Single Nephron Glomerular Filtration Rate Research Articles

Whole Genome and RNA Sequencing Identifies Genetic Factors involved in Nephron Deficiency in the HSRA Congenital Solitary Kidney Rat

The HSRA rat is a unique polygenic model of congenital abnormalities of the kidney and urogenital tract, with 50–75% of offspring developing a single kidney. The model also exhibits nephron deficiency (~20% less nephrons) in the remaining kidney, glomerular and kidney hypertrophy, and elevated single nephron GFR, which leads to progressive kidney injury and decline in kidney function with age. A number of animal studies have provided an understanding of the physiological mechanism of injury, but it remains unknown the specific genetic factors that cause failure of one kidney to develop, as well as the mechanism of nephron deficiency in the remaining kidney. A recent genome‐wide association study (GWAS) performed by others using the heterogeneous stock rat (progenitor of the HSRA model), identified genomic loci on chromosomes (RNO) 4, 10, and 14 associated with the solitary kidney trait. The confidence interval for the RNO4 locus is ~4.2 Mb containing 14 genes, the RNO10 interval is ~1.5Mb containing 15 genes, and the RNO14 locus is consistent with locus linked to solitary kidney in the ACI rat. The whole genome was sequenced to a depth of 18X coverage using the Illumina NextSeq500 platform. Sequence alignment and annotation [BN reference genome (UCSC Rn5)], identified 1,776,635 SNP, 314,786 INDEL, and 25,346 structural variants. RNA sequencing of embryonic kidney isolated at key stages of nephrogenesis (E14.5, 15.5, and 16.5) between single kidney and two‐kidney control identified dysregulation of genes linked to cellular movement (p‐value= 2.36E‐06 ‐ 1.49E‐02 n=78/451), cell morphology (p‐value= 1.86E‐05 ‐ 1.49E‐02 n=82/451) and cell‐to‐cell signaling and interaction (p‐value= 8.38E‐05 ‐1.49E‐02 n=68/451). The top canonical pathway was associated with Wnt/b‐catenin (p‐value= 4.89E‐05) which includes Wnt2/Nlk candidates genes and is highly relevant to kidney development. Based on the GWAS, whole genome sequencing, and RNA sequencing, the causative genomic loci were refined to specific genes [c‐Kit (RNO14), Fam58b and Nlk (RNO10), and Met and Wnt2 (RNO4)] that could play a role in the solitary kidney trait and nephron deficiency in the HSRA model. In summary, the identification of the specific gene defect, modifier genes, and signaling pathways that lead to loss of one kidney and nephron deficiency will provide a mechanistic link between the solitary kidney genotype and the development of kidney dysfunction exhibited by the HSRA rat.Support or Funding InformationSupported by HL094446 (Garrett), UMMC, and Robert Hearin Foundation. Genomics Core [P20 GM103476 [MS‐INBRE‐(Elarsi)], P30GM103328 (CPN‐COBRE) and P20GM104357 (Obesity‐COBRE)

Sodium-Glucose Linked Cotransporter-2 Inhibition Does Not Attenuate Disease Progression in the Rat Remnant Kidney Model of Chronic Kidney Disease.

Pharmacological inhibition of the proximal tubular sodium-glucose linked cotransporter-2 (SGLT2) leads to glycosuria in both diabetic and non-diabetic settings. As a consequence of their ability to modulate tubuloglomerular feedback, SGLT2 inhibitors, like agents that block the renin-angiotensin system, reduce intraglomerular pressure and single nephron GFR, potentially affording renoprotection. To examine this further we administered the SGLT2 inhibitor, dapagliflozin, to 5/6 (subtotally) nephrectomised rats, a model of progressive chronic kidney disease (CKD) that like CKD in humans is characterised by single nephron hyperfiltration and intraglomerular hypertension and where angiotensin converting enzyme inhibitors and angiotensin receptor blockers are demonstrably beneficial. When compared with untreated rats, both sham surgery and 5/6 nephrectomised rats that had received dapagliflozin experienced substantial glycosuria. Nephrectomised rats developed hypertension, heavy proteinuria and declining GFR that was unaffected by the administration of dapagliflozin. Similarly, SGLT2 inhibition did not attenuate the extent of glomerulosclerosis, tubulointerstitial fibrosis or overexpression of the profibrotic cytokine, transforming growth factor-ß1 mRNA in the kidneys of 5/6 nephrectomised rats. While not precluding beneficial effects in the diabetic setting, these findings indicate that SGLT2 inhibition does not have renoprotective effects in this classical model of progressive non-diabetic CKD.

