AJP: Renal Physiology current issue

Function and regulation of TRPP2 at the plasma membrane

Tsiokas, L. — 2009-06-24

The vast majority (~99%) of all known cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by naturally occurring mutations in two separate, but genetically interacting, loci, pkd1 and pkd2. pkd1 encodes a large multispanning membrane protein (PKD1) of unknown function, while pkd2 encodes a protein (TRPP2, polycystin-2, or PKD2) of the transient receptor potential (TRP) superfamily of ion channels. Biochemical, functional, and genetic studies support a model in which PKD1 physically interacts with TRPP2 to form an ion channel complex that conveys extracellular stimuli to ionic currents. However, the molecular identity of these extracellular stimuli remains elusive. Functional studies in cell culture show that TRPP2 can be activated in response to mechanical cues (fluid shear stress) and/or receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) activation at the cell surface. Recent genetic studies in Chlamydomonas reinhardtii show that CrPKD2 functions in a pathway linking cell-cell adhesion and Ca²⁺ signaling. The mode of activation depends on protein-protein interactions with other channel subunits and auxiliary proteins. Therefore, understanding the mechanisms underlying the molecular makeup of TRPP2-containing complexes is critical in delineating the mechanisms of TRPP2 activation and, most importantly, the mechanisms by which naturally occurring mutations in pkd1 or pkd2 lead not only to ADPKD, but also to other defects reported in model organisms lacking functional TRPP2. This review focuses on the molecular assembly, function, and regulation of TRPP2 as a cell surface cation channel and discusses its potential role in Ca²⁺ signaling and ADPKD pathophysiology.

Aquaporin-2 abundance in the renal collecting duct: new insights from cultured cell models

Hasler, U., Leroy, V., Martin, P.-Y., Feraille, E. — 2009-06-24

The renal cortico-papillary osmotic gradient is generated by sodium reabsorption in the thick ascending limb. The antidiuretic hormone arginine vasopressin (AVP) increases collecting duct water permeability by enhancing aquaporin-2 (AQP2) water channel insertion in the apical membrane of principal cells, allowing water to passively flow along the osmotic gradient from the tubule lumen to the interstitium. In addition to short-term AQP2 redistribution between intracellular compartments and the cell surface, AQP2 whole cell abundance is tightly regulated. AVP is a major transcriptional activator of the AQP2 gene, and stimulation of insulin- and calcium-sensing receptors respectively potentiate and reduce its action. Extracellular tonicity is another key factor that determines the levels of AQP2 abundance. Its effect is dependent on activation of the tonicity-responsive enhancer binding protein that reinforces AVP-induced AQP2 transcriptional activation. Conversely, activation of the NF-B transcriptional factor by proinflammatory factors reduces AQP2 gene transcription. Aldosterone additionally regulates AQP2 whole cell abundance by simultaneously reducing AQP2 gene transcription and stimulating AQP2 mRNA translation. These examples illustrate how cross talk between various stimuli regulates AQP2 abundance in collecting duct principal cells and consequently contributes to maintenance of body water homeostasis.

Low-dose carbon monoxide inhibits progressive chronic allograft nephropathy and restores renal allograft function

Nakao, A., Faleo, G., Nalesnik, M. A., Seda-Neto, J., Kohmoto, J., Murase, N. — 2009-06-24

Chronic allograft nephropathy (CAN) represents progressive deterioration of renal allograft function with fibroinflammatory changes. CAN, recently reclassified as interstitial fibrosis (IF) and tubular atrophy (TA) with no known specific etiology, is a major cause of late renal allograft loss and remains a significant deleterious factor of successful renal transplantation. Carbon monoxide (CO), an effector byproduct of heme oxygenase pathway, is known to have potent anti-inflammatory and antifibrotic functions. We hypothesized that inhaled CO would inhibit fibroinflammatory process of CAN and restore renal allograft function, even when the treatment was initiated after CAN was established. Lewis rat kidney grafts were orthotopically transplanted into binephrectomized allogenic Brown Norway rats under brief tacrolimus (0.5 mg/kg im, days 0–6). At day 60, CO (20 ppm) inhalation was initiated to recipients and continued until day 150 or animal death. Development of CAN was confirmed at day 60 with decreased creatinine clearance (CCr), significant proteinuria, and histopathological findings of TA, IF, and intimal arteritis. Air-treated control recipients continued to deteriorate with further declines of CCr and increases of urinary protein excretion and died with a median survival of 82 days. In contrast, progression of CAN was decelerated when recipients received CO on days 60–150, showing markedly improved graft histopathology, restored renal function, and improved recipient survival to a median of >150 days. CO significantly reduced intragraft mRNA levels for IFN- and TNF- at day 90. Expression of profibrotic TGF-β/Smad was significantly suppressed with CO, together with downregulation of ERK-MAPK pathways. Continuous CO (20 ppm) treatment for days 0–30, days 30–60, or days 0–90, or daily 1-h CO (250 ppm) treatment for days 0–90, also showed efficacy in inhibiting CAN. The study demonstrates that CO is able to inhibit progression of fibroinflammatory process of CAN, restore renal allograft function, and improve survival even when the treatment is started after CAN is diagnosed.

