Iron Is Filtered by the Kidney and Is Reabsorbed by the Proximal Tubule

The aim of this study was to determine the iron (Fe) concentration profile within the lumen of the S2 renal proximal convoluted tubule (PCT) and to resolve whether this nephron segment transported Fe. To do this, we performed in vivo renal micropuncture on Wistar rats, collected PCT tubular fluid from superficial nephrons, and measured Fe concentration. The Fe concentration profile along the S2 PCT suggested significant Fe reabsorption. Proximal tubules were also microperfused in vivo with physiological solutions containing Fe and Zn, Cu, Mn, or Cd. PCTs perfused with 12μmol.l−1 55FeCl3 reabsorbed 105.2±12.7 fmol.mm−1.min−1 Fe, 435±52pmol.mm-1.min−1 Na, and 2.7±0.2nl.mm−1.min−1 water (mean ± SEM; n=19). Addition of ascorbate (1mmol.l−1) to the perfusate did not significantly alter Fe, Na, or water reabsorption. Supplementing the control perfusate with 60μmol.l−1 FeSO4 significantly decreased 55Fe uptake. Recalculating for the altered molar activity following addition of unlabeled Fe revealed a three-fold increase in Fe flux. Addition to the perfusate 12μmol.l−1 CuSO4, MnSO4, CdSO4, or ZnSO4 did not affect Fe, Na, or water flux. In conclusion, (1) in vivo, S2 PCTs of rat reabsorb Fe and (2) Fe is reabsorbed along the PCT via a pathway that is insensitive to Cu, Mn, Cd, or Zn. Together, these data demonstrate for the first time the hitherto speculated process of renal Fe filtration and subsequent tubular Fe reabsorption in a living mammal.

Role of connecting tubule glomerular feedback in obesity related renal damage

Zucker obese rats (ZOR) have higher glomerular capillary pressure (PGC) that can cause renal damage. PGC is controlled by afferent (Af-Art) and efferent arteriole (Ef-Art) resistance. Af-Art resistance is regulated by factors that regulate other arterioles, such as myogenic response. In addition, it is also regulated by 2 intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to increased NaCl in the macula densa and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation in response to an increase in NaCl transport in the connecting tubule via the epithelial sodium channel. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that increased CTGF contributes to TGF attenuation, which in turn increases PGC in ZOR. We performed a renal micropuncture experiment and measured stop-flow pressure (PSF), which is an indirect measurement of PGC in ZOR. Maximal TGF response at 40 nl/min was attenuated in ZOR (4.47 ± 0.60 mmHg) in comparison to the Zucker lean rats (ZLR; 8.54 ± 0.73 mmHg, P < 0.05), and CTGF was elevated in ZOR (5.34 ± 0.87 mmHg) compared with ZLR (1.12 ± 1.28 mmHg, P < 0.05). CTGF inhibition with epithelial sodium channel blocker normalized the maximum PSF change in ZOR indicating that CTGF plays a significant role in TGF attenuation (ZOR, 10.67 ± 1.07 mmHg vs. ZLR, 9.5 ± 1.53 mmHg). We conclude that enhanced CTGF contributes to TGF attenuation in ZOR and potentially contribute to progressive renal damage.

Abstract 066: Regulation of Nephron Afferent Arteriole Resistance in Obesity: Role of Insulin and Connecting Tubule Glomerular Feedback

Introduction: In obesity, increased glomerular capillary pressure (P GC ) may participate in renal damage.P GC is controlled in part by afferent arteriolar ( Af-Art ) resistance that in turn is regulated by two renal intrinsic feedback mechanisms, the vasoconstrictor Tubulo-Glomerular Feedback (TGF), and vasodilator Connecting Tubule Glomerular Feedback (CTGF). CTGF is initiated by an increase in NaCl transport by the epithelial sodium channel (ENaC) in the connecting tubule (CNT). Interestingly, obesity is strongly associated with hyperinsulinemia and insulin is a potent ENaC activator. Hypothesis: In obesity, hyperinsulinemia increases CTGF via activation of the ENaC, this increase, in turn, contributes to TGF attenuation leading to increased P GC and renal damage. Methods: In vivo : In zucker obese rats (ZOR) and zucker lean rats (ZLR), we measured TGF and CTGF using renal micropuncture at 9-10 weeks of age. We quantify stop-flow pressure (P SF ) as an index of P GC . We measured proteinuria as a marker of renal damage in both ZOR and ZLR. In vitro: Microdissected rabbit Af-Arts and their adherent CNTs were perfused with NaCl, insulin or ENaC inhibitor (Benzamil; BZ) to investigate the role of insulin on TGF and CTGF. Results: In-vivo : Maximal TGF response was significantly less in ZOR (6.11 ± 0.75 mmHg) in comparison to the ZLR (9.5 ± 1.1 mmHg, p<0.05). CTGF inhibition by BZ normalized the TGF response in ZOR similar to ZLR (ZOR, 14.01±1.70 mmHg vs ZLR, 11.46± 2.25 mmHg) suggesting CTGF playing a key role in TGF resetting in ZOR. Additionally, ZOR develops proteinuria (mg/24h) at 12 weeks of age (ZOR; 24.85±3.02 vs ZLR; 7.21±1.09, p<0.05 ). In-vitro microperfusion of NaCl in the CNT that elicited a half-maximal response (EC 50 , mmol/L) of Af-Art dilation was 25.0±0.8; an addition of insulin 10 -7 mol/l to the CNT lumen decreased the EC 50 to 8.1±0.8 ( P <0.05) suggesting insulin potentiates CTGF. BZ blocked the insulin-mediated CTGF (Insulin EC 50 : 7.8±0.9 vs . Insulin+BZ, EC 50 : 19.7±5.5; P <0.05). Conclusion: In-vivo : TGF is reset in ZOR due to enhanced CTGF before they develop proteinuria. In-vitro : Insulin increased CTGF during microperfusion experiments. Perspective: Insulin-induced increased in CTGF may explain higher P GC and renal damage in obesity.