Searching for the genetic basis and mechanism of nephrogenesis defects in the HSRA congenital solitary kidney rat

Our lab recently developed a unique rat model (HSRA rat) with 50‐75% of offspring developing with a single kidney. The HSRA model exhibits ~20% less nephrons in the remaining kidney (in addition to the loss of one kidney), early glomerular and kidney hypertrophy, and elevated single nephron GFR, leading to progressive kidney injury and decline in kidney function. The HSRA model is also highly susceptible to hypertension induced kidney injury. With a detailed understanding of the physiological mechanism of long‐term cardiovascular and renal hemodynamics, we now seek to establish the mechanism of nephron deficiency in the solitary kidney as well as identify the exact genetic basis leading to the failure of one kidney to develop. RNA sequencing of embryonic kidney isolated at key stages of nephrogenesis (E14.5, 15.5, and 16.5) between embryos exhibiting a single kidney and two‐kidney control identified genes/pathways that may explain decreased nephrons exhibited in the solitary kidney. A recent genome‐wide association study performed by others using the heterogeneous stock (HS) rat (progenitor of HSRA model) identified genomic loci on chromosome 4, 10, and 14 associated with the solitary kidney. Whole genome sequencing of the HSRA model is currently planned, which when combined with identified loci, will expedite identification of genes contributing to solitary kidney. In summary, the identification of the specific gene defect, modifier genes, and signaling pathways that lead to loss of one kidney and nephron deficiency in the remaining kidney will provide a mechanistic link between solitary kidney genotype and the development of kidney dysfunction exhibited by the HSRA.

Sodium–glucose linked transporter-2 inhibitors: potential for renoprotection beyond blood glucose lowering?

The proximal tubule's sodium-glucose linked transporter-2 (SGLT2) accounts for the vast majority of glucose reabsorption by the kidney. Its selective inhibition, accordingly, leads to substantial glycosuria, lowering blood glucose, and facilitating weight loss in individuals with diabetes. During the past year, two SGLT2 inhibitors, canagliflozin and dapagliflozin, have been approved for the treatment of type 2 diabetes. Beyond their anti-hyperglycemic properties, however, this new class of drugs has several other attributes that provide a theoretical basis for kidney protection. Like agents that block the renin-angiotensin system, SGLT2 inhibitors also reduce single-nephron glomerular filtration rate (SNGFR) in the chronically diseased kidney, though by quite different mechanisms. Additional potentially beneficial effects of SGLT2 inhibition include modest reductions in blood pressure and plasma uric acid. Finally, cell culture studies indicate that glucose uptake from the tubular lumen, as well as from the basolateral compartment, can contribute to proximal tubular production of extracellular matrix proteins. Whether such attributes will translate into reducing the progression of chronic kidney disease will require the undertaking of long-term, dedicated studies.

