AJP: Renal Physiology recent issues

Intrarenal oxygenation: unique challenges and the biophysical basis of homeostasis

Evans, R. G., Gardiner, B. S., Smith, D. W., O'Connor, P. M. — 2008-11-05

The kidney is faced with unique challenges for oxygen regulation, both because its function requires that perfusion greatly exceeds that required to meet metabolic demand and because vascular control in the kidney is dominated by mechanisms that regulate glomerular filtration and tubular reabsorption. Because tubular sodium reabsorption accounts for most oxygen consumption (Vo₂) in the kidney, renal Vo₂ varies with glomerular filtration rate. This provides an intrinsic mechanism to match changes in oxygen delivery due to changes in renal blood flow (RBF) with changes in oxygen demand. Renal Vo₂ is low relative to supply of oxygen, but diffusional arterial-to-venous (AV) oxygen shunting provides a mechanism by which oxygen superfluous to metabolic demand can bypass the renal microcirculation. This mechanism prevents development of tissue hyperoxia and subsequent tissue oxidation that would otherwise result from the mismatch between renal Vo₂ and RBF. Recent evidence suggests that RBF-dependent changes in AV oxygen shunting may also help maintain stable tissue oxygen tension when RBF changes within the physiological range. However, AV oxygen shunting also renders the kidney susceptible to hypoxia. Given that tissue hypoxia is a hallmark of both acute renal injury and chronic renal disease, understanding the causes of tissue hypoxia is of great clinical importance. The simplistic paradigm of oxygenation depending only on the balance between local perfusion and Vo₂ is inadequate to achieve this goal. To fully understand the control of renal oxygenation, we must consider a triad of factors that regulate intrarenal oxygenation: local perfusion, local Vo₂, and AV oxygen shunting.

Role of three-dimensional architecture in the urine concentrating mechanism of the rat renal inner medulla

Pannabecker, T. L., Dantzler, W. H., Layton, H. E., Layton, A. T. — 2008-11-05

Recent studies of three-dimensional architecture of rat renal inner medulla (IM) and expression of membrane proteins associated with fluid and solute transport in nephrons and vasculature have revealed structural and transport properties that likely impact the IM urine concentrating mechanism. These studies have shown that 1) IM descending thin limbs (DTLs) have at least two or three functionally distinct subsegments; 2) most ascending thin limbs (ATLs) and about half the ascending vasa recta (AVR) are arranged among clusters of collecting ducts (CDs), which form the organizing motif through the first 3–3.5 mm of the IM, whereas other ATLs and AVR, along with aquaporin-1-positive DTLs and urea transporter B-positive descending vasa recta (DVR), are external to the CD clusters; 3) ATLs, AVR, CDs, and interstitial cells delimit interstitial microdomains within the CD clusters; and 4) many of the longest loops of Henle form bends that include subsegments that run transversely along CDs that lie in the terminal 500 µm of the papilla tip. Based on a more comprehensive understanding of three-dimensional IM architecture, we distinguish two distinct countercurrent systems in the first 3–3.5 mm of the IM (an intra-CD cluster system and an inter-CD cluster system) and a third countercurrent system in the final 1.5–2 mm. Spatial arrangements of loop of Henle subsegments and multiple countercurrent systems throughout four distinct axial IM zones, as well as our initial mathematical model, are consistent with a solute-separation, solute-mixing mechanism for concentrating urine in the IM.

Evidence for increased postprandial distal nephron calcium delivery in hypercalciuric stone-forming patients

2008-11-05

A main mechanism of idiopathic hypercalciuria (IH) in calcium stone-forming patients (IHSF) is postprandial reduction of renal tubule calcium reabsorption that cannot be explained by selective reduction of serum parathyroid hormone levels; the nephron site(s) responsible are not as yet defined. Using fourteen 1-h measurements of the clearances of sodium, calcium, and endogenous lithium during a three-meal day in the University of Chicago General Clinical Research Center, we found reduced postprandial proximal tubule reabsorption of sodium and calcium in IHSF vs. normal subjects. The increased distal sodium delivery is matched by increased distal reabsorption so that urine sodium excretions do not differ, but distal calcium reabsorption does not increase enough to match increased calcium delivery, so hypercalciuria results. In fact, urine calcium excretion and overall renal fractional calcium reabsorption both are high in IHSF vs. normal when adjusted for distal calcium delivery, strongly suggesting a distal as well as proximal reduction of calcium reabsorption. The combination of reduced proximal tubule and distal nephron calcium reabsorption in IHSF is a new finding and indicates that IH involves a complex, presumably genetic, variation of nephron function. The increased calcium delivery into the later nephron may play a role in stone formation via deposition of papillary interstitial apatite plaque.

Effects of terlipressin on the aquaretic system: evidence of antidiuretic effects

Krag, A., Bendtsen, F., Pedersen, E. B., Holstein-Rathlou, N.-H., Moller, S. — 2008-11-05

The vasopressin analog terlipressin is believed to cause vasoconstriction selectively by V₁ receptor stimulation. However, a possible antidiuretic effect by V₂ receptor stimulation has never been ruled out. Twenty-two patients with ascites, including seven with refractory ascites, were included. The subjects were studied during a 400 ml/h oral water load before and after infusion of 2 mg of terlipressin (18 patients) or placebo infusion (4 patients). Effects on the V₂ receptors were assessed by evaluating aquaporin (AQP)2 excretion, free water clearance (C_H₂O), urine osmolality (U_osm), and fractional distal water excretion (DFeH₂O). After terlipressin the excretion of AQP2 increased by 89% [144 ng/mmol creatinine, 95% confidence interval (CI) 73–214 ng/mmol creatinine, P = 0.001]. C_H₂O decreased 1.05 ml/min (from 0.17 to –0.89 ml/min, P = 0.001), and DFeH₂O decreased 37% (19 vs. 12; 95% CI 2–11, P = 0.01). U_osm increased by 27% (93 mosmol/kgH₂O, 95% CI 23–164 mosmol/kgH₂O, P = 0.02). Plasma sodium decreased 1.1 mmol/l (P < 0.01). An increase in AQP2 excretion and a decrease in C_H₂O and distal water excretion after terlipressin despite water loading is a clear indication of activation of the antidiuretic system (V₂ receptor effect).

Shuttling of calcium between endoplasmic reticulum and mitochondria in the renal vasculature

Kanwar, Y. S., Sun, L. — 2008-11-05

Uncoupling of ER-mitochondrial calcium communication by transforming growth factor-{beta}

Pacher, P., Sharma, K., Csordas, G., Zhu, Y., Hajnoczky, G. — 2008-11-05

Transforming growth factor-β (TGF-β) has been implicated as a key factor in mediating many cellular processes germane to disease pathogenesis, including diabetic vascular complications. TGF-β alters cytosolic [Ca²⁺] ([Ca²⁺]_c) signals, which in some cases may result from the downregulation of the IP₃ receptor Ca²⁺ channels (IP3R). Ca²⁺ released by IP3Rs is effectively transferred from endoplasmic reticulum (ER) to the mitochondria to stimulate ATP production and to allow feedback control of the Ca²⁺ mobilization. To assess the effect of TGF-β on the ER-mitochondrial Ca²⁺ transfer, we first studied the [Ca²⁺]_c and mitochondrial matrix Ca²⁺ ([Ca²⁺]_m) signals in single preglomerular afferent arteriolar smooth muscle cells (PGASMC). TGF-β pretreatment (24 h) decreased both the [Ca²⁺]_c and [Ca²⁺]_m responses evoked by angiotensin II or endothelin. Strikingly, the [Ca²⁺]_m signal was more depressed than the [Ca²⁺]_c signal and was delayed. In permeabilized cells, TGF-β pretreatment attenuated the rate but not the magnitude of the IP₃-induced [Ca²⁺]_c rise, yet caused massive depression of the [Ca²⁺]_m responses. ER Ca²⁺ storage and mitochondrial uptake of added Ca²⁺ were not affected by TGF-β. Also, TGF-β had no effect on mitochondrial distribution and on the ER-mitochondrial contacts assessed by two-photon NAD(P)H imaging and electron microscopy. Downregulation of both IP3R1 and IP3R3 was found in TGF-β-treated PGASMC. Thus, TGF-β causes uncoupling of mitochondria from the ER Ca²⁺ release. The sole source of this would be suppression of the IP3R-mediated Ca²⁺ efflux, indicating that the ER-mitochondrial Ca²⁺ transfer depends on the maximal rate of Ca²⁺ release. The impaired ER-mitochondrial coupling may contribute to the vascular pathophysiology associated with TGF-β production.