Abstract 062: Regulation of Nephron Afferent Arteriole Resistance in Obesity: Role of Connecting Tubule Glomerular Feedback

In obesity, renal damage is caused by increase in renal blood flow (RBF), glomerular capillary pressure (P GC ), and single nephron glomerular filtration rate but the mechanism behind this alteration in renal hemodynamics is unclear. P GC is controlled mainly by the afferent arteriole (Af-Art) resistance. Af-Art resistance is regulated by mechanism similar to that in other arterioles and in addition, it is regulated by two intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to an increase in sodium chloride (NaCl) in the macula densa, via sodium–potassium-2-chloride cotransporter-2 (NKCC2) and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation and is mediated by connecting tubule via epithelial sodium channel (ENaC). CTGF is blocked by the ENaC inhibitor benzamil. Attenuation of TGF reduces Af-Art resistance and allows systemic pressure to get transmitted to the glomerulus that causes glomerular barotrauma/damage. In the current study, we tested the hypothesis that TGF is attenuated in obesity and that CTGF contributes to this effect. We used Zucker obese rats (ZOR) while Zucker lean rats (ZLR) served as controls. We performed in-vivo renal micropuncture of individual rat nephrons while measuring stop-flow pressure (P SF ), an index of P GC. TGF response was measured as a decrease in P SF induced by changing the rate of late proximal perfusion from 0 to 40nl/min in stepwise manner.CTGF was calculated as the difference of P SF value between vehicle and benzamil treatment, at each perfusion rate. Maximal TGF response was significantly less in ZOR (6.16 ± 0.52 mmHg) when compared to the ZLR (8.35 ± 1.00mmHg), p<0.05 , indicating TGF resetting in the ZOR. CTGF was significantly higher in ZOR (6.33±1.95 mmHg) when compared to ZLR (1.38±0.89 mmHg), p<0.05 . When CTGF was inhibited with the ENaC blocker Benzamil (1μM), maximum P SF decrease was 12.30±1.72 mmHg in ZOR and 10.60 ± 1.73 mmHg in ZLR, indicating that blockade of CTGF restored TGF response in ZOR. These observations led us to conclude that TGF is reset in ZOR and that enhanced CTGF contributes to this effect. Increase in CTGF may explain higher renal blood flow, increased P GC and higher glomerular damage in obesity.

Mouse Models and the Urinary Concentrating Mechanism in the New Millennium

Our understanding of urinary concentrating and diluting mechanisms at the end of the 20th century was based largely on data from renal micropuncture studies, isolated perfused tubule studies, tissue analysis studies and anatomical studies, combined with mathematical modeling. Despite extensive data, several key questions remained to be answered. With the advent of the 21st century, a new approach, transgenic and knockout mouse technology, is providing critical new information about urinary concentrating processes. The central goal of this review is to summarize findings in transgenic and knockout mice pertinent to our understanding of the urinary concentrating mechanism, focusing chiefly on mice in which expression of specific renal transporters or receptors has been deleted. These include the major renal water channels (aquaporins), urea transporters, ion transporters and channels (NHE3, NKCC2, NCC, ENaC, ROMK, ClC-K1), G protein-coupled receptors (type 2 vasopressin receptor, prostaglandin receptors, endothelin receptors, angiotensin II receptors), and signaling molecules. These studies shed new light on several key questions concerning the urinary concentrating mechanism including: 1) elucidation of the role of water absorption from the descending limb of Henle in countercurrent multiplication, 2) an evaluation of the feasibility of the passive model of Kokko-Rector and Stephenson, 3) explication of the role of inner medullary collecting duct urea transport in water conservation, 4) an evaluation of the role of tubuloglomerular feedback in maintenance of appropriate distal delivery rates for effective regulation of urinary water excretion, and 5) elucidation of the importance of water reabsorption in the connecting tubule versus the collecting duct for maintenance of water balance.