Abstract 220: Nephron Deficient Rats are Highly Susceptible to Hypertension Induced Kidney Injury

It is well known that hypertension can promote decline in renal function, but there is a paucity of information on how hypertension influences single kidney individuals. The HSRA model exhibits a high incidence of congenital single kidney (50-75% offspring) that is characterized by low nephron number (~20% less in solitary kidney vs. control kidney), glomerular and kidney hypertrophy, elevated single nephron glomerular filtration rate (GFR), which culminates in kidney injury and decline in kidney function. We hypothesized that renal hemodynamic stressors, such as DOCA induced hypertension, would accelerate onset and progression of kidney injury in rats born with a solitary kidney (HSRA-S) compared to two-kidney littermates (HSRA-C) or rats that have undergone unilateral nephrectomy (HSRA-UNX). Time course evaluation of blood pressure, renal hemodynamics, and kidney injury was performed in 6 animal groups: (1-2) HSRA-S+DOCA or placebo; (3-4) HSRA-C+DOCA or placebo; and (5-6) HSRA-UNX+DOCA or placebo. At the conclusion of the study (5 weeks), HSRA-S+DOCA treated rats demonstrated a significant (ANOVA; p<0.05) rise in blood pressure (192±4 mmHg), proteinuria (205±31 mg/24hr) and decline in GFR (600+42 μl/min/gKW) relative to HSRA-UNX+DOCA (173±3 mmHg, 76±26 mg/24hr, 963+36 μl/min/gKW) and HSRA-C+DOCA (154±2 mmHg, 7±1 mg/24hr, 1484+121 μl/min/gKW). Animals in placebo groups showed no significant change in blood pressure, proteinuria or GFR from week 13-18 between all three groups. To elucidate the molecular mechanism of nephron deficiency in the HSRA model and better understand how elevated blood pressure promotes rapid decline in renal function, whole transcriptome analysis (RNA sequencing) of embryonic kidney isolated at key stages in nephrogenesis (E14.5, 15.5, and 16.5) was performed which identified a number of novel genes involved in nephrogenesis and kidney development. We are currently working to understand the functional role of these genes and pathways using an in vitro whole organ kidney culture system. In summary, nephron deficient rats are highly susceptible to hypertension induced kidney injury and several novel genes/pathways involved in nephrogenesis likely program the model to develop significant kidney disease with age.

Theoretical assessment of renal autoregulatory mechanisms

A mathematical model of renal hemodynamics was used to assess the individual contributions of the tubuloglomerular feedback (TGF) mechanism and the myogenic response to glomerular filtration rate regulation in the rat kidney. The model represents an afferent arteriole segment, glomerular filtration, and a short loop of Henle. The afferent arteriole model exhibits myogenic response, which is activated by hydrostatic pressure variations to induce changes in membrane potential and vascular muscle tone. The tubule model predicts tubular fluid and Cl(-) transport. Macula densa Cl(-) concentration is sensed as the signal for TGF, which acts to constrict or dilate the afferent arteriole. With this configuration, the model afferent arteriole maintains stable glomerular filtration rate within a physiologic range of perfusion pressure (80-180 mmHg). The contribution of TGF to overall autoregulation is significant only within a narrow band of perfusion pressure values (80-110 mmHg). Model simulations of ramp-like perfusion pressure perturbations agree well with findings by Flemming et al. (Flemming B, Arenz N, Seeliger E, Wronski T, Steer K, Persson PB. J Am Soc Nephrol 12: 2253-2262, 2001), which indicate that changes in vascular conductance are markedly sensitive to pressure velocity. That asymmetric response is attributed to the rate-dependent kinetics of the myogenic mechanism. Moreover, simulations of renal autoregulation in diabetes mellitus predict that, due to the impairment of the voltage-gated Ca(2+) channels of the afferent arteriole smooth muscle cells, the perfusion pressure range in which single-nephron glomerular filtration rate remains stable is reduced by ~70% and that TGF gain is reduced by nearly 40%, consistent with experimental findings.

Compensatory responses to nephron deficiency: Adaptive or maladaptive?