The stoichiometry of the Na-K pump: one plus one doesn't equal one

Pressley, T. A. — 2008-11-05

{beta}-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

Clifford, R. J., Kaplan, J. H. — 2008-11-05

In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase - and β-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase β₁-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged β₁-subunits (β₁flag) or Myc-tagged β₁-subunits (β₁myc) under the control of a tetracycline-dependent promoter. The induction of β₁flag subunit synthesis in MDCK cells, which increases β₁-subunit expression at the plasma membrane by more than twofold, while maintaining stable ₁ expression levels, revealed that all mature β₁-subunits associate with ₁-subunits, and no evidence of "free" β₁-subunits was obtained. Consequently, the ratio of assembled β₁- to ₁-subunits is significantly increased when "extra" β-subunits are expressed. An increased β₁/₁ stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured β₁myc-expressing and β₁flag-expressing MDCK cells show colocalization of β₁myc and β₁flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the β-subunits. Immunoprecipitation using MDCK cells constitutively expressing β₁myc and tetracycline-regulated β₁flag subunits confirmed β-β-subunit interactions. These results demonstrate that the equimolar ratio of assembled β₁/₁-subunits of the Na-K-ATPase in kidney cells is not fixed by the inherent properties of the interacting subunits. It is likely that cellular mechanisms are present that regulate the individual Na-K-ATPase subunit abundance.

TRPV1 mediates the uterine capsaicin-induced NMDA NR2B-dependent cross-organ reflex sensitization in anesthetized rats

2008-11-05

Spinal cord-mediated cross-organ sensitization between the uterus and the lower urinary tract may underlie the high concurrence of obstetrical/gynecological inflammation and chronic pelvic pain syndrome characterized by urogenital pain. However, the neural pathway and the neurotransmitters involved are still unknown. We tested the hypothesis that the excitation of capsaicin-sensitive primary afferent fibers arising from the uterus through the stimulation of transient receptor potential vanilloid 1 (TRPV₁) induces cross-organ sensitization on the pelvic-urethra reflex activity. Capsaicin (1–1,000 µM, 0.05 ml) was instilled into the uterus to induce cross-organ reflex sensitization. Activation of capsaicin-sensitive primary afferent fibers by capsaicin instillation into the uterine horn sensitized the pelvic-urethra reflex activity that was reversed by an intrauterine pretreatment with capsaizepine, a TRPV₁-selective antagonist. Intrathecal injection of AP5, a glutamatergic N-methyl-d-aspartate (NMDA) antagonist, and Co-101244, an NMDA NR2B-selective antagonist, both abolished the cross-organ reflex sensitization caused by capsaicin instillation. These results demonstrated that TRPV₁ plays a crucial role in contributing to the capsaicin-sensitive primary afferent fibers mediating the glutamatergic NMDA-dependent cross-organ sensitization between the uterus and the lower urinary tract when there is a tissue injury.

Urea transporters UT-A1 and UT-A3 accumulate in the plasma membrane in response to increased hypertonicity

Blessing, N. W., Blount, M. A., Sands, J. M., Martin, C. F., Klein, J. D. — 2008-11-05

The UT-A1 and UT-A3 urea transporters are expressed in the terminal inner medullary collecting duct (IMCD) and play an important role in the production of concentrated urine. We showed that both hyperosmolarity and vasopressin increase urea permeability in perfused rat terminal IMCDs and that UT-A1 and UT-A3 accumulate in the plasma membrane in response to vasopressin. In this study, we investigated whether hyperosmolarity causes UT-A1 and/or UT-A3 to accumulate in the plasma membrane or represents a complimentary stimulatory pathway. Rat IMCD suspensions were incubated in 450 vs. 900 mosM solutions. We biotinylated the IMCD surface proteins, collected, and analyzed them. Membrane accumulation was assessed by Western blotting of the biotinylated protein pool probed with anti-UT-A1 or anti-UT-A3. We studied the effect of NaCl, urea, and sucrose as osmotic agents. Membrane-associated UT-A1 and UT-A3 increased relative to control levels when either NaCl (UT-A1 increased 37 ± 6%; UT-A3 increased 46 ± 13%) or sucrose (UT-A1 increased 81 ± 13%; UT-A3 increased 60 ± 8%) was used to increase osmolarity. There was no increase in membrane UT-A1 or UT-A3 when urea was added. Analogously, UT-A1 phosphorylation was increased in NaCl- and sucrose- but not in urea-based hyperosmolar solutions. Hypertonicity also increased UT-A3 phosphorylation. We conclude that the increase in the urea permeability in response to hyperosmolarity reflects both UT-A1 and UT-A3 movement to the plasma membrane and may be a direct response to tonicity. Furthermore, this movement is accompanied by, and may require, increased phosphorylation in response to hypertonicity.

Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na+/H+ exchanger in proximal S3 segment: role of cytosolic calcium

Leite-Dellova, D. C. A., Oliveira-Souza, M., Malnic, G., Mello-Aires, M. — 2008-11-05

The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na⁺/H⁺ exchanger and on the [Ca²⁺]_i were investigated in isolated rat S3 segment. Aldosterone [10^–12, 10^–10, or 10^–8 M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10^–6 M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca²⁺]_i and after 6-min pi there was a new increase of [Ca²⁺]_i that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca²⁺]_i. RU 486 prevented the stimulatory effect of aldosterone (10^–12 M, 15- or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10^–6 M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca²⁺]_i to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca²⁺]_i and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca²⁺]_i in the lower range (at 10^–12 M aldosterone) and inhibition by increases at high levels (at 10^–6 M aldosterone) or decreases in [Ca²⁺]_i (at 10^–6 M aldosterone plus RU 486).

A mathematical model of distal nephron acidification: diuretic effects

Weinstein, A. M. — 2008-11-05

Through their action on the distal nephron (DN), diuretics may produce systemic acid-base disturbances: metabolic alkalosis with thiazides or loop diuretics and metabolic acidosis with amiloride. Enhanced acid excretion may be due to a local effect on the diuretic target cell (a shift of Na⁺ reabsorption from NaCl transport to Na⁺/H⁺ exchange), or an effect at a distance: namely, increases in luminal fluid flow or luminal Na⁺ concentration may enhance more distal proton secretion. Both local and distance effects are supported by micropuncture data. In the present work, mathematical models of the distal convoluted tubule (DCT)/connecting tubule (CNT) (Weinstein AM, Am J Physiol Renal Physiol 289: F721–F741, 2005), and cortical and medullary collecting ducts (CD) (Weinstein AM, Am J Physiol Renal Physiol 283: F1237–F1251, 2002) have been concatenated to yield a model of rat DN. Among the segments of this DN, the DCT-CNT is responsible for the major portion of distal acidification. Predictions from the model calculations include the following. 1) With increasing distal Na⁺ delivery, there is little change in net acid excretion, but a shift in acidification locus from shared DCT and CNT contributions, to DCT prominence. 2) Urinary acidification by thiazides is primarily local (in the DCT) via the shift in Na⁺ reabsorption from NaCl cotransport to entry via NHE2. Increased NaCl delivery to the CNT increases β-cell HCO₃^– secretion, and thus blunts urine acidification. 3) In contrast to conclusions drawn from the isolated CD model, inclusion of the CNT now reproduces the observed distal acidification defect found with ENaC block, so that this action of amiloride appears to be sufficient to produce "voltage-dependent" distal renal tubular acidosis. 4) The effect of furosemide to enhance distal urinary acidification is not reproduced by the model without major upregulation of CNT -cell transport, perhaps as a result of increased luminal flow.

hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics

2008-11-05

Telomere-dependent replicative senescence is one of the mechanisms that limit the number of population doublings of normal human cells. By overexpression of telomerase, cells of various origins have been successfully immortalized without changing the phenotype. While a limited number of telomerase-immortalized cells of epithelial origin are available, none of renal origin has been reported so far. Here we have established simple and safe conditions that allow serial passaging of renal proximal tubule epithelial cells (RPTECs) until entry into telomere-dependent replicative senescence. As reported for other cells, senescence of RPTECs is characterized by arrest in G₁ phase, shortened telomeres, staining for senescence-associated β-galactosidase, and accumulation of -H2AX foci. Furthermore, ectopic expression of the catalytic subunit of telomerase (TERT) was sufficient to immortalize these cells. Characterization of immortalized RPTEC/TERT1 cells shows characteristic morphological and functional properties like formation of tight junctions and domes, expression of aminopeptidase N, cAMP induction by parathyroid hormone, sodium-dependent phosphate uptake, and the megalin/cubilin transport system. No genomic instability within up to 90 population doublings has been observed. Therefore, these cells are proposed as a valuable model system not only for cell biology but also for toxicology, drug screening, biogerontology, as well as tissue engineering approaches.

