine not attributable to creatinine filtration for accurately determining the glomerular filtration rate in mice. To measure this we compared creatinine filtration with the simultaneous measurement of inulin clearance using both single-bolus fluorescein isothiocyanate (FITC)-inulin elimination kinetics and standard FITC-inulin infusion. During anesthesia, creatinine filtration was found to be systematically higher than inulin clearance in both male and female C57BL/6J mice.

Enhanced tubuloglomerular feedback in mice with vascular overexpression of A1 adenosine receptors.

Adenosine 1 receptors (A1AR) in the kidney are expressed in the vasculature and the tubular system. Pharmacological inhibition or global genetic deletion of A1AR causes marked reductions or abolishment of tubuloglomerular feedback (TGF) responses. To assess the function of vascular A1AR in TGF, we generated transgenic mouse lines in which A1AR expression in smooth muscle was augmented by placing A1AR under the control of a 5.

Dense-core vesicle proteins IA-2 and IA-2{beta} affect renin synthesis and secretion through the {beta}-adrenergic pathway.

IA-2 and IA-2beta, major autoantigens in type 1 diabetes, are transmembrane proteins in dense-core vesicles, and their expression influences the secretion of hormones and neurotransmitters. The present experiments were performed to examine whether IA-2 and IA-2beta modulate the release of renin from dense-core vesicles of juxtaglomerular granular cells in the kidney. Plasma renin concentration (PRC; ng angiotensin I.

Salt sensitivity of blood pressure in NKCC1-deficient mice.

NKCC1 is a widely expressed isoform of the Na-2Cl-K cotransporter that mediates several direct and indirect vascular effects and regulates expression and release of renin. In this study, we used NKCC1-deficient (NKCC1-/-) and wild-type (WT) mice to assess day/night differences of blood pressure (BP), locomotor activity, and renin release and to study the effects of high (8%) or low (0.03%) dietary NaCl intake on BP, activity, and the renin/aldosterone system.

Persistence of circadian variation in arterial blood pressure in beta1/beta2-adrenergic receptor-deficient mice.

The beta-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in beta1/beta2-adrenergic receptor-deficient mice (beta1/beta2ADR-/-) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four-hour recordings of MAP, heart rate (HR), and locomotor activity were made in conscious 16- to 17-wk-old mice [wild-type, (WT), n = 7; beta1/beta2ADR-/-, n = 10] by telemetry.

Lack of A1 adenosine receptors augments diabetic hyperfiltration and glomerular injury.

Intraglomerular hypertension and glomerular hyperfiltration likely contribute to the pathogenesis of diabetic nephropathy, and tubuloglomerular feedback (TGF) has been suggested to play a role in diabetic hyperfiltration. A1 adenosine receptor (A1AR) null mice lack a TGF response, so this model was used to investigate the contribution of TGF to hyperfiltration in diabetic Ins2(+/-) Akita mice. TGF responses in Ins2(+/-) A1AR(-/-) double mutants were abolished, whereas they were attenuated in Ins2(+/-) mice.

Tubuloglomerular feedback and renin secretion in NTPDase1/CD39-deficient mice.

Studies in mice with null mutations of adenosine 1 receptor or ecto-5'-nucleotidase genes suggest a critical role of adenosine and its precursor 5'-AMP in tubulovascular signaling. To assess whether the source of juxtaglomerular nucleotides can be traced back to ATP dephosphorylation, experiments were performed in mice with a deficiency in NTPDase1/CD39, an ecto-ATPase catalyzing the formation of AMP from ATP and ADP. Urine osmolarity and glomerular filtration rate (GFR) were indistinguishable between NTPDase1/CD39(-/-) and wild-type (WT) mice.

Skeletal abnormalities and extra-skeletal ossification in mice with restricted Gsalpha deletion caused by a renin promoter-Cre transgene.

We have recently generated a transgenic mouse line (termed hRen-Cre) that expresses Cre-recombinase under the control of a 12.2-kb fragment of the human renin promoter. In the present study, we have crossed hRen-Cre mice with a mouse strain in which exon 1 of the Gnas gene is flanked by loxP sites.

Regulation of renin secretion and expression in mice deficient in beta1- and beta2-adrenergic receptors.

The present experiments were performed in beta1/beta2-adrenergic receptor-deficient mice (beta1/beta2ADR(-/-)) to assess the role of beta-adrenergic receptors in basal and regulated renin expression and release. On a control diet, plasma renin concentration (in ng angiotensin I per mL per hour), determined in tail vein blood, was significantly lower in beta1/beta2ADR(-/-) than in wild-type (WT) mice (222+/-65 versus 1456+/-335; P<0.01).