Compensatory renal growth is a characteristic adaptation to reduced renal mass that appears to recapitulate the normal pattern of maturation of the kidney during the postnatal period. Hypertrophy of tubules (predominantly the proximal tubule) and glomeruli is accompanied by increased single nephron glomerular filtration rate and tubular reabsorption of sodium. We propose that the very factors, which contribute to the increase in growth and function of the renal tubular system, are, in the long term, the precursors to the development of hypertension in those with a nephron deficit. The increase in single nephron glomerular filtration rate is dependent on multiple factors, including reduced renal vascular resistance associated with an increased influence of nitric oxide, and a rightward shift in the tubuloglomerular feedback curve, both of which contribute to the normal maturation of renal function. The increased influence of nitric oxide appears to contribute to the reduction in tubuloglomerular feedback sensitivity and facilitate the initial increase in glomerular filtration rate. The increased single-nephron filtered load associated with nephron deficiency may promote hypertrophy of the proximal tubule and so increased reabsorption of sodium, and thus a rightward shift in the pressure natriuresis relationship. Normalization of sodium balance can then only occur at the expense of chronically increased arterial pressure. Therefore, alterations/adaptations in tubules and glomeruli in response to nephron deficiency may increase the risk of hypertension and renal disease in the long-term.

Salt sensitivity of tubuloglomerular feedback in the early remnant kidney

We previously reported internephron heterogeneity in the tubuloglomerular feedback (TGF) response 1 wk after subtotal nephrectomy (STN), with 50% of STN nephrons exhibiting anomalous TGF (Singh P, Deng A, Blantz RC, Thomson SC. Am J Physiol Renal Physiol 296: F1158-F1165, 2009). Presently, we tested the theory that anomalous TGF is an adaptation of the STN kidney to facilitate increased distal delivery when NaCl balance forces the per-nephron NaCl excretion to high levels. To this end, the effect of dietary NaCl on the TGF response was tested by micropuncture in STN and sham-operated Wistar rats. An NaCl-deficient (LS) or high-salt NaCl diet (HS; 1% NaCl in drinking water) was started on day 0 after STN or sham surgery. Micropuncture followed 8 days later with measurements of single-nephron GFR (SNGFR), proximal reabsorption, and tubular stop-flow pressure (PSF) obtained at both extremes of TGF activation, while TGF was manipulated by microperfusing Henle's loop (LOH) from the late proximal tubule. Activating TGF caused SNGFR to decline by similar amounts in Sham-LS, Sham-HS and STN-LS [ΔSNGFR (nl/min) = -16 ± 2, -11 ± 3, -11 ± 2; P = not significant by Tukey]. Activating TGF in STN-HS actually increased SNGFR by 5 ± 2 nl/min (P < 0.0005 vs. each other group by Tukey). HS had no effect on the PSF response to LOH perfusion in sham [ΔPSF (mmHg) = -9.6 ± 1.1 vs. -9.8 ± 1.0] but eliminated the PSF response in STN (+0.3 ± 0.9 vs. -5.7 ± 1.0, P = 0.0002). An HS diet leads to anomalous TGF in the early remnant kidney, which facilitates NaCl and fluid delivery to the distal nephron.

Fluid flow shear stress over podocytes is increased in the solitary kidney

Glomerular hyperfiltration is emerging as the key risk factor for progression of chronic kidney disease (CKD). Podocytes are exposed to fluid flow shear stress (FFSS) caused by the flow of ultrafiltrate within Bowman's space. The mechanism of hyperfiltration-induced podocyte injury is not clear. We postulated that glomerular hyperfiltration in solitary kidney increases FFSS over podocytes. Infant Sprague-Dawley rats at 5 days of age and C57BL/6J 14-week-old adult mice underwent unilateral nephrectomy. Micropuncture and morphological studies were then performed on 20- and 60-day-old rats. FFSS over podocytes in uninephrectomized rats and mice was calculated using the recently published equation by Friedrich et al. which includes the variables-single nephron glomerular filtration rate (SNGFR), filtration fraction (f), glomerular tuft diameter (2RT) and width of Bowman's space (s). Glomerular hypertrophy was observed in uninephrectomized rats and mice. Uninephrectomized rats on Day 20 showed a 2.0-fold increase in SNGFR, 1.0-fold increase in 2RT and 2.1-fold increase in FFSS, and on Day 60 showed a 1.9-fold increase in SNGFR, 1.3-fold increase in 2RT and 1.5-fold increase in FFSS, at all values of modeled 's'. Similarly, uninephrectomized mice showed a 2- to 3-fold increase in FFSS at all values of modeled SNGFR. FFSS over podocytes is increased in solitary kidneys in both infant rats and adult mice. This increase is a consequence of increased SNGFR. We speculate that increased FFSS caused by reduced nephron number contributes to podocyte injury and promotes the progression of CKD.

