Concurrent activation of multiple vasoactive signaling pathways in vasoconstriction caused by tubuloglomerular feedback: a quantitative assessment.

Tubuloglomerular feedback (TGF) describes the negative relationship between (a) NaCl concentration at the macula densa and (b) glomerular filtration rate or glomerular capillary pressure. TGF-induced vasoconstriction of the afferent arteriole results from the enhanced effect of several vasoconstrictors with an effect size sequence of adenosine = 20-HETE > angiotensin II > thromboxane = superoxide > renal nerves > ATP. TGF-mediated vasoconstriction is limited by the simultaneous release of several vasodilators with an effect size sequence of nitric oxide > carbon monoxide = kinins > adenosine. The sum of the constrictor effects exceeds that of the dilator effects by the magnitude of the TGF response. The validity of the additive model used in this analysis can be tested by determining the effect of combined inhibition of some or all agents contributing to TGF. Multiple independent contributors to TGF are consistent with the variability of TGF and of the factors contributing to TGF resetting.

Defective renal autoregulation in the chronic bile duct ligation model of liver failure.

BACKGROUND: Cirrhosis of the liver is often associated with an impairment of renal function that is usually not associated with consistent structural abnormalities of the renal parenchyma, but is thought to be the functional consequence of arterial underfilling and reduced arterial blood pressure. METHOD: We have used the cirrhosis model of chronic bile duct ligation (BDL) to assess the response of renal blood flow to a change of blood pressure. We have measured renal haemodynamics in BDL rats. RESULT: Three weeks after BDL, rats showed elevated levels of total bilirubin, AST, and ALT as well as reduced arterial blood pressure. Creatinine clearance was significantly reduced, and plasma creatinine and urea nitrogen were elevated. Renal blood flow at baseline blood pressure was significantly lower in the BDL group than in the sham group. Clamp-induced reductions of renal perfusion pressure caused significantly greater changes of renal blood flow in BDL than control rats. The autoregulatory index over a comparable blood pressure range averaged 0.28 ± 0.35 in control rats and 1.26 ± 0.6 in BDL rats (p = 0.0004) indicating impairment of renal autoregulation in liver cirrhosis. CONCLUSION: Tubuloglomerular feedback (TGF) responses were significantly attenuated in BDL rats, especially in the subnormal flow range. Impairment of renal blood flow autoregulation, to some extent mediated by reduced TGF-mediated vasodilatation, may contribute to the renal vascular constrictor state in liver cirrhosis by preventing the full dilatory response to the blood pressure reduction.

Profound hypothermia after adenosine kinase inhibition in A1AR-deficient mice suggests a receptor-independent effect of intracellular adenosine.

Administration of the nucleoside adenosine has been shown to induce hypothermia in a number of species, an effect mediated predominantly by the adenosine 1 receptor (A1AR) subtype. The present experiments were performed to explore the possibility that the rise of intracellular adenosine levels expected to accompany adenosine administration may contribute to the hypothermic effect of adenosine independent of A1AR activation. Since phosphorylation of adenosine by adenosine kinase (ADK) is causal in the maintenance of low intracellular adenosine, we have examined the effect of ADK inhibition on core body temperature (CBT). Our data show that inhibition of ADK by A-134974 causes a long-lasting deep hypothermia in wild-type mice. Since there was an about 4-fold increase of adenosine plasma levels, experiments were repeated in A1AR-/- mice. ADK inhibition caused deep hypothermia despite the absence of A1AR, although the effect was significantly reduced compared to WT. Furthermore, the dose-dependent hypothermia caused by adenosine administration in WT mice was found to be reduced, but not abolished in A1AR-/- mice. To assess the possible role of A2AR and A3AR activation in our experimental setting, we compared the effects of the agonists CPA (A1AR), CGS21680 (A2AR), and IB-MECA (A3AR) on CBT. Hypothermia induced by CPA was much greater than that caused by CGS21680 or IB-MECA indicating that A1AR activation is the major receptor-dependent pathway for adenosine-induced hypothermia under our experimental conditions. Induction of deep hypothermia by inhibition of ADK, maintenance of this effect in A1AR-/- mice, and maintenance of adenosine-induced hypothermia in A1AR-deficient mice suggest that a receptor-independent action of adenosine requiring intact function of adenosine kinase contributes importantly to the hypothermia induced by adenosine.