Ischemia-reperfusion reduces cystathionine-{beta}-synthase-mediated hydrogen sulfide generation in the kidney

Xu, Z., Prathapasinghe, G., Wu, N., Hwang, S.-Y., Siow, Y. L., O, K. — 2009-06-24

Cystathionine-β-synthase (CBS) catalyzes the rate-limiting step in the transsulfuration pathway for the metabolism of homocysteine (Hcy) in the kidney. Our recent study demonstrates that ischemia-reperfusion reduces the activity of CBS leading to Hcy accumulation in the kidney, which in turn contributes to renal injury. CBS is also capable of catalyzing the reaction of cysteine with Hcy to produce hydrogen sulfide (H₂S), a gaseous molecule that plays an important role in many physiological and pathological processes. The aim of the present study was to examine the effect of ischemia-reperfusion on CBS-mediated H₂S production in the kidney and to determine whether changes in the endogenous H₂S generation had any impact on renal ischemia-reperfusion injury. The left kidney of Sprague-Dawley rat was subjected to 45-min ischemia followed by 6-h reperfusion. The ischemia-reperfusion caused lipid peroxidation and cell death in the kidney. The CBS-mediated H₂S production was decreased, leading to a significant reduction in the renal H₂S level. The activity of cystathionine--lyase, another enzyme responsible for endogenous H₂S generation, was not significantly altered in the kidney upon ischemia-reperfusion. Partial restoration of CBS activity by intraperitoneal injection of the nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide not only increased renal H₂S levels but also alleviated ischemia-reperfusion-induced lipid peroxidation and reduced cell damage in the kidney tissue. Furthermore, administration of an exogenous H₂S donor, NaHS (100 µg/kg), improved renal function. Taken together, these results suggest that maintenance of tissue H₂S level may offer a renal protective effect against ischemia-reperfusion injury.

MUPP1 complexes renal K+ channels to alter cell surface expression and whole cell currents

2009-06-24

We previously found that the Ca²⁺-sensing receptor (CaR) interacts with and inactivates the inwardly rectifying K⁺ channel Kir4.2 that is expressed in the kidney cortex and that has a COOH-terminal PDZ domain. To identify potential scaffolding proteins that could organize a macromolecular signaling complex involving the CaR and Kir4.2, we used yeast two-hybrid cloning with the COOH-terminal 125 amino acids (AA) of Kir4.2 as bait to screen a human kidney cDNA library. We identified two independent partial cDNAs corresponding to the COOH-terminal 900 AA of MUPP1, a protein containing 13 PDZ binding domains that is expressed in the kidney in tight junctions and lateral borders of epithelial cells. When expressed in human embryonic kidney (HEK)-293 cells, Kir4.2 coimmunoprecipitates reciprocally with MUPP1 but not with a Kir4.2 construct lacking the four COOH-terminal amino acids, Kir5.1, or the CaR. MUPP1 and Kir4.2 coimmunoprecipitate reciprocally from rat kidney cortex extracts. Coexpression of MUPP1 with Kir4.2 in HEK-293 cells leads to reduced cell surface expression of Kir4.2 as assessed by cell surface biotinylation. Coexpression of MUPP1 and Kir4.2 in Xenopus oocytes results in reduced whole cell currents compared with expression of Kir4.2 alone, whereas expression of Kir4.2PDZ results in minimal currents and is not affected by coexpression with MUPP1. Immunofluorescence studies of oocytes demonstrate that MUPP1 reduces Kir4.2 membrane localization. These results indicate that Kir4.2 interacts selectively with MUPP1 to affect its cell surface expression. Thus MUPP1 and Kir4.2 may participate in a protein complex in the nephron that could regulate transport of K⁺ as well as other ions.

Basic fibroblast growth factor causes urinary bladder overactivity through gap junction generation in the smooth muscle

2009-06-24

Overactive bladder is a highly prevalent clinical condition that is often caused by bladder outlet obstruction (BOO). Increased coupling of bladder smooth muscle cells (BSMC) via gap junctions has been hypothesized as a mechanism for myogenic bladder overactivity in BOO, although little is known about the regulatory system underlying such changes. Here, we report the involvement of basic fibroblast growth factor (bFGF) and connexin 43, a bladder gap junction protein, in bladder overactivity. BOO created by urethral constriction in rats resulted in elevated bFGF and connexin 43 levels in the bladder urothelium and muscle layer, respectively, and muscle strips from these bladders were more sensitive than those from sham-operated controls to a cholinergic agonist. In vitro bFGF treatment increased connexin 43 expression in cultured rat BSMC via the ERK 1/2 pathway. This finding was supported by another in vivo model, where bFGF released from gelatin hydrogels fixed on rat bladder walls caused connexin 43 upregulation and gap junction formation in the muscle layer. Bladder muscle strips in this model showed increased sensitivity to a cholinergic agonist that was blocked by inhibition of gap junction function with -glycyrrhetinic acid. Cystometric analyses of this model showed typical features of detrusor overactivity such as significantly increased micturition frequency and decreased bladder capacity. These findings suggest that bFGF from the urothelium could induce bladder hypersensitivity to acetylcholine via gap junction generation in the smooth muscle, thereby contributing to the myogenic overactivity of obstructed bladders.