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.

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.

Nephron filtration rate and proximal tubular fluid reabsorption in the Akita mouse model of type I diabetes mellitus

An increase of glomerular filtration rate (hyperfiltration) is an early functional change associated with type I or type II diabetes mellitus in patients and animal models. The causes underlying glomerular hyperfiltration are not entirely clear. There is evidence from studies in the streptozotocin model of diabetes in rats that an increase of proximal tubular reabsorption results in the withdrawal of a vasoconstrictor input exerted by the tubuloglomerular feedback (TGF) mechanism. In the present study, we have used micropuncture to assess single nephron function in wild type (WT) mice and in two strains of type I diabetic Ins2+/- mice in either a C57Bl/6 (Akita) or an A1AR-/- background (Akita/A1AR-/-) in which TGF is non-functional. Kidney glomerular filtration rate (GFR) of anesthetized mice was increased by 25% in Akita mice and by 52% in Akita/A1AR-/-, but did not differ between genotypes when corrected for kidney weight. Single nephron GFR (SNGFR) measured by end-proximal fluid collections averaged 11.8 ± 1 nl/min (n=17), 13.05 ± 1.1 nl/min (n=23; p=0.27), and 15.4 ± 0.84 nl/min (n=26; p=0.009 compared to WT; p=0.09 compared to Akita) in WT, Akita, and Akita/A1AR-/- mice respectively. Proximal tubular fluid reabsorption was not different between WT and diabetic mice and correlated with SNGFR in all genotypes. We conclude that glomerular hyperfiltration is a primary event in the Akita model of type I diabetes, perhaps driven by an increased filtering surface area, and that it is ameliorated by TGF to the extent that this regulatory system is functional.

Development of a Physiologically Based Computational Kidney Model to Describe the Renal Excretion of Hydrophilic Agents in Rats

A physiologically based kidney model was developed to analyze the renal excretion and kidney exposure of hydrophilic agents, in particular contrast media, in rats. In order to study the influence of osmolality and viscosity changes, the model mechanistically represents urine concentration by water reabsorption in different segments of kidney tubules and viscosity dependent tubular fluid flow. The model was established using experimental data on the physiological steady state without administration of any contrast media or drugs. These data included the sodium and urea concentration gradient along the cortico-medullary axis, water reabsorption, urine flow, and sodium as well as urea urine concentrations for a normal hydration state. The model was evaluated by predicting the effects of mannitol and contrast media administration and comparing to experimental data on cortico-medullary concentration gradients, urine flow, urine viscosity, hydrostatic tubular pressures and single nephron glomerular filtration rate. Finally the model was used to analyze and compare typical examples of ionic and non-ionic monomeric as well as non-ionic dimeric contrast media with respect to their osmolality and viscosity. With the computational kidney model, urine flow depended mainly on osmolality, while osmolality and viscosity were important determinants for tubular hydrostatic pressure and kidney exposure. The low diuretic effect of dimeric contrast media in combination with their high intrinsic viscosity resulted in a high viscosity within the tubular fluid. In comparison to monomeric contrast media, this led to a higher increase in tubular pressure, to a reduction in glomerular filtration rate and tubular flow and to an increase in kidney exposure. The presented kidney model can be implemented into whole body physiologically based pharmacokinetic models and extended in order to simulate the renal excretion of lipophilic drugs which may also undergo active secretion and reabsorption.