Renal function in mice with targeted disruption of the A isoform of the Na-K-2Cl co-transporter.

Three different full-length splice isoforms of the Na-K-2Cl co-transporter (NKCC2/BSC1) are expressed along the thick ascending limb of Henle (TAL), designated NKCC2A, NKCC2B, and NKCC2F. NKCC2F is expressed in the medullary, NKCC2B mainly in the cortical, and NKCC2A in medullary and cortical portions of the TAL. NKCC2B and NKCC2A were shown to be coexpressed in the macula densa (MD) segment of the mouse TAL.

Low plasma renin and reduced renin secretory responses to acute stimuli in conscious COX-2-deficient mice.

In the current experiments, we determined the response of plasma renin concentration (PRC) to acute intraperitoneal administration of furosemide (40 mg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), candesartan (50 microg), or quinaprilate (50 microg) in conscious wild-type (WT) and cyclooxygenase (COX)-2-/- mice on three different genetic backgrounds (mixed, C57BL/6, 129J). PRC was measured in plasma obtained by tail vein puncture. Basal PRC was significantly lower in COX-2-/- than WT mice independent of genetic background (51, 10, and 17% of WT in mixed, 129J, and C57BL/6).

Regulation of renin in mice with Cre recombinase-mediated deletion of G protein Gsalpha in juxtaglomerular cells.

By crossing mice with expression of Cre recombinase under control of the endogenous renin promoter (Sequeira Lopez ML, Pentz ES, Nomasa T, Smithies O, Gomez RA. Dev Cell 6: 719-728, 2004) with mice in which exon 1 of the Gnas gene was flanked by loxP sites (Chen M, Gavrilova O, Liu J, Xie T, Deng C, Nguyen AT, Nackers LM, Lorenzo J, Shen L, Weinstein LS. Proc Natl Acad Sci USA), we generated animals with preferential and nearly complete excision of Gsalpha in juxtaglomerular granular (JG) cells.

Macula densa control of renin secretion and preglomerular resistance in mice with selective deletion of the B isoform of the Na,K,2Cl co-transporter.

Na,K,2Cl co-transporter (NKCC2), the primary NaCl uptake pathway in the thick ascending limb of Henle, is expressed in three different full-length splice variants, called NKCC2F, NKCC2A, and NKCC2B. These variants, derived by differential splicing of the variable exon 4, show a distinct distribution pattern along the loop of Henle, but the functional significance of this organization is unclear. By introduction of premature stop codons into exon 4B, specific for the B isoform, mice with an exclusive NKCC2B deficiency were generated.

Effect of apocynin treatment on renal expression of COX-2, NOS1, and renin in Wistar-Kyoto and spontaneously hypertensive rats.

Macula densa (MD) cells of the juxtaglomerular apparatus (JGA) synthesize type 1 nitric oxide synthase (NOS1) and type 2 cyclooxygenase (COX-2). Both nitric oxide (NO) and prostaglandins have been considered to mediate or modulate the control of renin secretion. Reactive oxygen species (ROS) produced locally by NADPH oxidase may influence NO bioavailability.

Reporter gene recombination in juxtaglomerular granular and collecting duct cells by human renin promoter-Cre recombinase transgene.

To assess the feasibility of using the renin promoter for expressing Cre recombinase in juxtaglomerular (JG) cells only, we generated five independent transgenic mouse lines (designated hRen-Cre) expressing Cre recombinase under control of a 12.2-kb human renin promoter. In the kidneys of adult mice Cre mRNA (RT-PCR) was found in the renal cortex, with Cre protein (immunohistochemistry) being localized in afferent arterioles and to a lower degree in interlobular arteries.

Adenosine as a mediator of macula densa-dependent inhibition of renin secretion.

Adenosine acting through A(1) adenosine receptors (A1AR) has been shown previously to be required for the vasoconstriction elicited by high luminal NaCl concentrations at the macula densa (MD). The present experiments were performed to investigate a possible role of A1AR in MD control of renin secretion in conscious wild-type (WT) and A1AR-deficient mice. The intravenous injection of NaCl (5% body wt) reduced plasma renin concentration (PRC; ng ANG I x ml(-1) x h(-1)) from 1,479 +/- 129 to 711 +/- 77 (P < 0.