Bombesin-like receptor 3 regulates blood pressure and heart rate via a central sympathetic mechanism.

Bombesin-like receptor 3 (BRS-3) is an orphan G protein-coupled receptor that regulates energy expenditure, food intake, and body weight. We examined the effects of BRS-3 deletion and activation on blood pressure and heart rate. In free-living, telemetered Brs3 null mice the resting heart rate was 10% lower than wild-type controls, while the resting mean arterial pressure was unchanged. During physical activity, the heart rate and blood pressure increased more in Brs3 null mice, reaching a similar heart rate and higher mean arterial pressure than control mice. When sympathetic input was blocked with propranolol, the heart rate of Brs3 null mice was unchanged, while the heart rate in control mice was reduced to the level of the null mice. The intrinsic heart rate, measured after both sympathetic and parasympathetic blockade, was similar in Brs3 null and control mice. Intravenous infusion of the BRS-3 agonist MK-5046 increased mean arterial pressure and heart rate in wild-type but not in Brs3 null mice, and this increase was blocked by pretreatment with clonidine, a sympatholytic, centrally acting α2-adrenergic agonist. In anesthetized mice, hypothalamic infusion of MK-5046 also increased both mean arterial pressure and heart rate. Taken together, these data demonstrate that BRS-3 contributes to resting cardiac sympathetic tone, but is not required for activity-induced increases in heart rate and blood pressure. The data suggest that BRS-3 activation increases heart rate and blood pressure via a central sympathetic mechanism.

Targeting proximal tubule mitochondrial dysfunction attenuates the renal disease of methylmalonic acidemia.

Isolated methylmalonic acidemia (MMA), caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT), is often complicated by end stage renal disease that is resistant to conventional therapies, including liver transplantation. To establish a viable model of MMA renal disease, Mut was expressed in the liver of Mut(-/-) mice as a stable transgene under the control of an albumin (INS-Alb-Mut) promoter. Mut(-/-);Tg(INS-Alb-Mut) mice, although completely rescued from neonatal lethality that was displayed by Mut(-/-) mice, manifested a decreased glomerular filtration rate (GFR), chronic tubulointerstitial nephritis and ultrastructural changes in the proximal tubule mitochondria associated with aberrant tubular function, as demonstrated by single-nephron GFR studies. Microarray analysis of Mut(-/-);Tg(INS-Alb-Mut) kidneys identified numerous biomarkers, including lipocalin-2, which was then used to monitor the response of the GFR to antioxidant therapy in the mouse model. Renal biopsies and biomarker analysis from a large and diverse patient cohort (ClinicalTrials.gov identifier: NCT00078078) precisely replicated the findings in the animals, establishing Mut(-/-);Tg(INS-Alb-Mut) mice as a unique model of MMA renal disease. Our studies suggest proximal tubular mitochondrial dysfunction is a key pathogenic mechanism of MMA-associated kidney disease, identify lipocalin-2 as a biomarker of increased oxidative stress in the renal tubule, and demonstrate that antioxidants can attenuate the renal disease of MMA.

One physiology does not fit all: a path from data variability to "physiogenetics"?