Relationships between glomerular filtration rate and kidney volume in low-birth-weight neonates

Low birth weight (LBW), defined as birth weight below 2,500 g, is an important risk factor for the development of hypertension and renal disease in adult life. LBW is associated with a reduced nephron number, which results in hyperfiltration. The objective of this study was to compare the glomerular filtration rates (GFRs) of LBW and normal-birth-weight (NBW) term infants relative to their kidney volumes. Term infants (born after 37 weeks of gestation) who had been admitted to Townsville Hospital's neonatal unit were recruited for this study. Serum cystatin C was used to calculate gfr. a kidney ultrasound was used to measure renal volume. all assessments were performed during the first week of life. Data from 39 infants (17 male, 22 female; 13 LBW, 26 NBW) were analyzed. There were no significant differences in the median cystatin C (1.36 mg/L, inter quartile range [IQR] = 1.12 - 1.41, vs. 1.17 mg/L, IQR = 1.10 - 1.39; p = 0.39) and gestational age. There was no significant difference in the median GFR (53.0 ml/min per 1.73 m2, IQR = 50.8-66.9, vs. 63.2 ml/min per m2, IQR = 51.8-69.5; p = 0.39) between LBW and NBW infants, but LBW infants had smaller total renal volume compared with NBW infants (18.0 ± 4.7 mL vs. 24.4 ± 6.2 mL; p = 0.002). Within 6 days, LBW infants achieved a similar GFR to NBW infants, despite 25% smaller kidney volumes. Thus, the single-nephron glomerular filtration rate must be increased in LBW infants. Prior to this study, it was unclear when hyperfiltration begins, but our results demonstrate that hyperfiltration begins in early life.

Glucagon-like peptide-1 receptor stimulation increases GFR and suppresses proximal reabsorption in the rat.

The incretin hormone glucagon-like peptide-1 (GLP-1) is released from the gut in response to fat or carbohydrate and contributes to negative feedback control of blood glucose by stimulating insulin secretion, inhibiting glucagon, and slowing gastric emptying. GLP-1 receptors (GLP-1R) are also expressed in the proximal tubule, and possibly elsewhere in the kidney. Presently, we examined the effect of a GLP-1R agonist on single-nephron glomerular filtration rate (GFR; SNGFR), proximal reabsorption (Jprox), tubuloglomerular feedback (TGF) responses, and urine flow rate in hydropenic male Wistar and Wistar-Froemter rats. Micropuncture and whole-kidney data were obtained before and during infusion of the GLP-1 agonist exenatide (1 nmol/h iv). SNGFR and Jprox were measured by late proximal collection at both extremes of TGF activation, which was achieved by perfusing Henle's loop at 0 or 50 nl/min. Primary changes in Jprox were revealed by analysis of covariance for Jprox with SNGFR as a covariate. Effects on TGF activation were determined in a separate set of experiments by comparing early distal and late proximal collections. Exenatide increased SNGFR by 33-50%, suppressed proximal tubular reabsorption by 20-40%, doubled early distal flow rate, and increased urine flow rate sixfold without altering the efficiency of glomerulotubular balance, TGF responsiveness, or the tonic influence of TGF. This implies that exenatide is both a proximal diuretic and a renal vasodilator. Since the natural agonist for the GLP-1R is regulated by intake of fat and carbohydrate, but not by salt or fluid, the control of salt excretion by the GLP-1R system departs from the usual negative-feedback paradigm for regulating salt balance.