Dissociation of NEPH1 from nephrin is involved in development of a rat model of focal segmental glomerulosclerosis

2008-11-05

Focal segmental glomerulosclerosis (FSGS) is a disease showing severe proteinuria, and the disease progresses to end-stage kidney failure in many cases. However, the pathogenic mechanism of FSGS is not well understood. The slit diaphragm (SD), which bridges the neighboring foot processes of glomerular epithelial cells, is understood to function as a barrier of the glomerular capillary wall. To investigate the role of SD dysfunction in the development of FSGS, we analyzed the expression of SD-associated molecules in rat adriamycin-induced nephropathy, a mimic of FSGS. The staining of the SD molecules nephrin, podocin, and NEPH1 had already shifted to a discontinuous dotlike pattern at the initiation phase of the disease, when neither proteinuria nor any morphological alterations were detected yet. The alteration of NEPH1 expression was the most evident among the molecules examined, and NEPH1 was dissociated from nephrin at the initiation phase. On day 28, when severe proteinuria was detected and sclerotic changes were already observed, alteration of the expressions of nephrin, podocin, and NEPH1 worsened, but no alteration in the expression of other SD-associated molecules or other podocyte molecules was detected. It is postulated that the dissociation of NEPH1 from nephrin initiates proteinuria and that the SD alteration restricted in these molecules plays a critical role in the development of sclerotic changes in FSGS.

Role of Kruppel-like factor 6 in transforming growth factor-{beta}1-induced epithelial-mesenchymal transition of proximal tubule cells

2008-11-05

Krüppel-like factor 6 (KLF6) is a DNA-binding protein containing a triple zinc-fingered motif and plays a key role in the regulation of cell proliferation, differentiation, and development. More recently it has been implicated in hepatic fibrosis via its binding to the transforming growth factor (TGF)-β control element. In the kidney, epithelial-mesenchymal transition (EMT) is a major contributor to the pathogenesis of renal fibrosis, with TGF-β1 being a key mediator of EMT. The present study aimed to determine the role of KLF6 and TGF-β1 in EMT in proximal tubule cells. To determine the relevance in clinical disease, KLF6 was measured in kidneys of streptozotocin-induced diabetic Ren-2 rats and in cells exposed to high (30 mM) glucose. TGF-β1 was confirmed to induce EMT by morphological change, loss of E-cadherin, and gain in vimentin expression. KLF6 mRNA expression was concomitantly measured. To determine the role of KLF6 in EMT, the above markers of EMT were determined in KLF6-silenced (small interfering RNA) and KLF6-overexpressing proximal tubule cells. KLF6 overexpression significantly promoted a phenotype consistent with EMT. High glucose induced KLF6 in proximal tubule cells (P < 0.05). This increase in KLF6 in response to high glucose was TGF-β1 mediated. In an in vivo model of diabetic nephropathy KLF6 increased at week 8 (P < 0.05). KLF6 plays a permissive role in TGF-β1-induced EMT in proximal tubule cells. Its upregulation in in vivo models of diabetic nephropathy suggests it as a potential therapeutic target.

Complement activation by tubular cells is mediated by properdin binding

2008-11-05

Activation of filtered complement products on the brush border of the tubular epithelium is thought to be a key factor underlying proteinuria-induced tubulointerstitial injury. However, the mechanism of tubular complement activation is still unclear. Recent studies on mechanisms of complement activation indicate a key role for properdin in the initiation of an alternative pathway. We hypothesized that properdin serves as a focal point for complement activation on the tubulus. We observed a strong staining for properdin on the luminal surface of the tubules in kidney biopsies from patients with proteinuric renal disease. In vitro experiments revealed dose-dependent binding of properdin to proximal tubular epithelial cells (PTEC), whereas no significant binding to endothelial cells was detected. Exposure of PTEC with normal human serum as a source of complement resulted in complement activation with deposition of C3 and generation of C5b-9. These effects were virtually absent with properdin-deficient serum. Preincubation of PTEC with properdin before addition of properdin-depleted serum fully restored complement activation on the cells, strongly suggesting a key role for properdin in the activation of complement at the tubular surface. In proteinuric renal disease, filtered properdin may bind to PTEC and act as a focal point for alternative pathway activation. We propose that this contribution of properdin is pivotal in tubular complement activation and subsequent damage. Interference with properdin binding to tubular cells may provide an option for the treatment of proteinuric renal disease.

Bradykinin B2 receptor null mice harboring a Ser23-to-Ala substitution in the p53 gene are protected from renal dysgenesis

El-Dahr, S. S., Aboudehen, K., Dipp, S. — 2008-11-05

A physiological cross talk operates between the tumor suppressor protein p53 and the bradykinin B₂ receptor (BdkrB2) during renal organogenesis. Thus, although BdkrB2 is a target for p53-mediated transcriptional activation, BdkrB2 is required to restrict p53 proapoptotic activity. We previously demonstrated that BdkrB2^–/– embryos exposed to gestational salt stress develop renal dysgenesis as a result of p53-mediated apoptosis of nephron progenitors and repression of the terminal differentiation program. Compared with wild-type kidneys, BdkrB2^–/– express abnormally high levels of the Checkpoint kinase (Chk1), which activates p53 via Ser²³ phosphorylation. To define the functional relevance of p53^S23 phosphorylation, we generated a compound strain of BdkrB2^–/– mice harboring a homozygous Ser²³-to-Ala (S23A) mutation in the p53 gene by crossing BdkrB2^–/– with p53^S23A knockin mice. Unlike salt-stressed BdkrB2^–/– pups, which exhibit renal dysgenesis, homozygous S23A;BdkrB2^–/– littermates are protected and have normal renal development. Heterozygous S23A;BdkrB2^–/– mice have an intermediate phenotype. The p53-S23A substitution was associated with amelioration of apoptosis and restored markers of nephrogenesis and tubulogenesis. Real-time quantitative RT-PCR of terminal differentiation genes demonstrated that the S23A substitution restored normal expression patterns of aquaporin-2, Na-Cl cotransporter, Na-K-2Cl cotransporter, Na-bicarbonate cotransporter, and Sglt1. We conclude that p53 phosphorylation on Ser²³ is an essential step in the signaling pathway mediating the susceptibility of BdkrB2^–/– mutants to renal dysgenesis.

Interleukin-18 binding protein transgenic mice are protected against ischemic acute kidney injury

2008-11-05

IL-18 function is neutralized in IL-18 binding protein transgenic (IL-18BP Tg) mice. First, we determined whether IL-18BP Tg mice are protected against ischemic acute kidney injury (AKI). Ischemic AKI was induced by bilateral renal pedicle clamping. IL-18BP Tg mice were functionally and histologically protected against ischemic AKI as determined by blood urea nitrogen, serum creatinine, and acute tubular necrosis score. We have demonstrated that the injurious effect of IL-18 in the kidney is independent of neutrophils and lymphocytes. Thus the effect of IL-18 inhibition on renal macrophage infiltration was determined. The number of macrophages was significantly reduced in IL-18BP Tg compared with wild-type kidneys. To determine the cytokines and chemokines that are dependent on IL-18, we performed flow cytometry based assays. Multiple chemokines/cytokines, IL-3, IL-6, IL-15, IL-18, leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1 were significantly increased in AKI vs. sham kidneys. Only CXCL1 (also known as KC or IL-8) was significantly increased in AKI vs. sham kidneys and significantly reduced in IL-18BP Tg AKI vs. wild-type AKI kidneys. To determine whether macrophages are the source of CXCL1 in the kidney, we depleted macrophages with liposomal encapsulated clodronate. CXCL1 was significantly decreased in macrophage-depleted vs. control AKI mice. In summary, in ischemic AKI in mice, 1) IL-18BP Tg mice are functionally and histologically protected, 2) macrophage infiltration in the kidney and CXCL1 are significantly reduced in IL-18BP Tg mice, and 3) macrophage depletion significantly reduces CXCL1 in the kidney. In conclusion, protection against ischemic AKI in IL-18BP Tg mice is associated with less macrophage infiltration and less production of CXCL1 in the kidney.