Data variability is a costly complication of biomedical experimentation because the same experiment must be repeated a sufficient number of times so that the sample mean becomes a credible representation of the entire population. Since sampling is ideally done randomly in populations normalized for environmental and genetic backgrounds, data variability is viewed as a purely statistical issue reflecting the distribution in the population and captured as the standard deviation of the sampled data. The factors contributing to data variability are not analyzed by statistical methods; for want of a better explanation, data scatter is simply attributed to random noise and/or methodological limitations. In this commentary, evidence is discussed that documents an important role of interindividual biological diversity as a cause for data variability based on studies in which repeated sampling in the same individual permitted statistical comparisons between individuals in the same sample. Significant differences were found for proximal fluid reabsorption and plasma renin concentration between sample means of individuals of the same population. Furthermore, arterial blood pressure varied significantly between individual mice independently of strain and sex. Recognition of the extent of interindividual variability has important implications for data reproducibility, data collection, and data presentation in physiological research. Such nonrandom data variability may have different causes, but DNA modifications by genetic or epigenetic mechanisms could generate phenotype variants without being associated with disease symptoms. Exploration of the heritability of phenotypical diversity in physiology may be defined as "physiogenetics," and it would thus be the physiological corollary of pharmacogenetics and pharmacogenomics.

Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity.

Adenosine is a potent anticonvulsant acting on excitatory synapses through A1 receptors. Cellular release of ATP, and its subsequent extracellular enzymatic degradation to adenosine, could provide a powerful mechanism for astrocytes to control the activity of neural networks during high-intensity activity. Despite adenosine's importance, the cellular source of adenosine remains unclear. We report here that multiple enzymes degrade extracellular ATP in brain tissue, whereas only Nt5e degrades AMP to adenosine. However, endogenous A1 receptor activation during cortical seizures in vivo or heterosynaptic depression in situ is independent of Nt5e activity, and activation of astrocytic ATP release via Ca(2+) photolysis does not trigger synaptic depression. In contrast, selective activation of postsynaptic CA1 neurons leads to release of adenosine and synaptic depression. This study shows that adenosine-mediated synaptic depression is not a consequence of astrocytic ATP release, but is instead an autonomic feedback mechanism that suppresses excitatory transmission during prolonged activity.

Comparison of serum creatinine and serum cystatin C as biomarkers to detect sepsis-induced acute kidney injury and to predict mortality in CD-1 mice.

Acute kidney injury (AKI) dramatically increases sepsis mortality, but AKI diagnosis is delayed when based on serum creatinine (SCr) changes, due in part, to decreased creatinine production. During experimental sepsis, we compared serum cystatin C (sCysC), SCr, and blood urea nitrogen (BUN) to inulin glomerular filtration rate (iGFR) before or 3-18 h after cecal ligation and puncture (CLP)-induced sepsis in CD-1 mice. sCysC had a faster increase and reached peak levels more rapidly than SCr in both sepsis and bilateral nephrectomy (BiNx) models. sCysC was a better surrogate of iGFR than SCr during sepsis. Combining sCysC with SCr values into a composite biomarker improved correlation with iGFR better than any biomarker alone or any other combination. We determined the renal contribution to sCysC handling with BiNx. sCysC and SCr were lower post-BiNx/CLP than post-BiNx alone, despite increased inflammatory and nonrenal organ damage biomarkers. Sepsis decreased CysC production in nephrectomized mice without changing body weight or CysC space. Sepsis decreased sCysC production and increased nonrenal clearance, similar to effects of sepsis on SCr. sCysC, SCr, and BUN were measured 6 h postsepsis to link AKI with mortality. Mice with above-median sCysC, BUN, or SCr values 6 h postsepsis died earlier than mice with below-median values, corresponding to a substantial AKI association with sepsis mortality in this model. sCysC performs similarly to SCr in classifying mice at risk for early mortality. We conclude that sCysC detects AKI early and better reflects iGFR in CLP-induced sepsis. This study shows that renal biomarkers need to be evaluated in specific contexts.

Tubular control of renin synthesis and secretion.