PPAR{gamma} agonists inhibit vasopressin-mediated anion transport in the MDCK-C7 cell line

2009-06-24

PPAR agonists are synthetic ligands for the peroxisome proliferator-activated receptor- (PPAR). These agents have insulin-sensitizing properties but can cause fluid retention, thereby limiting their usefulness in patients at risk for cardiovascular disease. The side effect etiology is unknown, but the nature of presentation suggests modulation of renal salt and water homeostasis. In a well-characterized cell culture model of the principal cell type [Madin-Darby canine kidney (MDCK)-C7], PPAR agonists inhibit vasopressin-stimulated Cl^– secretion with agonist dose-response relationships that mirror receptor transactivation profiles. Analyses of the components of the vasopressin-stimulated intracellular signaling pathway indicated no PPAR agonist-induced changes in basolateral membrane conductances, intracellular cAMP, protein kinase A, or total cellular adenine nucleotides. The PPAR agonist-induced decrease in anion secretion is the result of decreased mRNA of the final effector in the pathway, the apically located cystic fibrosis transmembrane regulator (CFTR). These data showing that CFTR is a target for PPAR agonists may provide new insights into the physiology of PPAR agonist-induced fluid retention.

Sp1 trans-activates the murine H+-K+-ATPase {alpha}2-subunit gene

Yu, Z., Li, M., Zhang, D., Xu, W., Kone, B. C. — 2009-06-24

The H⁺-K⁺-ATPase ₂ (HK2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-B and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the –144/–135 Sp element influences basal HK2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for –154/–127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HK2 gene in mIMCD3 cells in vivo. HK2 minimal promoter-luciferase constructs with point mutations in the –144/–135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HK2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HK2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HK2 promoter without altering the binding or regulatory influence of NF-B p65 or CREB-1 on the proximal HK2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HK2 gene expression in mIMCD3 cells in vivo and in vitro.

Organic anion transporter OAT1 is involved in renal handling of citrulline

Nakakariya, M., Shima, Y., Shirasaka, Y., Mitsuoka, K., Nakanishi, T., Tamai, I. — 2009-06-24

Because citrulline plasma concentration is elevated in kidney failure, citrulline could be a biomarker of renal insufficiency, although the mechanism regulating its disposition in the kidney has not been clarified. In rat kidney slices, citrulline uptake was apparently Na⁺ dependent, saturable with K_m 556 µM, and significantly inhibited by anionic (PAH) and cationic (TEA) compounds, but not by probenecid at 1 mM. Preincubation of kidney slices with glutarate increased citrulline uptake, while such an increase was not observed after preincubation of the slices in Na⁺-free buffer. This result suggested that a sodium-dependent dicarboxylate cotransporter is involved in citrulline uptake by rat kidney slices. In studies using transporter-overexpressing cells, human organic anion transporter 1 (OAT1) and rat Oat1 exhibited citrulline transport activity with K_m values of 238 and 373 µM, respectively, while other OATs and organic cation transporters (OCTs) did not transport citrulline. Based on the relative activity factor method, the contribution of rat Oat1 to the overall uptake of citrulline in rat kidney slices was ~70%. Moreover, the interaction among citrulline, PAH, and probenecid uptakes via rat Oat1 suggested that there are multiple functional sites on Oat1 and that the citrulline site may be distinct from the PAH and probenecid site. Thus OAT1/Oat1 appears to be one of the major contributors to renal basolateral uptake of citrulline, and impaired activities of these transporters may contribute substantially to the increase in plasma citrulline in renal failure. Accordingly, citrulline may be useful for diagnosis of kidney function as is creatinine.

Sexual dimorphism in glomerular arginine transport affects nitric oxide generation in old male rats

2009-06-24

Animal models suggest that decreased renal endothelial nitric oxide synthase (eNOS) activity in old males promotes renal injury, whereas females are protected. We aimed to explore whether aging alters glomerular arginine uptake by CAT-1, the selective arginine supplier to eNOS in rats. Arginine uptake by glomeruli from young males (3 mo) was significantly higher than in young females. Old males (19 mo) exhibited a significant decrease in arginine transport compared with young males, whereas no differences were observed between old and young females. CAT-1 abundance remained unchanged in all experimental groups. The abundance of PKC (CAT-1 inhibitor) was significantly augmented in young females vs. young males, old vs. young males, and in old females vs. old males. No differences in PKC content were detected between old and young females. Phosphorylated PKC was significantly increased in old rats from both genders. Tocopherol, a PKC inhibitor, produced a significant increase in arginine transport and restored NO generation in old males only. Ex vivo incubation of glomeruli from old males with PMA (PKC stimulant) significantly attenuated the effect of tocopherol on arginine uptake. In conclusion, attenuated glomerular arginine transport by CAT-1 contributes to the age-dependent, NO-deficient state in old male rats through upregulation of PKC. In old females glomerular arginine transport is protected from the effects of PKC by an unknown mechanism.

The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-{beta}, increases podocyte motility and albumin permeability

2009-06-24

The role of monocyte chemoattractant protein-1 (MCP-1) in diabetic nephropathy is typically viewed through the lens of inflammation, but MCP-1 might exert noninflammatory effects on the kidney cells directly. Glomerular podocytes in culture, verified to express the marker nephrin, were exposed to diabetic mediators such as high glucose or angiotensin II and assayed for MCP-1. Only transforming growth factor-β (TGF-β) significantly increased MCP-1 production, which was prevented by SB431542 and LY294002, indicating that signaling proceeded through the TGF-β type I receptor kinase and the phosphatidylinositol 3-kinase pathway. The TGF-β-induced MCP-1 was found to activate the podocyte's cysteine-cysteine chemokine receptor 2 (CCR2) and, as a result, enhance the cellular motility, cause rearrangement of the actin cytoskeleton, and increase podocyte permeability to albumin in a Transwell assay. The preceding effects of TGF-β were replicated by treatment with recombinant MCP-1 and blocked by a neutralizing anti-MCP-1 antibody or a specific CCR2 inhibitor, RS102895. In conclusion, this is the first description that TGF-β signaling through PI3K induces the podocyte expression of MCP-1 that can then operate via CCR2 to increase cellular migration and alter albumin permeability characteristics. The pleiotropic effects of MCP-1 on the resident kidney cells such as the podocyte may exacerbate the disease process of diabetic albuminuria.

