Tumor necrosis factor-alpha is an endogenous inhibitor of Na+-K+-2Cl- cotransporter (NKCC2) isoform A in the thick ascending limb.

The effects of TNF gene deletion on renal Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) expression and activity were determined. Outer medulla from TNF(-/-) mice exhibited a twofold increase in total NKCC2 protein expression compared with wild-type (WT) mice. This increase was not observed in TNF(-/-) mice treated with recombinant human TNF (hTNF) for 7 days. Administration of hTNF had no effect on total NKCC2 expression in WT mice. A fourfold increase in NKCC2A mRNA accumulation was observed in outer medulla from TNF(-/-) compared with WT mice; NKCC2F and NKCC2B mRNA accumulation was similar between genotypes. The increase in NKCC2A mRNA accumulation was attenuated when TNF(-/-) mice were treated with hTNF. Bumetanide-sensitive O(2) consumption, an in vitro correlate of NKCC2 activity, was 2.8 ± 0.2 nmol·min(-1)·mg(-1) in medullary thick ascending limb tubules from WT, representing ∼40% of total O(2) consumption, whereas, in medullary thick ascending limb tubules from TNF(-/-) mice, it was 5.6 ± 0.3 nmol·min(-1)·mg(-1), representing ∼60% of total O(2) consumption. Administration of hTNF to TNF(-/-) mice restored the bumetanide-sensitive component to ∼30% of total O(2) consumption. Ambient urine osmolality was higher in TNF(-/-) compared with WT mice (2,072 ± 104 vs. 1,696 ± 153 mosmol/kgH(2)O, P < 0.05). The diluting ability of the kidney, assessed by measuring urine osmolality before and after 1 h of water loading also was greater in TNF(-/-) compared with WT mice (174 ± 38 and 465 ± 81 mosmol/kgH(2)O, respectively, P < 0.01). Collectively, these findings suggest that TNF plays a role as an endogenous inhibitor of NKCC2 expression and function.

A cell-based screen for inhibitors of flagella-driven motility in Chlamydomonas reveals a novel modulator of ciliary length and retrograde actin flow.

Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical-biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time-scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose-dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound "ciliabrevin" due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose-dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug.

Differential regulation of the bumetanide-sensitive cotransporter (NKCC2) by ovarian hormones.

The Na-K-2Cl cotransporter (NKCC2) regulates sodium transport along the thick ascending limb of Henle's loop and is important in control of sodium balance, renal concentrating ability and renin release. To determine if there are sex differences in NKCC2 abundance and/or distribution, and to evaluate the contribution of ovarian hormones to any such differences, we performed semiquantitative immunoblotting and immunoperoxidase immunohistochemistry for NKCC2 in the kidney of Sprague Dawley male, female and ovariectomized (OVX) rats with and without 17-beta estradiol or progesterone supplementation. Intact females demonstrated greater NKCC2 protein in homogenates of whole kidney (334+/-29%), cortex (219+/-20%) and outer medulla (133+/-9%) compared to males. Ovarian hormone supplementation to OVX rats regulated NKCC2 in the outer medulla only, with NKCC2 protein abundance decreasing slightly in response to progesterone but increasing in response to 17-beta estradiol. Immunohistochemistry demonstrated prominent NKCC2 labeling in the apical membrane of thick ascending limb cells. Kidney section NKCC2 labeling confirmed regionalized regulation of NKCC2 by ovarian hormones. Localized regulation of NKCC2 by ovarian hormones may have importance in controlling sodium and water balance over the lifetime of women as the milieu of sex hormones varies.

Vasopressin V2 receptor expression along rat, mouse, and human renal epithelia with focus on TAL.

In renal epithelia, vasopressin influences salt and water transport, chiefly via vasopressin V(2) receptors (V(2)Rs) linked to adenylyl cyclase. A combination of vasopressin-induced effects along several distinct portions of the nephron and collecting duct system may help balance the net effects of antidiuresis in cortex and medulla. Previous studies of the intrarenal distribution of V(2)Rs have been inconclusive with respect to segment- and cell-type-related V(2)R expression. Our study therefore aimed to present a high-resolution analysis of V(2)R mRNA expression in rat, mouse, and human kidney epithelia, supplemented with immunohistochemical data. Cell types of the renal tubule were identified histochemically using specific markers. Pronounced V(2)R signal in thick ascending limb (TAL) was corroborated functionally; phosphorylation of Na(+)-K(+)-2Cl(-) cotransporter type 2 (NKCC2) was established in cultured TAL cells from rabbit and in rats with diabetes insipidus that were treated with the V(2)R agonist desmopressin. We found solid expression of V(2)R mRNA in medullary TAL (MTAL), macula densa, connecting tubule, and cortical and medullary collecting duct and weaker expression in cortical TAL and distal convoluted tubule in all three species. Additional V(2)R immunostaining of kidneys and rabbit TAL cells confirmed our findings. In agreement with strong V(2)R expression in MTAL, kidneys from rats with diabetes insipidus and cultured TAL cells revealed sharp, selective increases in NKCC2 phosphorylation upon desmopressin treatment. Macula densa cells constitutively showed strong NKCC2 phosphorylation. Results suggest comparably significant effects of vasopressin-induced V(2)R signaling in MTAL and in connecting tubule/collecting duct principal cells across the three species. Strong V(2)R expression in macula densa may be related to tubulovascular signal transfer.