Localization of phosphatidylinositol phosphate kinase II{gamma} in kidney to a membrane trafficking compartment within specialized cells of the nephron

Clarke, J. H., Emson, P. C., Irvine, R. F. — 2008-11-05

PIP4Ks (type II phosphatidylinositol 4-phosphate kinases) are phosphatidylinositol 5-phosphate (PtdIns5P) 4-kinases, believed primarily to regulate cellular PtdIns5P levels. In this study, we investigated the expression, localization, and associated biological activity of the least-studied PIP4K isoform, PIP4K. Quantitative RT-PCR and in situ hybridization revealed that compared with PIP4K and PIP4Kβ, PIP4K is expressed at exceptionally high levels in the kidney, especially the cortex and outer medulla. A specific antibody was raised to PIP4K, and immunohistochemistry with this and with antibodies to specific kidney cell markers showed a restricted expression, primarily distributed in epithelial cells in the thick ascending limb and in the intercalated cells of the collecting duct. In these cells, PIP4K had a vesicular appearance, and transfection of kidney cell lines revealed a partial Golgi localization (primarily the matrix of the cis-Golgi) with an additional presence in an unidentified vesicular compartment. In contrast to PIP4K, bacterially expressed recombinant PIP4K was completely inactive but did have the ability to associate with active PIP4K in vitro. Overall our data suggest that PIP4K may have a function in the regulation of vesicular transport in specialized kidney epithelial cells.

Treatment with pyrrolidine dithiocarbamate improves proteinuria, oxidative stress, and glomerular hypertension in overload proteinuria

2008-11-05

We evaluated whether the blockade of the proinflammatory transcription factor NF-B would modify the oxidative stress, inflammation, and structural and hemodynamic alterations found in the kidney as a result of massive proteinuria. Twenty male Sprague-Dawley rats were injected with 2 g of BSA intraperitoneally daily for 2 wk. Ten of them received in addition the inhibitor of NF-B activation pyrrolidine dithiocarbamate (PDTC; 200 mg·kg^–1·day^–1 sc) and the rest received vehicle. Seven rats that received intraperitoneal saline were used as controls. Glomerular hemodynamics were studied after 14 days. Markers of oxidative stress (NF-B subunit p65+ cells, 3-nitrotyrosine, and 4-hydroxynonenal), inflammation (cortical CD68+ cells and NOS-II), and afferent arteriole damage were assessed by immunohistochemistry and morphometry. Activity of antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase was evaluated in renal cortex and medulla. Albumin overload induced massive proteinuria, oxidative stress with reduced activity of antioxidant enzymes, NF-B activation, inflammatory cell infiltration, a significant presence of proteinaceous casts, systemic and glomerular hypertension, as well as arteriolar remodeling. Treatment with PDTC prevented or improved all of these findings. In this model of nephrotic syndrome, we demonstrate a key role for oxidative stress and inflammation in causing systemic and glomerular hypertension and proteinuria. Oxidative stress and inflammation may have a key role in accelerating renal injury associated with intense proteinuria.

An evolutionarily conserved N-terminal Sgk1 variant with enhanced stability and improved function

Raikwar, N. S., Snyder, P. M., Thomas, C. P. — 2008-11-05

Sgk1 is an aldosterone-induced kinase that regulates epithelial sodium channel (ENaC)-mediated Na⁺ transport in the collecting duct and connecting tubule of the kidney. The NH₂ terminus of Sgk1 contains instability motifs that direct the ubiquitination of Sgk1 resulting in a rapidly degraded protein. By bioinformatic analysis, we identified a 5' variant alternate transcript of human Sgk1 (Sgk1_v2) that is widely expressed, is conserved from rodent to humans, and is predicted to encode an Sgk1 isoform, Sgk1_i2, with a different NH₂ terminus. When expressed in HEK293 cells, Sgk1_i2 was more abundant than Sgk1 because of an increased protein half-life and this correlated with reduced ubiquitination of Sgk1_i2 and enhanced surface expression of ENaC. Immunocytochemical studies demonstrated that in contrast to Sgk1, Sgk1_i2 is preferentially targeted to the plasma membrane. When coexpressed with ENaC subunits in FRT epithelia, Sgk1_i2 had a significantly greater effect on amiloride-sensitive Na⁺ transport compared with Sgk1. Together, the data demonstrate that a conserved NH₂-terminal variant of Sgk1 shows improved stability, enhanced membrane association, and greater stimulation of epithelial Na⁺ transport in a heterologous expression system.

Continuously measured renal blood flow does not increase in diabetes if nitric oxide synthesis is blocked

Bell, T. D., DiBona, G. F., Biemiller, R., Brands, M. W. — 2008-11-05

This study used 16 h/day measurement of renal blood flow (RBF) and arterial pressure (AP) to determine the role of nitric oxide (NO) in mediating the renal vasodilation caused by onset of type 1 diabetes. The AP and RBF power spectra were used to determine the autoregulatory efficiency of the renal vasculature. Rats were instrumented with artery and vein catheters and a Transonic flow probe on the left renal artery and were divided randomly into four groups: control (C), diabetes (D), control plus nitro-l-arginine methyl ester (l-NAME; CL), and diabetes plus l-NAME (DL). Mean AP averaged 90 ± 1 and 121 ± 1 mmHg in the D and DL groups, respectively, during the control period, and RBF averaged 5.9 ± 1.2 and 5.7 ± 0.7 ml/min, respectively. Respective C and CL groups were not different. Onset of diabetes (streptozotocin 40 mg/kg iv) in D rats increased RBF gradually, but it averaged 55% above control by day 14. In DL rats, on the other hand, RBF remained essentially constant, tracking with RBF in the nondiabetic C and CL groups for the 2-wk period. Diabetes did not change mean AP in any group. Transfer function analysis revealed impaired dynamic autoregulation of RBF overall, including the frequency range of tubuloglomerular feedback (TGF), and l-NAME completely prevented those changes as well. These data strongly support a role for NO in causing renal vasodilation in diabetes and suggest that an effect of NO to blunt RBF autoregulation may play an important role.

Dietary doses of nitrite restore circulating nitric oxide level and improve renal injury in L-NAME-induced hypertensive rats

2008-11-05

We have reported that pharmacological doses of oral nitrite increase circulating nitric oxide (NO) and exert hypotensive effects in N-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive rats. In this study, we examined the effect of a chronic dietary dose of nitrite on the hypertension and renal damage induced by chronic l-NAME administration in rats. The animals were administered tap water containing l-NAME (1 g/l) or l-NAME + nitrite (low dose: 0.1 mg/l, medium dose: 1 mg/l, high dose: 10 mg/l) for 8 wk. We evaluated blood NO levels as hemoglobin-NO adducts (iron-nitrosyl-hemoglobin), using an electron paramagnetic resonance method. Chronic administration of l-NAME for 8 wk induced hypertension and renal injury and reduced the blood iron-nitrosyl-hemoglobin level (control 38.8 ± 8.9 vs. l-NAME 6.0 ± 3.1 arbitrary units). Coadministration of a low dose of nitrite with l-NAME did not change the reduced iron-nitrosyl-hemoglobin signal and did not improve the l-NAME-induced renal injury. The blood iron-nitrosyl-hemoglobin signals of the medium dose and high dose of nitrite were significantly higher than that of l-NAME alone. Chronic administration of a medium dose of nitrite attenuated l-NAME-induced renal histological changes and proteinuria. A high dose of nitrite also attenuated l-NAME-induced renal injury. These findings suggest that dietary doses of nitrite that protect the kidney are associated with significant increase in iron-nitrosyl-hemoglobin levels. We conclude that dietary nitrite-derived NO generation may serve as a backup system when the nitric oxide synthase/l-arginine-dependent NO generation system is compromised.