Expression of the RNA-stabilizing protein HuR in ischemia-reperfusion injury of rat kidney

Ayupova, D. A., Singh, M., Leonard, E. C., Basile, D. P., Lee, B. S. — 2009-06-24

The RNA-binding protein human antigen R (HuR) participates in the posttranscriptional regulation of mRNAs bearing 3' AU-rich and U-rich elements, which HuR can stabilize under conditions of cellular stress. Using the LLC-PK₁ proximal tubule cell line model, we recently suggested a role for HuR in protecting kidney epithelia from injury during ischemic stress (Jeyaraj S, Dakhlallah D, Hill SR, Lee BS. J Biol Chem 280: 37957–37964, 2005; Jeyaraj SC, Dakhlallah D, Hill SR, Lee BS. Am J Physiol Renal Physiol 291: F1255–F1263, 2006). Here, we have extended this work to show that small interfering RNA-mediated suppression of HuR in LLC-PK₁ cells increased apoptosis during energy depletion, while overexpression of HuR diminished apoptosis. Suppression of HuR also resulted in diminished levels of key cell survival proteins such as Bcl-2 and Hsp70. Furthermore, rat kidneys were subjected in vivo to transient ischemia followed by varying periods of reperfusion. Ischemia and reperfusion (I/R) affected intensity and distribution of HuR in a nephron segment-specific manner. Cells of the proximal tubule, which are most sensitive to I/R injury, demonstrated a transient shift of HuR to the cytoplasm immediately following ischemia. Over a 14-day period following the onset of reperfusion, nuclear and total HuR protein gradually increased in cortical and medullary proximal tubules, but not in non-proximal tubule cells. HuR mRNA was expressed in two forms with alternate transcriptional start sites that increased over a 14-day I/R period, and in vitro studies suggest selective translatability of these two mRNAs. Baseline and I/R-stimulated levels of HuR mRNA did not parallel those of HuR protein, suggesting translational control of HuR expression, particularly in medullary proximal tubules. These findings suggest that alterations in distribution and expression of the antiaptotic protein HuR specifically in cells of the proximal tubule effect a protective mechanism during and following I/R injury in kidney.

Niacin ameliorates oxidative stress, inflammation, proteinuria, and hypertension in rats with chronic renal failure

Cho, K.-h., Kim, H.-j., Rodriguez-Iturbe, B., Vaziri, N. D. — 2009-06-24

Significant reduction of renal mass causes progressive deterioration of renal function and structure which is mediated by systemic and glomerular hypertension, hyperfiltration, oxidative stress, inflammation, and dyslipidemia. Niacin is known to improve lipid metabolism and exert antioxidant/anti-inflammatory actions. Therefore, we considered that niacin supplementation may attenuate oxidative stress, inflammation, and tissue injury in the remnant kidney. To this end, 56 nephrectomized [chronic kidney disease (CKD)] rats were randomly assigned to niacin-treated (50 mg·kg^–1·day^–1 in the drinking water for 12 wk) and untreated groups. Sham-operated rats served as controls. The untreated CKD rats exhibited azotemia, hypertension, hypertriglyceridemia, proteinuria, glomerulosclerosis, tubulointerstitial damage, upregulation of MCP-1, plasminogen activator inhibitor-1 (PAI-1), transforming growth factor (TGF)-β, cyclooxygenase (COX)-1, COX-2, and NAD(P)H oxidase (NOX-4, gp91^phox, p47^phox and p22^phox subunits) and activation of NF-B (IB phosphorylation). Niacin administration reduced MCP-1, PAI-1, TGF-β, p47^phox, p22^phox, COX-1, and NF-B activation, ameliorated hypertension, proteinuria, glomerulosclerosis, and tubulointerstitial injury. Although niacin lowered serum creatinine and raised creatinine clearance, the differences did not reach statistical significance. Thus niacin supplementation helps to attenuate histological injury and mitigate upregulation of oxidative and inflammatory systems in the remnant kidney.

Pressure activates epidermal growth factor receptor leading to the induction of iNOS via NF{kappa}B and STAT3 in human proximal tubule cells

Broadbelt, N. V., Chen, J., Silver, R. B., Poppas, D. P., Felsen, D. — 2009-06-24

Ureteral obstruction leads to increased pressure and inducible nitric oxide synthase (iNOS) expression. This study examined the involvement of epidermal growth factor (EGF) receptor (EGFR), nuclear factor-B (NFB), and signal transducers and activators of transcription 3 (STAT3) in iNOS induction in human proximal tubule (HKC-8) cells in response to pressure or EGF. HKC-8 cells were subjected to 60 mmHg pressure or treated with EGF for 0–36 h. iNOS was more rapidly induced in response to EGF than pressure. The addition of EGFR, NFB, and STAT3 inhibitors significantly suppressed pressure- or EGF-stimulated iNOS mRNA and protein expression. Analysis of the activated states of EGFR, NFB p65, and STAT3 after exposure to both stimuli demonstrated phosphorylation within 2.5 min. Anti-EGF antibody inhibited iNOS induction in pressurized HKC-8 cells, providing evidence that endogenous EGF mediates the response to pressure. In ureteral obstruction, when pressure is elevated, phosphorylated EGFR was detected in the apical surface of the renal tubules, validating the in vitro findings. These data indicate that EGFR, NFB, and STAT3 are required for human iNOS gene induction in response to pressure or EGF, indicating a similar mechanism of activation.