The intratubular composition of fluid at the tubulovascular contact site of the juxtaglomerular apparatus serves as regulatory input for secretion and synthesis of renin. Experimental evidence, mostly from in vitro perfused preparations, indicates an inverse relation between luminal NaCl concentration and renin secretion. The cellular transduction mechanism is initiated by concentration-dependent NaCl uptake through the Na-K-2Cl cotransporter (NKCC2) with activation of NKCC2 causing inhibition and deactivation of NKCC2 causing stimulation of renin release. Changes in NKCC2 activity are coupled to alterations in the generation of paracrine factors that interact with granular cells. Among these factors, generation of PGE2 in a COX-2-dependent fashion appears to play a dominant role in the stimulatory arm of tubular control of renin release. [NaCl] is a determinant of local PG release over an appropriate concentration range, and blockade of COX-2 activity interferes with the NaCl dependency of renin secretion. The complex array of local paracrine controls also includes nNOS-mediated synthesis of nitric oxide, with NO playing the role of a modifier of the intracellular signaling pathway. A role of adenosine may be particularly important when [NaCl] is increased, and at least some of the available evidence is consistent with an important suppressive effect of adenosine at higher salt concentrations.

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. Animal means of PFR [% of glomerular filtration rate (GFR)] derived from TF/Piothalamate ratios in 12 mice in each of four groups [wild type (WT), Cldn2(-/-), AQP1(-/-), and DKO) were 45.8 ± 0.85 (51 tubules), 35.4 ± 1 (54 tubules; P < 0.01 vs. WT), 36.8 ± 1 (63 tubules; P < 0.05 vs. WT), and 33.9 ± 1.4 (69 tubules; P < 0.01 vs. WT). Kidney and single-nephron GFRs (SNGFR) were significantly reduced in all mutant strains. The direct relationship between PFR and SNGFR was maintained in mutant mice, but the slope of this relationship was reduced in the absence of Cldn2 and/or AQP1. Transtubular osmotic pressure differences were not different between WT and Cldn2(-/-) mice, but markedly increased in DKO. In conclusion, the deletion of Cldn2, AQP1, or of both Cldn2 and AQP1 reduces PFR by 22.7%, 19.6%, and 26%, respectively. Our data are consistent with an up to 25% paracellular contribution to PFR. The reduced osmotic water permeability caused by absence of AQP1 augments luminal hypotonicity. Aided by a fall in filtered load, the capacity of non-AQP1-dependent transcellular reabsorption is sufficient to maintain PFR without AQP1 and claudin-2 at 75% of control.

Direct assessment of tubuloglomerular feedback responsiveness in connexin 40-deficient mice.

Participation of connexin 40 (Cx40) in the regulation of renin secretion and in the tubuloglomerular feedback (TGF) component of renal autoregulation suggests that gap junctional coupling through Cx40 contributes to the function of the juxtaglomerular apparatus. In the present experiments, we determined the effect of targeted Cx40 deletion in C57BL/6 and FVB mice on TGF responsiveness. In C57BL/6 mice, stop-flow pressure (PSF) fell from 40.3 ± 2 to 34.5 ± 2 mmHg in wild-type (WT) and from 31 ± 1.06 to 26.6 ± 0.98 mmHg in Cx40-/- mice. PSF changes of 5.85 ± 0.67 mmHg in WT and of 4.3 ± 0.55 mmHg in Cx40-/- mice were not significantly different (P = 0.08). In FVB mice, PSF fell from 37.4 ± 1.5 to 31.6 ± 1.5 mmHg in WT and from 28.1 ± 1.6 to 25.4 ± 1.7 mmHg in Cx40-/-, with mean TGF responses being significantly greater in WT than Cx40-/- (5.5 ± 0.55 vs. 2.7 ± 0.84 mmHg; P = 0.002). In both genetic backgrounds, PSF values were significantly lower in Cx40-/- than WT mice at all flow rates. Arterial blood pressure in the animals prepared for micropuncture was not different between WT and Cx40-/- mice. We conclude that the TGF response magnitude in superficial cortical nephrons is reduced by 30-50% in mice without Cx40, but that with the exception of a small number of nephrons, residual TGF activity is maintained. Thus gap junctional coupling appears to modulate TGF, perhaps by determining the kinetics of signal transmission.