Periostin induces proliferation of human autosomal dominant polycystic kidney cells through {alpha}V-integrin receptor

2008-11-05

Progressive renal enlargement due to the growth of innumerable fluid-filled cysts is a central pathophysiological feature of autosomal dominant polycystic kidney disease (ADPKD). These epithelial neoplasms enlarge slowly and damage noncystic parenchyma by mechanisms that have not been clearly defined. In a microarray analysis of cultured human ADPKD cyst epithelial cells, periostin mRNA was overexpressed 15-fold compared with normal human kidney (NHK) cells. Periostin, initially identified in osteoblasts, is not expressed in normal adult kidneys but is expressed transiently during renal development. We found periostin in cyst-lining cells in situ in the extracellular matrix adjacent to the cysts and within cyst fluid. ADPKD cells secreted periostin across luminal and basolateral plasma membranes. Periostin increased proliferation of cyst epithelial cells 27.9 ± 3.1% (P < 0.001) above baseline and augmented in vitro cyst growth but did not affect proliferation of normal renal cells. Expression of _V-integrin, a periostin receptor, was ninefold higher in ADPKD cells compared with NHK cells, and antibodies that block _V-integrin inhibited periostin-induced cell proliferation. We conclude that periostin is a novel autocrine mitogen secreted by mural epithelial cells with the potential to accelerate cyst growth and promote interstitial remodeling in ADPKD.

WNK3 positively regulates epithelial calcium channels TRPV5 and TRPV6 via a kinase-dependent pathway

Zhang, W., Na, T., Peng, J.-B. — 2008-11-05

WNK3, a member of the With No Lysine (K) family of protein serine/threonine kinases, was shown to regulate members of the SLC12A family of cation-chloride cotransporters and the renal outer medullary K⁺ channel ROMK and Cl^– channel SLC26A9. To evaluate the effect of WNK3 on TRPV5, a renal epithelial Ca²⁺ channel that serves as a gatekeeper for active Ca²⁺ reabsorption, WNK3 and TRPV5 were coexpressed in Xenopus laevis oocytes and the function and expression of TRPV5 were subsequently examined. An 82.7 ± 7.1% increase in TRPV5-mediated Ca²⁺ uptake was observed when WNK3 was coexpressed. A similar increase in TRPV5-mediated Na⁺ current was observed with the voltage-clamp technique. WNK3 also enhanced Ca²⁺ influx and Na⁺ current mediated by TRPV6, which is the closest homolog of TRPV5 that mediates active intestinal Ca²⁺ absorption. The kinase domain of WNK3 alone was sufficient to increase TRPV5-mediated Ca²⁺ transport, and the positive regulatory effect was abolished by the kinase-inactive D294A mutation in WNK3, indicating a kinase-dependent mechanism. The complexly glycosylated TRPV5 that appears at the plasma membrane was increased by WNK3. The exocytosis of TRPV5 was increased by WNK3, and the effect of WNK3 on TRPV5 was abolished by the microtubule inhibitor colchicine. The increased plasma membrane expression of TRPV5 was likely due to the enhanced delivery of mature TRPV5 to the plasma membrane from its intracellular pool via the secretory pathway. These results indicate that WNK3 is a positive regulator of the transcellular Ca²⁺ transport pathway.

Visfatin: a new player in mesangial cell physiology and diabetic nephropathy

2008-11-05

Visfatin is an adipocytokine that improves insulin resistance and has an antidiabetic effect. However, the role of visfatin in the kidney has not yet been reported. In this experiment, the synthesis and physiological action of visfatin in cultured mesangial cells (MCs) were studied to investigate the role of visfatin in diabetic nephropathy. Visfatin was found synthesized in MCs as well as adipocytes. Visfatin synthesis was markedly increased, not by angiotensin II, but by high glucose stimuli. In addition, visfatin treatment induced a rapid uptake of glucose, peaking at 20 min after visfatin treatment in a dose-dependent manner. A small inhibiting RNA against insulin receptor significantly blocked visfatin-mediated glucose uptake. Visfatin stimuli also enhanced intracellular NAD levels, and treatment with FK866, which is a specific inhibitor of nicotinamide phosphoribosyltransferase (Nampt), significantly inhibited visfatin-induced NAD synthesis and glucose uptake. Visfatin treatment increased glucose transporter-1 (GLUT-1) protein expression in isolated cellular membranes, and pretreatment with cytochalasin B completely inhibited visfatin-induced glucose uptake. Moreover, immunofluorescent microscopy showed the migration of cytosolic GLUT-1 into cellular membranes after visfatin treatment. In accordance with these results, the activation of protein kinase B was detected after visfatin treatment. Furthermore, visfatin treatment dramatically increased the synthesis of profibrotic molecules including transforming growth factor-β1, plasminogen activator inhibitor-1, and type I collagen, and pretreatment with cytochalasin B completely inhibited visfatin-induced upregulation of profibrotic molecules. These results suggest that visfatin is produced in MCs, which are a novel target for visfatin, and play an important role in the pathogenesis of diabetic nephropathy.

Suppression of cytokine responses by indomethacin in podocytes: a mechanism through induction of unfolded protein response

2008-11-05

We found that, in murine podocytes, expression of monocyte chemoattractant protein 1 (MCP-1) in response to TNF- was suppressed by indomethacin but not by ibuprofen. This anti-inflammatory potential was correlated with induction of 78-kDa glucose-regulated protein (GRP78), a marker of unfolded protein response (UPR). Indomethacin, but not ibuprofen, also triggered expression of CHOP, another endogenous indicator of UPR, as well as repression of endoplasmic reticulum stress-responsive alkaline phosphatase, an exogenous indicator of UPR. Like ibuprofen, other nonsteroidal anti-inflammatory drugs including aspirin and sulindac also did not induce UPR, indicating that the induction of UPR by indomethacin was independent of cyclooxygenase inhibition. The induction of UPR by indomethacin was observed similarly in other cells including mesangial cells and tubular epithelial cells. In tumor necrosis factor (TNF)--treated cells, suppression of MCP-1 by indomethacin was inversely correlated with induction of UPR, and other inducers of UPR including tunicamycin, thapsigargin, and A23187 reproduced the suppressive effect. Reporter assays showed that indomethacin as well as thapsigargin attenuated activation of NF-B by TNF-, and it was associated with enhanced degradation of TNF receptor-associated factor 2 (TRAF2) and blunted degradation of IBβ. Subsequent experiments revealed that acute ablation of GRP78 protein by AB₅ subtilase cytotoxin caused reinforcement of MCP-1 induction and NF-B activation by TNF- and that transfection with GRP78 significantly suppressed the cytokine-induced activation of NF-B. These results suggested that indomethacin suppressed the response of podocytes to TNF- via UPR and that UPR-triggered induction of GRP78 and degradation of TRAF2 may be responsible, at least in part, for the suppressive effect of indomethacin.

Intact renal afferent arteriolar autoregulatory responsiveness in db/db mice

Park, S., Bivona, B. J., Feng, Y., Lazartigues, E., Harrison-Bernard, L. M. — 2008-11-05

The db/db mouse is a genetic model of type 2 diabetes that exhibits progressive renal disease. Obesity, hyperglycemia, and albuminuria (822 ± 365 vs. 28 ± 8 µg/day) are evident in 18-wk-old db/db compared with db/m (lean littermate control) mice. Our goal was to determine the blood pressure (BP) phenotype of the db/db mouse. Mean arterial BP measured in conscious mice by radiotelemetry was not different between db/db (n = 9) and db/m (n = 12) mice, averaging 113 ± 3 and 112 ± 2 mmHg, respectively. The circadian BP profile of db/db mice was shifted to the left and exhibited a significant reduction in amplitude compared with db/m mice. Heart rate (487 ± 9 vs. 542 ± 7 beats/min; P < 0.05) and locomotor activity were significantly reduced in db/db compared with db/m mice. We tested the hypothesis that intact afferent arteriole (AA) responsiveness to increases in renal artery pressure (RAP) and angiotensin (ANG) II sensitivity contributes to normal BP in this diabetic model. AA diameters of in vitro blood-perfused juxtamedullary nephrons of db/db mice (15.7 ± 0.5 µm; n = 38) were significantly larger than those of db/m mice (12.5 ± 0.4 µm; n = 37). AA responses to increases in RAP and ANG II were not different between kidneys of db/db and db/m mice. Significant AA vasoconstriction to 1 nM ANG II was observed in kidneys of db/db mice (–11 ± 4%), while 10 nM ANG II decreased AA diameter in both groups [db/db, –20 ± 4%, (n = 12); db/m, –26 ± 4% (n = 12)]. In summary, AA responses to increases in renal perfusion pressure and ANG II remain intact in db/db mice. Diabetic renal disease occurs in db/db mice independently of elevated BP.