Kidney ischemia-reperfusion injury induces caspase-dependent pulmonary apoptosis

Hassoun, H. T., Lie, M. L., Grigoryev, D. N., Liu, M., Tuder, R. M., Rabb, H. — 2009-06-24

Distant organ effects of acute kidney injury (AKI) are a leading cause of morbidity and mortality. While little is known about the underlying mechanisms, limited data suggest a role for inflammation and apoptosis. Utilizing a lung candidate gene discovery approach in a mouse model of ischemic AKI-induced lung dysfunction, we identified prominent lung activation of 66 apoptosis-related genes at 6 and/or 36 h following ischemia, of which 6 genes represent the tumor necrosis factor receptor (TNFR) superfamily, and another 23 genes are associated with the TNFR pathway. Given that pulmonary apoptosis is an important pathogenic mechanism of acute lung injury (ALI), we hypothesized that AKI leads to pulmonary proapoptotic pathways that facilitate lung injury and inflammation. Functional correlation with 1) terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and 2) active caspase-3 (aC3) activity, immunoblotting, and immunohistochemistry (IHC) identified kidney IRI-induced pulmonary apoptosis at 24 h, and colocalization studies with CD34 identified predominantly endothelial apoptosis. Mice were treated with the caspase inhibitor Z-VAD-FMK (0.25 mg ip) or vehicle 1 h before and 8 h after sham or kidney IRI, and bronchoalveolar lavage fluid protein was measured at 36 h as a surrogate for lung leak. Caspase inhibition reduced lung microvascular changes after kidney IRI. The pulmonary apoptosis seen in wild-type control mice during AKI was absent in TNFR^–/– mice. Using an initial genomic approach to discovery followed by a mechanistic approach to disease targeting, we demonstrate that pulmonary endothelial apoptosis is a direct mediator of the distant organ dysfunction during experimental AKI.

Antibodies to protein tyrosine phosphatase receptor type O (PTPro) increase glomerular albumin permeability (Palb)

2009-06-24

Glomerular capillary filtration barrier characteristics are determined in part by the slit-pore junctions of glomerular podocytes. Protein tyrosine phosphatase receptor-O (PTPro) is a transmembrane protein expressed on the apical surface of podocyte foot processes. Tyrosine phosphorylation of podocyte proteins including nephrin may control the filtration barrier. To determine whether PTPro activity is required to maintain glomerular macromolecular permeability, albumin permeability (P_alb) was studied after incubation of glomeruli from normal animals with a series of monoclonal (mAb) and polyclonal antibodies. Reagents included mAbs to rabbit and rat PTPro and polyclonal rabbit immune IgG to rat PTPro. mAb 4C3, specific to the amino acid core of PTPro, decreased its phosphatase activity and increased P_alb of rabbit glomeruli in a time- and concentration-dependent manner. In contrast, mAb P8E7 did not diminish phosphatase activity and did not alter P_alb. Preincubation of 4C3 with PTPro extracellular domain fusion protein blocked glomerular binding and abolished permeability activity. In parallel experiments, P_alb of rat glomeruli was increased by two mAbs (1B4 and 1D1) or by polyclonal anti-rat PTPro. We conclude that PTPro interaction with specific antibodies acutely increases P_alb. The identity of the normal ligand for PTPro and of its substrate, as well as the mechanism by which phosphatase activity of this receptor affects the filtration barrier, remain to be determined.

Ability of sat-1 to transport sulfate, bicarbonate, or oxalate under physiological conditions

Krick, W., Schnedler, N., Burckhardt, G., Burckhardt, B. C. — 2009-06-24

Tubular reabsorption of sulfate is achieved by the sodium-dependent sulfate transporter, NaSi-1, located at the apical membrane, and the sulfate-anion exchanger, sat-1, located at the basolateral membrane. To delineate the physiological role of rat sat-1, [³⁵S]sulfate and [¹⁴C]oxalate uptake into sat-1-expressing oocytes was determined under various experimental conditions. Influx of [³⁵S]sulfate was inhibited by bicarbonate, thiosulfate, sulfite, and oxalate, but not by sulfamate and sulfide, in a competitive manner with K_i values of 2.7 ± 1.3 mM, 101.7 ± 9.7 µM, 53.8 ± 10.9 µM, and 63.5 ± 38.7 µM, respectively. Vice versa, [¹⁴C]oxalate uptake was inhibited by sulfate with a K_i of 85.9 ± 9.5 µM. The competitive type of inhibition indicates that these compounds are most likely substrates of sat-1. Physiological plasma bicarbonate concentrations (25 mM) reduced sulfate and oxalate uptake by more than 75%. Simultaneous application of sulfate, bicarbonate, and oxalate abolished sulfate as well as oxalate uptake. These data and electrophysiological studies using a two-electrode voltage-clamp device provide evidence that sat-1 preferentially works as an electroneutral sulfate-bicarbonate or oxalate-bicarbonate exchanger. In kidney proximal tubule cells, sat-1 likely completes sulfate reabsorption from the ultrafiltrate across the basolateral membrane in exchange for bicarbonate. In hepatocytes, oxalate extrusion is most probably mediated either by an exchange for sulfate or bicarbonate.