Abstract 224: Differential Actions of Angiotensin II on Proximal Tubule Transport

Studies with the proximal tubule-specific angiotensin type 1 receptor (AT 1 R) knockout mouse have shown that the proximal tubule (PT) contributes to Ang II-induced hypertension. Ang II levels in the PT fluid are 100- to 1000-fold higher than in plasma, yet the function of the AT 1 -Rs on the luminal side of the PT is unclear. Moreover, hemodynamic actions of Ang II that raise the filtration fraction and the peritubular fluid uptake forces also stimulate PT fluid uptake. We tested the effects of Ang II and AT 1 -Rs on PT fluid uptake in rats. Ang II (200 ng/kg/min) delivered for 2 weeks increased MAP (Con: 95±9 vs Ang II: 140±5 mmHg, p&lt;0.001), single nephron GFR (snGFR)(Con: 36±3 vs Ang II: 43±3, p&lt;0.05) and absolute proximal reabsorption (APR) (Con: 20±3 vs Ang II: 34.5±6.5 nl/min, p&lt;0.01), measured by free flow collections in the S2 segments of the PT in Sprague Dawley (SD) rats. These effects were normalized by acute reduction of MAP via a supra-renal aortic clamp. In a separate group of Munich Wistar Frömter (MWF) rats, which have surface glomeruli, Ang II increased snGFR and APR in the S1 segment (Con: 19±4 vs AngII: 44±5 nl/min, p&lt;0.01; +125±9 %) substantially more than in the S2 segment (Con: 25±6 vs Ang II 37±7 nl/min, p&lt;0.01; +48±6 %). In microperfusion and recollection experiments, in which snGFR was controlled, fluid uptake (Jv) in the PT of SD rats was reduced dose-dependently by Ang II delivered directly into the lumen of S2 segments. Ang II (10 -9 M) at physiological levels reduced Jv by 60±7%. This effect on Jv was blocked by co-perfusion of an AT 1 R blocker. Ang II (10 -9 M), microperfused directly into the S2 reduced Jv in MWF rats as well by 44±5%. Ang II in cultured PT cells at higher concentrations (10 -7 to 10 -9 M) also reduced 22 Na + uptake by 20-35%. In conclusion, enhanced PT fluid reabsorption by Ang II is due to hemodynamic enhancement that likely entails peritubular fluid uptake primarily in the S1 segment. However, luminal Ang II acting on AT 1 -Rs in the S1 and S2 segments of the PT reduces fluid uptake. This effect could be overridden by the hemodynamic effect of systemic Ang II to increase reabsorption especially in the S1 segment.

Acute saline expansion increases nephron filtration and distal flow rate but maintains tubuloglomerular feedback responsiveness: role of adenosine A1 receptors

Temporal adaptation of tubuloglomerular feedback (TGF) permits readjustment of the relationship of nephron filtration rate [single nephron glomerular filtration rate (SNGFR)] and early distal tubular flow rate (V(ED)) while maintaining TGF responsiveness. We used closed-loop assessment of TGF in hydropenia and after acute saline volume expansion (SE; 10% body wt over 1 h) to determine whether 1) temporal adaptation of TGF occurs, 2) adenosine A(1) receptors (A(1)R) mediate TGF responsiveness, and 3) inhibition of TGF affects SNGFR, V(ED), or urinary excretion under these conditions. SNGFR was evaluated in Fromter-Wistar rats by micropuncture in 1) early distal tubules (ambient flow at macula densa), 2) recollected from early distal tubules while 12 nl/min isotonic fluid was added to late proximal tubule (increased flow to macula densa), and 3) from proximal tubules of same nephrons (zero flow to macula densa). SE increased both ambient SNGFR and V(ED) compared with hydropenia, whereas TGF responsiveness (proximal-distal difference in SNGFR, distal SNGFR response to adding fluid to proximal tubule) was maintained, demonstrating TGF adaptation. A(1)R blockade completely inhibited TGF responsiveness during SE and made V(ED) more susceptible to perturbation in proximal tubular flow, but did not alter ambient SNGFR or V(ED). Greater urinary excretion of fluid and Na(+) with A(1)R blockade may reflect additional effects on the distal nephron in hydropenia and SE. In conclusion, A(1)R-independent mechanisms adjust SNGFR and V(ED) to higher values after SE, which facilitates fluid and Na(+) excretion. Concurrently, TGF adapts and stabilizes early distal delivery at the new setpoint in an A(1)R-dependent mechanism.