Intrarenal RAS activity and urinary angiotensinogen excretion in anti-thymocyte serum nephritis rats

2008-11-05

The differential roles of circulating and intrarenal renin-angiotensin system (RAS) in glomerulonephritis have not been elucidated. In this study, we investigated the levels of circulating and intrarenal RAS activity and urinary angiotensinogen (AGT) excretion in anti-thymocyte serum (ATS) nephritis induced by an ATS injection (ATS group). The effect of olmesartan, an angiotensin II (ANG II) type 1 receptor blocker (ARB), on the development of nephritis was also examined (ATS+ARB group). In addition, the rats received a saline injection instead of ATS (control group). Mesangial proliferation with transient proteinuria, which peaked at day 7, was significantly increased in the ATS group compared with the control group. The levels of glomerular AGT mRNA, intrarenal ANG II, and urinary AGT excretion in the ATS group were increased significantly at day 7 compared with the control group. Administration of olmesartan (ATS+ARB group) significantly decreased the levels of renal lesions, proteinuria, and intrarenal RAS activity compared with the ATS group. In addition, the levels of urinary AGT excretion correlated with the levels of glomerular damage, urinary protein excretion, and immunoreactivity for AGT and ANG II in kidney. On the other hand, plasma renin activity was significantly lower in the ATS group compared with the control group and significantly higher in the ATS+ARB group than in the ATS group. These data suggest that an increase in kidney-specific RAS activity, which parallels urinary AGT excretion, plays an important role in the development of ATS nephritis.

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

Yu, L., Helms, M. N., Yue, Q., Eaton, D. C. — 2008-11-05

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (P_o) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is ~3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the P_o of ENaC for at least 6 h after exposure. A decrease in apical channel number and P_o was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC P_o. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.

MDM2 E3 ubiquitin ligase mediates UT-A1 urea transporter ubiquitination and degradation

2008-11-05

UT-A1 is the primary urea transporter in the apical plasma membrane responsible for urea reabsorption in the inner medullary collecting duct. Although the physiological function of UT-A1 has been well established, the molecular mechanisms that regulate its activity are less well understood. Analysis of the UT-A1 amino acid sequence revealed a potential MDM2 E3 ubiquitin ligase-binding motif in the large intracellular loop of UT-A1, suggesting that UT-A1 urea transporter protein may be regulated by the ubiquitin-proteasome pathway. Here, we report that UT-A1 is ubiquitinated and degraded by the proteasome but not the lysosome proteolytic pathway. Inhibition of proteasome activity causes UT-A1 cell surface accumulation and concomitantly increases urea transport activity. UT-A1 interacts directly with MDM2; the binding site is located in the NH₂-terminal p53-binding region of MDM2. MDM2 mediates UT-A1 ubiquitination both in vivo and in vitro. Overexpression of MDM2 promotes UT-A1 degradation. The mechanism is likely to be physiologically important as UT-A1 ubiquitination was identified in kidney inner medullary tissue. The ubiquitin-proteasome degradation pathway provides an important novel mechanism for UT-A1 regulation.

Coenzyme Q10 supplementation rescues renal disease in Pdss2kd/kd mice with mutations in prenyl diphosphate synthase subunit 2

2008-11-05

Homozygous mice carrying kd (kidney disease) mutations in the gene encoding prenyl diphosphate synthase subunit 2 (Pdss2^kd/kd) develop interstitial nephritis and eventually die from end-stage renal disease. The PDSS2 polypeptide in concert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q (Q or ubiquinone), a lipid quinone required for mitochondrial respiratory electron transport. We have shown that a deficiency in Q content is evident in Pdss2^kd/kd mouse kidney lipid extracts by 40 days of age and thus precedes the onset of proteinuria and kidney disease by several weeks. The presence of the kd (V117M) mutation in PDSS2 does not prevent its association with PDSS1. However, heterologous expression of the kd mutant form of PDSS2 together with PDSS1 in Escherichia coli recapitulates the Q deficiency observed in the Pdss2^kd/kd mouse. Dietary supplementation with Q₁₀ provides a dramatic rescue of both proteinuria and interstitial nephritis in the Pdss2^kd/kd mutant mice. The results presented suggest that Q may be acting as a potent lipid-soluble antioxidant, rather than by boosting kidney mitochondrial respiration. Such Q₁₀ supplementation may have profound and beneficial effects in treatment of certain forms of focal segmental glomerulosclerosis that mirror the renal disease of the Pdss2^kd/kd mouse.

A novel cell-permeable antioxidant peptide decreases renal tubular apoptosis and damage in unilateral ureteral obstruction

Mizuguchi, Y., Chen, J., Seshan, S. V., Poppas, D. P., Szeto, H. H., Felsen, D. — 2008-11-05

Unilateral ureteral obstruction (UUO) is characterized by decreases in renal function, increased interstitial fibrosis, tubular apoptosis, and cellular infiltration. It has been suggested that inhibition of tubular apoptosis may protect against renal damage in obstruction. We have recently developed a series of peptides which are concentrated in the inner mitochondrial membrane and prevent cell death. These peptides are also active in vivo, in myocardial infraction, ischemic brain injury, and amyotrophic lateral sclerosis models. We therefore used SS-31, a prototype of these peptides, and assessed its effects on renal damage and oxidative stress in a 14-day obstruction model. SS-31 (1 or 3 mg/kg) or saline was given 1 day before and throughout the 14 days of obstruction. Kidneys were harvested and assessed for apoptosis (terminal transferase-dUTP-nick-end labeling, caspase 3 expression), fibrosis (trichrome staining), macrophage infiltration, fibroblast expression (immunoperoxidase), and oxidative damage (8-OH deoxyguanosine and heme oxygenase-1 expression), cytokines, and signaling pathways (transforming growth factor-β, CCR-1, p38-MAPK, NF-B). SS-31 significantly attenuated the effects of obstruction on all aspects of renal damage which were examined, with both the 1 and 3 mg/kg doses showing efficacy. We noted increased oxidative stress in obstruction, which was also attenuated by SS-31 treatment. Signaling via NF-B and p38 MAPK pathways were both affected by SS-31 treatment. This study provides a proof of concept that peptides which protect mitochondria in vitro can provide protection from renal damage in a UUO model. The mechanism by which protection is afforded requires further studies both in vitro and in vivo.

Lipocalin-2-induced renal regeneration depends on cytokines

Vinuesa, E., Sola, A., Jung, M., Alfaro, V., Hotter, G. — 2008-11-05

This study investigated whether the renal regeneration occurring in the recovery phase of kidney ischemia-reperfusion (I/R) is mediated by endogenously generated lipocalin-2 (Lcn2). A second objective was to examine whether Lcn2-mediated cell effects could be regulated by the inflammatory cytokines in the environment through their action on Lcn2 receptors (Lcn2R and megalin). Male Swiss mice were subjected to 30 min of renal ischemia with a reperfusion period of 24 h (early reperfusion, expected time for maximum inflammation) and 96 h (late reperfusion, expected time for maximum regeneration). Different experimental groups underwent I/R, I/R with iv anti-mouse Lcn2 monoclonal antibody injected during the early/inflammatory or late/recovery phase, and I/R with proinflammatory cytokine cocktail administration (recombinant mouse IL-1β, TNF-, and IFN-). Compared with control nonischemic mice, the expression of three proliferation markers (stathmin, PCNA, and Ki-67, analyzed by quantitative RT-PCR) increased significantly in the I/R-treated animals. Blockade of Lcn2 by addition of anti-Lcn2 antibody significantly decreased the expression of these three proliferation markers when administered in the late/reparative phase, but had the opposite effect when administered in the early/inflammatory phase. Proinflammatory cytokine cocktail administration reduced the proliferative effects of Lcn2, and repressed Lcn2R and megalin expression. In conclusion, endogenously generated Lcn2 induces renal cell regeneration depending on the inflammatory cytokines in kidney I/R.