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. Maximum flow stimulation reduced P(SF) in ENT1(-/-) compared with wild-type (WT) mice by 1.6 ± 0.4 mmHg (n = 28) and 5.8 ± 1.1 mmHg (n = 17; P < 0.001) in C57Bl/6 and by 1.4 ± 0.4 mmHg (n = 15) and 9 ± 1.5 mmHg (n = 9; P < 0.001) in SWR/J. Plasma concentrations of adenosine and inosine were markedly higher in ENT1(-/-) than WT mice (ado: 1,179 ± 78 and 225 ± 48 pmol/ml; ino: 179 ± 24 and 47.5 ± 9 pmol/ml). Renal mRNA expressions of the four adenosine receptors, ENT2, and adenosine deaminase were not significantly different between WT and ENT1(-/-) mice. No significant differences of glomerular filtration rate or mean arterial blood pressure were found while plasma renin concentration, and heart rates were significantly lower in ENT1(-/-) animals. In conclusion, TGF responsiveness is significantly attenuated in the absence of ENT1, pointing to a role of nucleoside transport in the NaCl-synchronous changes of extracellular adenosine levels in the juxtaglomerular apparatus interstitium.

A gene therapy approach for long-term normalization of blood pressure in hypertensive mice by ANP-secreting human skin grafts.

The use of bioengineered human skin as a bioreactor to deliver therapeutic factors has a number of advantages including accessibility that allows manipulation and monitoring of genetically modified cells. We demonstrate a skin gene therapy approach that can regulate blood pressure and treat systemic hypertension by expressing atrial natriuretic peptide (ANP), a hormone able to decrease blood pressure, in bioengineered human skin equivalents (HSE). Additionally, the expression of a selectable marker gene, multidrug resistance (MDR) type 1, is linked to ANP expression on a bicistronic vector and was coexpressed in the human keratinocytes and fibroblasts of the HSE that were grafted onto immunocompromised mice. Topical treatments of grafted HSE with the antimitotic agent colchicine select for keratinocyte progenitors that express both MDR and ANP. Significant plasma levels of human ANP were detected in mice grafted with HSE expressing ANP from either keratinocytes or fibroblasts, and topical selection of grafted HSE resulted in persistent high levels of ANP expression in vivo. Mice with elevated plasma levels of human ANP showed lower renin levels and, correspondingly, had lower systemic blood pressure than controls. Furthermore, mice with HSE grafts expressing human ANP did not develop elevated blood pressure when fed a high-salt diet. These findings illustrate the potential of this human skin gene therapy approach to deliver therapeutic molecules systemically for long-term treatment of diverse diseases.

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). Effective vascular deletion of A1AR was affirmed by absence of vasoconstrictor responses to adenosine or cyclohexyl adenosine (CHA) in microperfused AA. Elevation of loop of Henle flow from 0 to 30 nl/min caused a 22.1 ± 3.1% reduction of stop flow pressure in control mice and of 7.2 ± 1.5% in SmCre/A1ARff mice (P < 0.001). Maintenance of residual TGF activity despite absence of A1AR-mediated responses in AA suggests participation of extravascular A1AR in TGF. Support for this notion comes from the observation that deletion of A1ARff by nestin-driven cre causes an identical TGF response reduction (7.3 ± 2.4% in NestinCre/A1ARff vs. 20.3 ± 2.7% in controls), whereas AA responsiveness was reduced but not abolished. A1AR on AA smooth muscle cells are primarily responsible for TGF activation, but A1AR on extravascular cells, perhaps mesangial cells, appear to contribute to the TGF response.

Synthesis and secretion of renin in mice with induced genetic mutations.