Detection of low-frequency oscillations in renal blood flow

Siu, K. L., Sung, B., Cupples, W. A., Moore, L. C., Chon, K. H. — 2009-06-24

Detection of the low-frequency (LF; ~0.01 Hz) component of renal blood flow, which is theorized to reflect the action of a third renal autoregulatory mechanism, has been difficult due to its slow dynamics. In this work, we used three different experimental approaches to detect the presence of the LF component of renal autoregulation using normotensive and spontaneously hypertensive rats (SHR), both anesthetized and unanesthetized. The first experimental approach utilized a blood pressure forcing in the form of a chirp, an oscillating perturbation with linearly increasing frequency, to elicit responses from the LF autoregulatory component in anesthetized normotensive rats. The second experimental approach involved collection and analysis of spontaneous blood flow fluctuation data from anesthetized normotensive rats and SHR to search for evidence of the LF component in the form of either amplitude or frequency modulation of the myogenic and tubuloglomerular feedback mechanisms. The third experiment used telemetric recordings of arterial pressure and renal blood flow from normotensive rats and SHR for the same purpose. Our transfer function analysis of chirp signal data yielded a resonant peak centered at 0.01 Hz that is greater than 0 dB, with the transfer function gain attenuated to lower than 0 dB at lower frequencies, which is a hallmark of autoregulation. Analysis of the data from the second experiments detected the presence of ~0.01-Hz oscillations only with isoflurane, albeit at a weaker strength compared with telemetric recordings. With the third experimental approach, the strength of the LF component was significantly weaker in the SHR than in the normotensive rats. In summary, our detection via the amplitude modulation approach of interactions between the LF component and both tubuloglomerular feedback and the myogenic mechanism, with the LF component having an identical frequency to that of the resonant gain peak, provides evidence that 0.01-Hz oscillations may represent the third autoregulatory mechanism.

AT1 receptor activation regulates the mRNA expression of CAT1, CAT2, arginase-1, and DDAH2 in preglomerular vessels from angiotensin II hypertensive rats

Hultstrom, M., Helle, F., Iversen, B. M. — 2009-06-24

Previously, we found increased expression of l-arginine metabolizing enzymes in both kidneys from two-kidney, one-clip (2K1C) hypertensive rats (Helle F, Hultstrom M, Skogstrand T, Palm F, Iversen BM. Am J Physiol Renal Physiol 296: F78–F86, 2009). In the present study, we investigate whether AT₁ receptor activation can induce the changes observed in 2K1C. Four groups of rats were infused with 80 ng/min ANG II or saline for 14 days and/or given 60 mg·kg^–1·day^–1 losartan. Gene expression was studied in isolated preglomerular vessels by RT-PCR. Dose-responses to ANG II were studied in isolated preglomerular vessels with and without acute NOS inhibition [10^–4 mol/l N^G-nitro-l-arginine methyl ester (l-NAME)]. Expressions of endothelial nitric oxide synthase (eNOS), caveolin-1, and arginase-2 were not changed by ANG II infusion. CAT1 (0.3 8 ± 0.07 to 0.73 ± 0.12, P < 0.05), CAT2 (1.14 ± 0.29 to 2.74 ± 0.48), DDAH2 (1.09 ± 0.27 to 2.3 ± 0.46), and arginase-1 (1.08 ± 0.17 to 1.82 ± 0.22) were increased in ANG II-infused rats. This was prevented by losartan treatment, which reduced the expression of eNOS (0.97 ± 0.26 to 0.37 ± 0.11 in controls; 0.8 ± 0.16 to 0.36 ± 0.1 in ANG II-infused rats) and caveolin-1 (2.49 ± 0.59 to 0.82 ± 0.24 in controls and 2.59 ± 0.61 to 1.1 ± 0.25 in ANG II-infused rats). ANG II (10^–10 mol/l) caused vessels from ANG II-infused animals to contract to 53 ± 15% of baseline diameter and 90 ± 5% of baseline diameter in controls (P < 0.05) and was further enhanced by l-NAME to 4 ± 4% of baseline diameter (P < 0.05). In vivo losartan treatment reduced the reactivity of isolated vessels to 91 ± 2% of baseline in response to 10^–7 mol/l ANG II compared with 82 ± 3% in controls (P < 0.05) and prevented the increased responsiveness caused by ANG II infusion. In conclusion, CAT1, CAT2, DDAH2, and arginase-1 expression in renal resistance vessels is regulated through the AT₁ receptor. This finding may be of direct importance for NOS and the regulation of preglomerular vascular function.

Mice lacking the ADP ribosyl cyclase CD38 exhibit attenuated renal vasoconstriction to angiotensin II, endothelin-1, and norepinephrine

Thai, T. L., Arendshorst, W. J. — 2009-06-24

ADP ribosyl (ADPR) cyclases comprise a family of ectoenzymes recently shown to influence cytosolic Ca²⁺ concentration in a variety of cell types. At least two ADPR cyclase family members have been identified in mammals: CD38 and CD157. We recently found reduced renal vascular reactivity to angiotensin II (ANG II), endothelin-1 (ET-1), and norepinephrine (NE) in the presence of the broad ADPR cyclase inhibitor nicotinamide. We hypothesized that CD38 mediates effects attributed to ADPR cyclase. We found expression of ADPR cyclases CD38 and CD157 mRNA in spleen, thymus, skin, and preglomerular arterioles of wild-type (WT) animals. Mice lacking CD38 showed decreased CD157 expression in most tissues tested. No difference in systolic or mean arterial pressure was observed between strains in either conscious or anesthetized states, whereas heart rate was reduced 10–20% in CD38–/– animals (P < 0.05). During anesthesia, CD38–/– mice had reduced basal renal blood flow (RBF) and urine excretion (P < 0.05). RBF responses to intravenous injection of ANG II, ET-1, and NE were attenuated ~50% in CD38–/– vs. WT mice (P < 0.01 for all). The systemic pressor response to ANG II was decreased in the absence of CD38 (P < 0.01), whereas that to NE was normal (P > 0.05); ET-1 was administered at a nonpressor dose. Nicotinamide effectively inhibited ANG II-induced renal vasoconstriction in WT mice (P < 0.001), but had no effect on renal responses to ANG II in CD38–/– mice (P > 0.5). Overall, our observations indicate the presence of two ADPR cyclase family members in renal preglomerular resistance arterioles and the importance of CD38 participation in acute vascular responses to all three vasoconstrictors in the renal microcirculation.