Accelerated senescence in the kidneys of patients with type 2 diabetic nephropathy

2008-11-05

We examined the hypothesis that senescence represents a proximate mechanism by which the kidney is damaged in type 2 diabetic nephropathy (DN). As a first step, we studied whether the senescence-associated β-galactosidase (SA-β-Gal) and the cell cycle inhibitor p16^INK4A are induced in renal biopsies from patients with type 2 DN. SA-β-Gal staining was approximately threefold higher (P < 0.05) than in controls in the tubular compartment of diabetic kidneys and correlated directly with body mass index and blood glucose. P16^INK4A expression was significantly increased in tubules (P < 0.005) and in podocytes (P = 0.04). Nuclear p16^INK4A in glomeruli was associated with proteinuria (P < 0.002), while tubular p16^INK4A was directly associated with body mass index, LDL cholesterol, and HbA1c (P < 0.001–0.05). In a parallel set of experiments, proximal tubule cells passaged under high glucose presented a limited life span and an approximately twofold increase in SA-β-Gal and p16^INK4A protein. Mean telomere lengths decreased ~20% as an effect of replicative senescence. In addition, mean telomere decreased further by ~30% in cells cultivated under high glucose. Our results show that the kidney with type 2 diabetic nephropathy displays an accelerated senescent phenotype in defined renal cell types, mainly tubule cells and, to a lesser extent, podocytes. A similar senescent pattern was observed when proximal tubule cell cultures where incubated under high-glucose media. These changes are associated with shortening tubular telomere length in vitro. These findings indicate that diabetes may boost common pathways involving kidney cell senescence, thus reinforcing the role of the metabolic syndrome on biological aging of tissues.

Renoprotective mechanisms of soy protein intake in the obese Zucker rat

2008-11-05

We previously showed that long-term consumption of a soy protein diet (SoyP) reduces renal damage in obese Zucker (ObeseZ) rats by restoring urinary NO₂ and NO₃ excretion (UNO₂/NO₃V), suggesting that nitric oxide (NO) deficiency may contribute to the renal progression observed in this model. In addition, there is compelling evidence that hyperleptinemia produced deleterious effects on the kidney through its interaction with the short leptin receptor (ObRa). This study was designed to evaluate the contribution of the NO/endothelial NO synthase (eNOS) system, renal oxidative stress, and ObRa expression to the renoprotection conferred by the consumption of a SoyP in ObeseZ rats. Ten lean and ten male ObeseZ rats were included. One-half of each group was fed with a 20% SoyP and the other half with a 20% casein protein diet (CasP) over the course of 160 days. eNOS protein levels and phosphorylation, renal lipoperoxidation (rLPO), and antioxidant enzyme activity were assessed. In addition, renal ObRa, TGF-β, and kidney injury molecule (Kim-1) mRNA levels, as well as urinary Kim-1 levels, were measured. Renal injury observed in ObeseZ rats fed with CasP was not associated with changes in eNOS expression or phosphorylation. However, this group did present with increased rLPO, reduced catalase activity, and upregulation of ObRa, TGF-β1, and Kim-1. In contrast, ObeseZ rats fed with a SoyP exhibited a reduction in NOS-Thr495 phosphorylation and rLPO, as well as an enhanced catalase activity. These findings were associated with a significant reduction of ObRa, TGF-β1, and Kim-1 mRNA levels and urinary Kim-1 protein. Our results show that renoprotection by SoyP in ObeseZ rats is in part mediated by increased NO availability secondary to a reduction in eNOS-T495 phosphorylation and oxidative stress, together with a significant reduction in ObRa and TGF-β expression.

Functional immunoassay technology (FIT), a new approach for measuring physiological functions: application of FIT to measure glomerular filtration rate (GFR)

2008-11-05

This is the first description of functional immunoassay technology (FIT), which as a diagnostic tool has broad application across the whole spectrum of physiological measurements. In this paper, FIT is used to measure the renal clearance of an ultra low-dose administration of a clinically available contrast reagent for the purpose of obtaining an accurate glomerular filtration rate (GFR) measurement. Biomarker-based GFR estimates offer convenience, but are not accurate and are often misleading. FIT overcomes previous analytic barriers associated with obtaining an accurate GFR measurement. We present the performance characteristics of this diagnostic test and demonstrate the method by directly comparing GFR values obtained by FIT to those obtained by an FDA approved nuclear test in 20 adults. Two subjects were healthy volunteers and the remaining 18 subjects had diagnosed chronic kidney disease, with 12 being kidney transplant recipients. Measured GFR values were calculated by the classic UV/P method and by the blood clearance method. GFR obtained by FIT and the nuclear test correlated closely over a wide range of GFR values (10.9–102.1 ml·min^–1·1.73 m^–2). The study demonstrates that FIT-GFR provides an accurate and reproducible measurement. This nonradioactive, immunoassay-based approach offers many advantages, chiefly that most laboratories already have the equipment and trained personnel necessary to run an ELISA, and therefore this important diagnostic measurement can more readily be obtained. The FIT-GFR test can be used throughout the pharmaceutical development pipeline: preclinical and clinical trials.

Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance

Castrop, H., Schnermann, J. — 2008-10-09

The renal Na-K-2Cl cotransporter (NKCC2, BSC1) is selectively expressed in the apical membrane of cells of the thick ascending limb of the loop of Henle (TAL) and macula densa. NKCC2-dependent salt transport constitutes the major apical entry pathway for transepithelial salt reabsorption in the TAL. Although NKCC2 is encoded by a single gene (Slc12a1), differential splicing of the NKCC2 pre-mRNA results in the formation of several alternate transcripts. Thus three full-length splice isoforms of NKCC2 differ in their variable exon 4, resulting in transcripts for NKCC2B, NKCC2A, and NKCC2F. In addition to full-length isoforms, variants with truncated COOH-terminal ends have been described. The various splice isoforms of NKCC2 differ in their localization along the TAL and in their transport characteristics. Data in the literature are reviewed to assess the principles of NKCC2 differential splicing, the localization of NKCC2 splice isoforms along the TAL in various species, and the functional characteristics of the splice isoforms. In addition, we discuss the functional significance of NKCC2 isoforms for TAL salt retrieval and for the specific salt sensor function of macula densa cells based on studies using isoform-specific NKCC2-knockout mice. We suggest that different NKCC2 splice variants cooperate in salt retrieval along the TAL and that the coexpression of two splice variants (NKCC2B and NKCC2A) in the macula densa cells facilitates efficient salt sensing over wide ranges of fluctuating salt concentrations.