Glomerulotubular balance, dietary protein, and the renal response to glycine in diabetic rats

The glomerular filtration rate (GFR) normally increases during glycine infusion, which is a test of “renal reserve.” Renal reserve is absent in diabetes mellitus. GFR increases after protein feeding because of increased tubular reabsorption, which reduces the signal for tubuloglomerular feedback (TGF). Dietary protein restriction normalizes some aspects of glomerular function in diabetes. Renal micropuncture was performed in rats 4–5 wk after diabetes was induced by streptozotocin to determine whether renal reserve is lost as a result of altered tubular function and activation of TGF, whether 10 days of dietary protein restriction could restore renal reserve, and whether this results from effects of glycine on the tubule. TGF activation was determined by locating single-nephron GFR (SNGFR) in the early distal tubule along the TGF curve. The TGF signal was determined from the ionic content of the early distal tubule. In nondiabetic rats, SNGFR in the early distal tubule increased during glycine infusion because of primary vasodilation augmented by increased tubular reabsorption, which stabilized the TGF signal. In diabetic rats, glycine reduced reabsorption, thereby activating TGF, which was largely responsible for the lack of renal reserve. In protein-restricted diabetic rats, the tubular response to glycine remained abnormal, but renal reserve was restored by a vascular mechanism. Glycine affects GFR directly and via the tubule. In diabetes, reduced tubular reabsorption dominates. In low-protein diabetes, the vascular effect is enhanced and overrides the effect of reduced tubular reabsorption.

Renal tubular sites of increased phosphate transport and NaPi-2 expression in the juvenile rat

To determine the tubular sites and mechanisms involved in enhanced renal phosphate (Pi) reabsorption seen in the juvenile animal, renal micropuncture experiments were performed in acutely thyroparathyroidectomized adult (>14 wk old) and juvenile (4 wk old) male Wistar rats fed either a normal Pi diet (NPD, 0.6% Pi) or low Pi diet (0.07% Pi) for 2 days, in the presence and absence of parathyroid hormone (PTH). Pi reabsorption was greater in proximal convoluted (PCT) and straight tubules (PST) of the juvenile compared with adult rats fed NPD, whether or not PTH was present. These findings were consistent with a greater Piuptake in brush-border membrane (BBM) vesicles from both superficial (SC) and outer juxtamedullary (JMC) cortices of juvenile animals. Western blot analysis revealed a 2- and 1.8-fold higher amount of NaPi-2 protein in the SC and JMC, respectively, in juvenile rats. Immunofluorescence microscopy also indicated that NaPi-2 protein expression was present in the proximal tubule (PT) BBM to a greater extent in juvenile rats. Dietary Pi restriction in juvenile rats resulted in a significant increase in Pi reabsorption in the PCT and PST segments. NaPi-2 expression in the PT BBM was also increased, as was the expression of intracellular NaPi-2 protein. These studies indicate that Pi reabsorption in both the PCT and PST segments of the renal tubule contributes to the attenuated response to PTH in the normal juvenile animal. In addition, dietary Pi restriction in the juvenile rat upregulates BBM NaPi-2 expression, which is associated with a further increase in proximal tubular Pi reabsorption.

Alpha 2-adrenoceptors determine the response to nitric oxide inhibition in the rat glomerulus and proximal tubule.

Arginine-derived nitric oxide exerts control over the processes of glomerular filtration and tubular reabsorption. The tonic influence of nitric oxide over both of these is eliminated by renal denervation. The hypothesis that the renal nerves function, in this regard, via the activation of alpha 2-adrenoceptors was tested by renal micropuncture. The physical determinants of glomerular filtration and proximal tubular reabsorption were assessed in Munich-Wistar rats before and during the administration of the nitric oxide synthase inhibitor NG-monomethyl L-arginine (L-NMMA). In one set of studies, the systemic infusion of the alpha 2-agonist B-HT 933 rendered nephron GFR, nephron plasma flow, and proximal reabsorption sensitive to reduction by L-NMMA after renal denervation. In a second set of studies, the infusion of the alpha 2 receptor antagonist, yohimbine, to rats with renal nerves intact was found to suppress the effects of L-NMMA on nephron plasma flow and proximal reabsorption. The effects of L-NMMA on nephron GFR and nephron plasma flow, afferent and efferent arteriolar resistances, and proximal reabsorption correlated with the level of underlying alpha 2-adrenergic activity. The activation of renal alpha 2-adrenoceptors increases the influence of arginine-derived nitric oxide in the glomerulus and proximal tubule.