Disordered oxygen metabolism in early chronic kidney disease

Structural changes leading to chronic renal hypoxia, implicated in chronic kidney disease (CKD) progression, occur late and do not explain early intrarenal hypoxia. To examine the early functional adaptations that may increase oxygen demand leading to hypoxia, micropuncture and oxygen consumption (QO2) studies were conducted in rats 1 week after subtotal nephrectomy (STN).Although, oxygen delivery (DO2) was lower in STN, renal oxygen extraction (arterial‐venous O2 content) doubled in STN vs. control (0.014 vs. 0.007, p&lt;0.05). Both QO2 and (A‐V)O2 factored for GFR were significantly higher in STN. Single nephron GFR nearly doubled in STN vs. controls (p&lt;0.05) contributing to transport related QO2 but could not explain the 3‐fold increase in QO2/nephron. Proximal reabsorption was lower in STN shifting salt reabsorption to distal sites associated with increased energetic costs. Expression of NADPH oxidase was also increased in STN. Mitochondrial QO2 revealed lower state3 respiratory capacity and respiratory control ratio (RCR) in STN, suggestive of mitochondrial complex I dysfunction seen in hypoxia. Treatment with hypoxia inducible factor (HIF) inducer, DMOG, significantly improved several parameters (p&lt;0.05) including DO2, (A‐V)O2,(A‐V)O2/GFR, and mitochondrial state3respiratory capacity and RCR in STN. Major alterations in both mitochondrial and non‐mitochondrial QO2 in early STN, most of which were corrected with HIF induction.

Aberrant Tubuloglomerular Feedback and HIF-1α Confer Resistance to Ischemia after Subtotal Nephrectomy

Nephron loss in a diseased kidney invokes adaptations in the remaining nephrons. Whether and how these adaptations condition the response of the kidney to injury is not known. We examined the susceptibility of the kidney after subtotal (5/6th) nephrectomy (STN) to ischemic injury in rats. GFR in STN kidneys did not significantly change after ischemia reperfusion (IR), whereas GFR fell by 70% after IR in unilateral nephrectomy controls. In micropuncture experiments, single-nephron GFR responses mirrored the whole-kidney responses: in STN, single-nephron GFR decreased by 7% after IR compared with 28% in controls. Furthermore, we found that tubuloglomerular feedback, a mechanism that links proximal tubular injury to a fall in GFR, was inoperative in STN but was normal in controls. Restoration of normal feedback in STN attenuated the functional resistance to IR. In addition to the functional resilience, the morphology of the kidney was better preserved in STN. In STN kidneys, the S3 segment of the proximal tubule, normally injured after ischemia, constitutively expressed hypoxia-inducible factor-1α (HIF-1α), which is cytoprotective in ischemia. Inducing HIF before IR improved GFR in control animals, and inhibiting the HIF target heme-oxygenase-1 before IR reduced GFR in STN animals. Taken together, these data suggest that fewer functioning nephrons in a diseased kidney do not increase the susceptibility to injury, but rather, hemodynamic and molecular adaptations in the remnant nephrons precondition them against ischemic injury.

Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases

Exosomes are 40–100 nm membrane vesicles secreted into the extracellular space by numerous cell types. These structures can be isolated from body fluids including urine and plasma. Exosomes contain proteins, mRNAs, miRNAs, and signaling molecules that reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarker molecules. Aside from diagnostic uses, exosome-mediated transfer of proteins, mRNAs, miRNAs, and signaling molecules offer the promise that they may be used for therapeutic purposes. In this review, we integrate new knowledge about exosomes from outside the field of nephrology with recent progress by renal researchers in order to provide a basis for speculation about how the study of exosomes may affect the fields of nephrology and renal physiology in the next few years.