The juxtaglomerular (JG) cell product renin is rate limiting in the generation of the bioactive octapeptide angiotensin II. Rates of synthesis and secretion of the aspartyl protease renin by JG cells are controlled by multiple afferent and efferent pathways originating in the CNS, cardiovascular system, and kidneys, and making critical contributions to the maintenance of extracellular fluid volume and arterial blood pressure. Since both excesses and deficits of angiotensin II have deleterious effects, it is not surprising that control of renin is secured by a complex system of feedforward and feedback relationships. Mice with genetic alterations have contributed to a better understanding of the networks controlling renin synthesis and secretion. Essential input for the setting of basal renin generation rates is provided by ß-adrenergic receptors acting through cyclic adenosine monophosphate, the primary intracellular activation mechanism for renin mRNA generation. Other major control mechanisms include COX-2 and nNOS affecting renin through PGE2, PGI2, and nitric oxide. Angiotensin II provides strong negative feedback inhibition of renin synthesis, largely an indirect effect mediated by baroreceptor and macula densa inputs. Adenosine appears to be a dominant factor in the inhibitory arms of the baroreceptor and macula densa mechanisms. Targeted gene mutations have also shed light on a number of novel aspects related to renin processing and the regulation of renin synthesis and secretion.

Convergence of major physiological stimuli for renin release on the Gs-alpha/cyclic adenosine monophosphate signaling pathway.

Control of the renin system by physiological mechanisms such as the baroreceptor or the macula densa (MD) is characterized by asymmetry in that the capacity for renin secretion and expression to increase is much larger than the magnitude of the inhibitory response. The large stimulatory reserve of the renin-angiotensin system may be one of the causes for the remarkable salt-conserving power of the mammalian kidney. Physiological stimulation of renin secretion and expression relies on the activation of regulatory pathways that converge on the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway. Mice with selective Gs-alpha (Gsα) deficiency in juxtaglomerular granular cells show a marked reduction of basal renin secretion, and an almost complete unresponsiveness of renin release to furosemide, hydralazine, or isoproterenol. Cyclooxygenase-2 generating prostaglandin E(2) (PGE(2)) and prostacyclin (PGI(2)) in MD and thick ascending limb cells is one of the main effector systems utilizing Gsα-coupled receptors to stimulate the renin-angiotensin system. In addition, ß-adrenergic receptors are critical for the expression of high basal levels of renin and for its release response to lowering blood pressure or MD sodium chloride concentration. Nitric oxide generated by nitric oxide synthases in the MD and in endothelial cells enhances cAMP-dependent signaling by stabilizing cAMP through cyclic guanosine monophosphate-dependent inhibition of phosphodiesterase 3. The stimulation of renin secretion by drugs that inhibit angiotensin II formation or action results from the convergent activation of cAMP probably through indirect augmentation of the activity of PGE(2) and PGI(2) receptors, ß-adrenergic receptors, and nitric oxide.

Functional expression of the olfactory signaling system in the kidney.

Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (G(olf)), are expressed in the kidney. AC3 and G(olf) colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3(-/-) mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.

The Sweet Pee model for Sglt2 mutation.

Inhibiting renal glucose transport is a potential pharmacologic approach to treat diabetes. The renal tubular sodium-glucose transporter 2 (SGLT2) reabsorbs approximately 90% of the filtered glucose load. An animal model with sglt2 dysfunction could provide information regarding the potential long-term safety and efficacy of SGLT2 inhibitors, which are currently under clinical investigation. Here, we describe Sweet Pee, a mouse model that carries a nonsense mutation in the Slc5a2 gene, which results in the loss of sglt2 protein function. The phenotype of Sweet Pee mutants was remarkably similar to patients with mutations in the Scl5a2 gene. The Sweet Pee mutants had improved glucose tolerance, higher urinary excretion of calcium and magnesium, and growth retardation. Renal physiologic studies demonstrated a prominent distal osmotic diuresis without enhanced natriuresis. Sweet Pee mutants did not exhibit increased KIM-1 or NGAL, markers of acute tubular injury. After induction of diabetes, Sweet Pee mice had better overall glycemic control than wild-type control mice, but had a higher risk for infection and an increased mortality rate (70% in homozygous mutants versus 10% in controls at 20 weeks). In summary, the Sweet Pee model allows study of the long-term benefits and risks associated with inhibition of SGLT2 for the management of diabetes. Our model suggests that inhibiting SGLT2 may improve glucose control but may confer increased risks for infection, malnutrition, volume contraction, and mortality.