Expression and modulation of translocator protein and its partners by hypoxia reoxygenation or ischemia and reperfusion in porcine renal models

2009-06-24

Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is an 18-kDa drug- and cholesterol-binding protein localized to the outer mitochondrial membrane and implicated in a variety of cell and mitochondrial functions. To determine the role of TSPO in ischemia-reperfusion injury (IRI), we used both in vivo and in vitro porcine models: an in vivo renal ischemia model where different conservation modalities were tested and an in vitro model where TSPO-transfected porcine proximal tubule LLC-PK₁ cells were exposed to hypoxia and oxidative stress. The expression of TSPO and its partners in steroidogenic cells, steroidogenic acute regulatory protein (StAR) and cytochrome P-450 side chain cleavage CYP11A1, as well as the impact of TSPO overexpression and exposure to TSPO ligands in vitro in hypoxia-ischemia conditions were investigated. Hypoxia induced caspase activation, reduction of ATP content, and LLC-PK₁ cell death. Transfection and overexpression of TSPO rescued the cells from the detrimental effects of hypoxia and reoxygenation. Moreover, TSPO overexpression was accompanied by a reduction of H₂O₂-induced necrosis. TSPO drug ligands did not affect TSPO-mediated functions. In vivo, TSPO expression was modulated by IRI and during regeneration particularly in proximal tubule cells, which do not express this protein at the basal level. Under the same conditions, StAR and CYP11A1 protein and gene expression was reduced without apparent relation to TSPO changes. Pregnenolone was identified and measured in the pig kidney. Pregnenolone synthesis was not affected by the experimental conditions used. Taken together, these results indicate that changes in TSPO expression in kidney regenerating tissue could be important for renal protection and maintenance of kidney function.

Improvement of renal hemodynamics during hypertension-induced chronic renal disease: role of EGF receptor antagonism

2009-06-24

The present study investigated mechanisms of regression of renal disease after severe proteinuria by focusing on the interaction among EGF receptors, renal hemodynamics, and structural lesions. The nitric oxide (NO) inhibitor N^G-nitro-l-arginine-methyl ester (l-NAME) was administered chronically in Sprague-Dawley rats. When proteinuria exceeded 2 g/mmol creatinine, animals were divided into three groups for an experimental period of therapy of 2 wk; in one group, l-NAME was removed to allow reactivation of endogenous NO synthesis; in the two other groups, l-NAME removal was combined with EGF or angiotensin receptor type 1 (AT₁) antagonism. l-NAME removal partially reduced mean arterial pressure and proteinuria and increased renal blood flow (RBF), but not microvascular hypertrophy. Progression of structural damage was stopped, but not reversed. The administration of an EGF receptor antagonist did not have an additional effect on lowering blood pressure or on renal inflammation but did normalize RBF and afferent arteriole hypertrophy; the administration of an AT₁ antagonist normalized all measured functional and structural parameters. Staining with a specific marker of endothelial integrity indicated loss of functional endothelial cells in the l-NAME removal group; in contrast, in the animals treated with an EGF or AT₁ receptor antagonist, functional endothelial cells reappeared at levels equal to control animals. In addition, afferent arterioles freshly isolated from the l-NAME removal group showed an exaggerated constrictor response to endothelin; this response was blunted in the vessels isolated from the EGF or AT₁ receptor antagonist groups. The EGF receptor is an important mediator of endothelial dysfunction and contributes to the decline of RBF in the chronic kidney disease induced by NO deficiency. The EGF receptor antagonist-induced improvement of RBF is important but not sufficient for a complete reversal of renal disease, because it has little effect on renal inflammation. To achieve full recovery, it is necessary to apply AT₁ receptor antagonism.

Colchicine attenuates inflammatory cell infiltration and extracellular matrix accumulation in diabetic nephropathy