Biology of claudins

Angelow, S., Ahlstrom, R., Yu, A. S. L. — 2008-10-09

Claudins are a family of tight junction membrane proteins that regulate paracellular permeability of epithelia, likely by forming the lining of the paracellular pore. Claudins are expressed throughout the renal tubule, and mutations in two claudin genes are now known to cause familial hypercalciuric hypomagnesemia with nephrocalcinosis. In this review, we discuss recent advances in our understanding of the physiological role of various claudins in normal kidney function, and in understanding the fundamental biology of claudins, including the molecular basis for selectivity of permeation, claudin interactions in tight junction formation, and regulation of claudins by protein kinases and other intracellular signals.

cGMP decreases surface NKCC2 levels in the thick ascending limb: role of phosphodiesterase 2 (PDE2)

Ares, G. R., Caceres, P., Alvarez-Leefmans, F. J., Ortiz, P. A. — 2008-10-09

NaCl absorption in the medullary thick ascending limb of the loop of Henle (THAL) is mediated by the apical Na/K/2Cl cotransporter (NKCC2). Hormones that increase cGMP, such as nitric oxide (NO) and natriuretic peptides, decrease NaCl absorption by the THAL. However, the mechanism by which cGMP decreases NaCl absorption in THALs is not known. We hypothesized that cGMP decreases surface NKCC2 levels in the THAL. We used surface biotinylation to measure surface NKCC2 levels in rat THAL suspensions. We tested the effect of the membrane-permeant cGMP analog dibutyryl-cGMP (db-cGMP) on surface NKCC2 levels. Incubating THALs with db-cGMP for 20 min decreased surface NKCC2 levels in a concentration-dependent manner (basal = 100%; db-cGMP 100 µM = 77 ± 7%; 500 µM = 54 ± 10% and 1,000 µM = 61 ± 8%). A different cGMP analog 8-bromo-cGMP (8-Br-cGMP) also decreased surface NKCC2 levels by 25%, (basal = 100%; 8-Br-cGMP = 75 ± 5%). Incubation of isolated, perfused THALs with db-cGMP decreased apical surface NKCC2 labeling levels as measured by immunofluorescence and confocal microscopy. cGMP-stimulated phosphodiesterase 2 (PDE2) mediates the inhibitory effect of NO on NaCl absorption by THALs. Thus we examined the role of PDE2 and found that PDE2 inhibitors blocked the effect of db-cGMP on surface NKCC2. Also, a nonstimulatory concentration of db-cAMP blocked the cGMP-induced decrease in surface NKCC2. Finally, db-cGMP inhibited THAL net Cl absorption by 48 ± 4%, and this effect was completely blocked by PDE2 inhibition. We conclude that cGMP decreases NKCC2 levels in the apical membrane of THALs and that this effect is mediated by PDE2. This is an important mechanism by which cGMP inhibits NaCl absorption by the THAL.

USP10: the nexus between nexin and vasopressin

Butterworth, M. B., Johnson, J. P. — 2008-10-09

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

2008-10-09

Adjustment of Na⁺ balance in extracellular fluids is achieved by regulated Na⁺ transport involving the amiloride-sensitive epithelial Na⁺ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na⁺ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na⁺ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of - and -ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD_cl1 cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC.

Carbon monoxide: vasoconstrictor or vasodilator? That's the question

Volti, G. L., Vanella, L., Gazzolo, D., Galvano, F. — 2008-10-09

Heme oxygenase induction attenuates afferent arteriolar autoregulatory responses

Botros, F. T., Prieto-Carrasquero, M. C., Martin, V. L., Navar, L. G. — 2008-10-09

Heme oxygenases (HO-1, HO-2) catalyze conversion of heme to iron, carbon monoxide (CO), and biliverdin/bilirubin. We studied the effects of renal HO-1 induction on afferent arteriole (Aff-Art) autoregulatory responses to increases in renal perfusion pressure (RPP). Rats were treated with hemin and SnCl₂ to induce HO-1, and Aff-Art autoregulatory responses were evaluated using the rat blood-perfused juxtamedullary nephron preparation. Renal HO-1 expression was significantly increased in hemin- and SnCl₂-treated rats, while HO-2 was not altered. Aff-Art autoregulatory constrictor responses to increases in RPP from 100 to 150 mmHg were attenuated in hemin- and SnCl₂-treated rats compared with control rats (+1.1 ± 3.3, n = 9 and +4.4 ± 5.3, n = 9 vs. –14.2 ± 1.5%, n = 10, respectively) (P < 0.05). Acute HO inhibition with chromium mesoporphyrin (CrMP; 15 µmol/l) restored Aff-Art autoregulatory responses in hemin- and SnCl₂-treated rats. Superfusing Aff-Arts from control rats with 100 µmol/l biliverdin did not alter autoregulatory responses; however, superfusion with 1 mmol/l CO significantly attenuated autoregulatory responses to increases in RPP from 100 to 150 mmHg (+3.3 ± 5.4 vs. –16.6 ± 3.8%, n = 6) (P < 0.05). Acute soluble guanylate cyclase inhibition with 10 µmol/l ODQ restored Aff-Art autoregulatory responses in hemin-treated rats. Immunohistochemistry shows HO-2 to be expressed mainly in epithelial cells with weak staining in proximal tubules, interlobular arteries, and Aff-Arts. In hemin- and SnCl₂-treated rats, HO-1 was induced in tubular epithelial cells but not interlobular arteries and Aff-Arts. We conclude that induction of renal HO-1 attenuates Aff-Art constrictor responses to increases in RPP via increasing CO production from tubular epithelial cells, suggesting that an augmented HO system in pathophysiological conditions modulates renal autoregulation.

Inhibition of cystathionine-{beta}-synthase activity during renal ischemia-reperfusion: role of pH and nitric oxide

Prathapasinghe, G. A., Siow, Y. L., Xu, Z., O, K. — 2008-10-09

Our recent study (Prathapasinghe GA, Siow YL, O K. Am J Physiol Renal Physiol 292: F1354–F1363, 2007) indicates that homocysteine (Hcy) plays a detrimental role in ischemia-reperfusion-induced renal injury. Elevation of renal Hcy concentration during ischemia-reperfusion is attributed to reduced activity of cystathionine-β-synthase (CBS) that catalyzes the rate-limiting step in the transsulfuration pathway for the metabolism of the majority of Hcy in the kidney. However, the mechanisms of impaired CBS activity in the kidney are unknown. The aim of this study was to investigate the effects of pH and nitric oxide (NO) on the CBS activity in the kidney during ischemia-reperfusion. The left kidney of a Sprague-Dawley rat was subjected to ischemia-reperfusion. The CBS activity was significantly reduced in kidneys subjected to ischemia alone (15–60 min) or subjected to ischemia followed by reperfusion for 1–24 h. The pH was markedly reduced in kidneys upon ischemia. Injection of alkaline solution into the kidney partially restored the CBS activity during ischemia. Further analysis revealed that reduction of CBS activity during reperfusion was accompanied by an elevation of NO metabolites (nitrate and nitrite) in the kidney tissue. Injection of a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), restored the CBS activity in the kidneys subjected to ischemia-reperfusion. Treatment with PTIO could abolish ischemia-reperfusion-induced lipid peroxidation and prevent cell death in the kidney. These results suggested that metabolic acidosis during ischemia and accumulation of NO metabolites during reperfusion contributed, in part, to reduced CBS activity leading to an elevation of renal Hcy levels, which in turn, played a detrimental role in the kidney.

Acute anal stretch inhibits NMDA-dependent pelvic-urethra reflex potentiation via spinal GABAergic inhibition in anesthetized rats

2008-10-09

The impact of acute anal stretch on the pelvic-urethra reflex potentiation was examined in urethane-anesthetized rats by recording the external urethra sphincter electromyogram activity evoked by the pelvic afferent stimulation. Test stimulation (1 stimulation/30 s) evoked a baseline reflex activity with a single action potential that was abolished by gallamine (5 mg/kg iv). On the other hand, the repetitive stimulation (1 stimulation/1 s) induced spinal reflex potentiation (SRP) that was attenuated by intrathecal 6-cyano-7-nitroquinoxaline-2,4-dione (a glutamatergic -amino-3-hydroxy-5-methyl-4-isoxazoleproprionat receptor antagonist, 100 µM, 10 µl) and d-2-amino-5-phosphonovalerate [a glutamatergic N-methyl-d-aspartate (NMDA) antagonist, 100 µM, 10 µl]. Acute anal stretch using a mosquito clamp with a distance of 4 mm exhibited no effect, whereas distances of 8 mm attenuated and 12 mm abolished the repetitive stimulation-induced SRP. Intrathecal NMDA (100 µM, 10 µl) reversed the abolition on SRP caused by anal stretch. On the other hand, pretreated bicuculline [-aminobutyric acid (GABA) A receptor antagonist, 100 µM, 10 µl] but not hydroxysaclofen (GABA_B receptor antagonist) counteracted the abolition on the repetitive stimulation-induced SRP caused by the anal stretch. All of the results suggested that anal stretch may be used as an adjunct to assist voiding dysfunction in patients with overactive urethra sphincter and that GABAergic neurotransmission is important in the neural mechanisms underlying external urethra sphincter activity inhibited by anal stretch.