Maintained tubuloglomerular feedback responses during acute inhibition of P2 purinergic receptors in mice.

Tubuloglomerular feedback (TGF), the change of afferent arteriolar resistance initiated by changes of luminal NaCl concentration, is thought to be related to NaCl-dependent release of ATP by macula densa cells. In the present study, we have explored the possibility that the released ATP may directly interact with vasoconstrictor P2 purinergic receptors in the vicinity of the glomerular vascular pole. In two different strains of wild-type mice (SWR/J and FVB), TGF responses were determined in vivo by measuring the stop flow pressure (P(SF)) change caused by a saturating increase in loop of Henle flow rate before and during the administration of the P2 receptor inhibitors PPADS (12 mg/kg + 35 mg·kg(-1)·h(-1) iv) or suramin (50 mg/kg + 150 mg·kg(-1)·h(-1)). Both agents significantly reduced the blood pressure response to the P2X agonist α,ß-methylene ATP. In SWR/J and FVB mice, elevating flow to 30 nl/min reduced P(SF) by 16.4 ± 2.2 and 17.1 ± 1.8%. During infusion of PPADS, P(SF) fell by 18.8 ± 2 (P = 0.4) and 16.5 ± 1.5% (P = 0.82) in the two strains of mice. During suramin infusion, P(SF) decreased by 14.7 ± 2.4 (P = 0.62) and 15 ± 1.3% (P = 0.4) in SWR/J and FVB mice, respectively. Including PPADS (10(-4) M) in the loop perfusate did not significantly alter the P(SF) response (18.9 ± 1.8%; P = 0.54). Arterial blood pressure was not systematically affected by the P2 inhibitors. As measured by free-flow micropuncture, PPADS significantly reduced proximal tubular fluid reabsorption in both fractional and absolute terms. These results indicate that the direct activation of P2 purinergic receptors by ATP is not a major cause of TGF-induced vasoconstriction in vivo.

Renin expression in large renal vessels during fetal development depends on functional beta1/beta2-adrenergic receptors.

During nephrogenesis, renin expression shifts from large renal arteries toward smaller vessels in a defined spatiotemporal pattern, finally becoming restricted to the juxtaglomerular position. Chronic stimulation in adult kidneys leads to a recruitment of renin expression in the upstream vasculature. The mechanisms that control this characteristic switch-on and switch-off in the immature and adult kidney are not well-understood. Previous studies in mice with juxtaglomerular cell-specific deletion of the adenylyl cyclase-stimulatory G protein Gsα suggested that signaling along the cAMP pathway plays an essential role for renin expression during nephrogenesis and in the adult kidney. To identify the Gsα-dependent receptor that might be involved in activating this pathway, the present studies were performed to compare renin expression in wild types with that in mice with targeted deletions of ß(1) and ß(2)-adrenoceptors. The sympathetic nervous system is an important regulator of the renin system in the adult kidney so that activation of ß-adrenenoceptors may also participate in the activation of renin expression along the developing arterial tree and in upstream vasculature in adulthood. Compared with wild-types, renin expression was found to be significantly lower at all developmental stages in the kidneys of ß(1)/ß(2) Adr(-/-) mice. Three-dimensional analysis showed reduced renin expression in all segments of the vascular tree in mutants and a virtual absence of renin expression in the large arcuate arteries. Adult mutant kidneys showed the typical upstream renin expression after chronic stimulation. Tyrosine hydroxylase staining in fetal and postnatal kidneys revealed that sympathetic innervation of renin-producing cells occurs early in fetal development. Our data indicate that genetic disruption of ß-adrenergic receptors reduces basal renin expression along the developing preglomerular tree and in adult kidneys. Furthermore, ß-adrenergic receptor input is critical for the expression of renin in large renal vessels during early fetal development.