2009-06-24

Recent studies have demonstrated that an inflammatory mechanism contributes to the pathogenesis of diabetic nephropathy (DN). It is also known that colchicine (Col) can prevent various renal injuries via its anti-inflammatory action. However, the effect of colchicine on DN has never been explored. This study was undertaken to elucidate the effect of colchicine on inflammation and extracellular matrix accumulation in DN. In vivo, 64 rats were injected with diluent (C; n = 32) or streptozotocin intraperitoneally (DM, n = 32). Sixteen rats from each group were treated with Col. In vitro, rat mesangial cells and NRK-52E cells were cultured in media with 5.6 mM glucose (NG) or 30 mM glucose (HG) with or without 10^–8 M Col. Monocyte chemotactic protein-1 (MCP-1) mRNA expression was determined by real-time PCR (RT-PCR), and the levels of MCP-1 in renal tissue and culture media were measured by ELISA. RT-PCR and Western blotting were also performed for intercellular adhesion molecule-1 (ICAM-1) and fibronectin (FN) mRNA and protein expression, respectively, and immunohistochemical staining (IHC) for ICAM-1, FN, and ED-1 with renal tissue. Twenty-four-hour urinary albumin excretion at 6 wk and 3 mo were significantly higher in DM compared with C rats (P < 0.05), and colchicine treatment significantly reduced albuminuria in DM rats (P < 0.05). Col significantly inhibited the increase in MCP-1 mRNA expression and protein levels under diabetic conditions both in vivo and in vitro. ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. IHC revealed that the number of ED-1(+) cells were significantly higher in DM compared with C kidney (P < 0.005), and this increase was significantly attenuated by Col treatment (P < 0.01). In conclusion, Col prevents not only inflammatory cell infiltration via inhibition of enhanced MCP-1 and ICAM-1 expression but also ECM accumulation in DN. These findings provide a new perspective on the renoprotective effects of Col in DN.

{beta}1-Integrin is required for kidney collecting duct morphogenesis and maintenance of renal function

2009-06-24

Deletion of integrin-β1 (Itgb1) in the kidney collecting system led to progressive renal dysfunction and polyuria. The defect in the concentrating ability of the kidney was concomitant with decreased medullary collecting duct expression of aquaporin-2 and arginine vasopressin receptor 2, while histological examination revealed hypoplastic renal medullary collecting ducts characterized by increased apoptosis, ectasia and cyst formation. In addition, a range of defects from small kidneys with cysts and dilated tubules to bilateral renal agenesis was observed. This was likely due to altered growth and branching morphogenesis of the ureteric bud (the progenitor tissue of the renal collecting system), despite the apparent ability of the ureteric bud-derived cells to induce differentiation of the metanephric mesenchyme. These data not only support a role for Itgb1 in the development of the renal collecting system but also raise the possibility that Itgb1 links morphogenesis to terminal differentiation and ultimately collecting duct function and/or maintenance.

The lymphocyte migration inhibitor FTY720 attenuates experimental hypertensive nephropathy

2009-06-24

The lymphocyte migration inhibitor FTY720 attenuates experimental hypertensive nephropathy. Infiltration with lymphocytes is found in both immune and nonimmune chronic kidney diseases. In a rat model of immune-initiated progressive glomerulosclerosis, selective inhibition of lymphocyte infiltration by FTY720 showed significant beneficial effects on renal fibrosis. To test whether this translates into hypertensive nephropathy (HN), the lymphocyte migration inhibitor was administered to rats following nephrectomy. Two days after surgery, male Wistar rats were allocated to the following groups: Sham surgery, nephrectomy (HN), and HN + FTY720 (0.3 mg/kg body wt). Therapy was continued for 6 wk. Treatment with FTY720 was found to selectively reduce blood lymphocyte counts by 85% (P < 0.001 vs. HN) and renal lymphocyte infiltration (CD-3 positive cells) by 63% (P < 0.01 vs. HN) as was anticipated. Lymphocyte depletion went along with a significant reduction in proteinuria (–28%), whereas hypertensive systemic blood pressure remained unchanged (160 ± 5 vs. 161 ± 5 mmHg, P = not significant). The markedly increased histological tubulointerstitial and glomerular matrix protein accumulation, collagen, laminin, and fibronectin deposition were all significantly impeded in the FTY720-treated animals. The anti-fibrotic effects of FTY720 were paralleled by significant reductions in renal transforming growth factor (TGF)-β overexpression, macrophage infiltration, and cell proliferation. In conclusion, the lymphocyte migration inhibitor FTY720 significantly limits histological and molecular fibrosis in a model of hypertensive nephropathy without affecting increased systemic blood pressure. Prevention of renal lymphocytes' infiltration by FTY720 was followed by significant reductions in TGF-β overexpression, macrophage infiltration, and renal cell proliferation. These results suggest that infiltrating lymphocytes play an active, profibrotic role in the progression of hypertensive renal tissue injury.

Novel use of ultrasound to examine regional blood flow in the mouse kidney

2009-06-24

Conventional methods used for measuring regional renal blood flow, such as laser-Doppler flowmetry, are highly invasive, and each measurement is restricted to a discrete location. The aim of this study was to determine whether ultrasound imaging in conjunction with enhanced contrast agent (microbubbles; Vevo MicroMarker, VisualSonics) could provide a viable noninvasive alternative. This was achieved by determining changes in renal cortical and medullary rate of perfusion in response to a bolus injection of endothelin-1 (ET-1; 0.6, 1.0, or 2.0 nmol/kg) and comparing these responses to those observed in separate groups of mice with conventional laser-Doppler methods. Intravenous infusion of ET-1 in anesthetized male C57bl/6 mice resulted in a dose-dependent increase in mean arterial pressure and a dose-dependent decrease in total renal blood flow as measured by pulse-wave Doppler. ET-1 infusion resulted in a dose-dependent decrease in regional kidney perfusion as measured by both ultrasound with enhanced contrast agent and laser-Doppler measurements, verifying the use of ultrasound to measure regional kidney perfusion. Noted limitations of ultrasound imaging compared with laser-Doppler flowmetry included a lower degree of sensitivity to changes in tissue perfusion and the inability to assess rapid or transient changes in tissue perfusion. In conclusion, ultrasound represents an effective and noninvasive method for the measurement of relatively short-term, steady-state changes in regional blood flow in the mouse kidney.