Lithium treatment inhibits renal GSK-3 activity and promotes cyclooxygenase 2-dependent polyuria.

The use of LiCl in clinical psychiatry is routinely complicated by overt nephrogenic diabetes insipidus (NDI), the mechanism of which is incompletely understood. In vitro studies indicate that lithium can induce renal medullary interstitial cell cyclooxygenase 2 (COX2) protein expression via inhibition of glycogen synthase kinase-3beta (GSK-3beta). Both COX1 and COX2 are expressed in the kidney. Renal prostaglandins have been suggested to play an important role in lithium-induced polyuria. The present studies examined whether induction of the COX2 isoform contributes to LiCl-induced polyuria. Four days after initiation of lithium treatment in C57 BL/6J mice, urine volume increased in LiCl-treated mice by fourfold compared with controls (P < 0.0001) and was accompanied by decreased urine osmolality. This was temporally associated with increased renal COX2 protein expression and increased urinary PGE(2) excretion, whereas COX1 levels remained unchanged. COX2 inhibition significantly blunted lithium-induced polyuria (P < 0.0001) and reduced urinary PGE(2) levels. Lithium-associated polyuria was also seen in COX1-/- mice and was associated with increased urinary PGE(2). COX2 inhibition completely prevented polyuria and PGE(2) excretion in COX1-/- mice, suggesting that COX2, but not COX1, plays a critical role in lithium-induced polyuria. Lithium also induced renal medullary COX2 protein expression in congenitally polyuric antidiuretic hormone (AHD)-deficient rats, demonstrating that lithium-induced COX2 protein expression is not secondary to altered ADH levels or polyuria. Lithium also decreased renal medullary GSK-3beta activity, and this was temporally related to increased COX2 expression in the kidney from lithium-treated mice, consistent with a tonic in vivo suppression of COX2 expression by GSK-3 activity. In conclusion, these findings temporally link decreased GSK-3 activity to enhanced renal COX2 expression and COX2-derived urine PGE(2) excretion. Suppression of COX2-derived PGE(2) blunts lithium-associated polyuria.

Membrane-associated PGE synthase-1 (mPGES-1) is coexpressed with both COX-1 and COX-2 in the kidney.

BACKGROUND: Prostaglandin E2 (PGE2) plays an important role in many physiologic and pathophysiologic processes in the kidney. Multiple enzymes are involved in PGE2 biosynthesis, including phospholipases, cyclooxygenases (COX), and the PGE2 synthases (PGES). The present studies were aimed at determining the intrarenal localization of mPGES-1 and whether it is coexpressed with COX-1 or COX-2. METHODS: Rabbit mPGES-1 and COX-1 cDNAs were cloned using reverse transcription-polymerase chain reaction (RT-PCR) and screening a cDNA library. RNase protection assay and immunoblotting were used to examine mPGES-1 expression levels. In situ hybridization and immunostaining were used to determine the intrarenal localization of mPGES-1 and cyclooxygenases. RESULTS: Rabbit mPGES-1 shares high sequence similarity to the human homolog. Nuclease protection studies showed that the kidney expresses among the highest level of mPGES-1 of any rabbit tissue. In situ hybridization showed COX-1 and mPGES-1 mRNA was highly expressed in renal medullary collecting ducts (MCD), and to a lesser extent in cortical collecting ducts (CCD). Fainter mPGES-1 expression was also observed in macula densa (MD) and medullary interstitial cells (RMICs), where COX-2 is highly expressed. Double-labeling studies (immunostaining plus in situ hybridization) and immunohistochemistry of mouse tissues confirmed that mPGES-1 predominantly colocalizes with COX-1 in distal convoluted tubule (DCT), CCD, and MCD, and is coexpressed with COX-2 at lower levels in MD and RMICs. CONCLUSION: Together, these studies suggest mPGES-1 colocalizes with both COX-1 and COX-2 to mediate the biosynthesis of PGE2 in the kidney.

Effects of oxalate on IMCD cells: a line of mouse inner medullary collecting duct cells.

Oxalate, a metabolic end product and a major constituent of the majority of renal stones, has been shown to be toxic to renal epithelial cells of cortical origin. However, it is unknown whether inner medullary collecting duct (IMCD) cells that are physiologically exposed to higher concentrations of oxalate also behave in a similar manner. In the present study, we examined the effects of oxalate on IMCD cells. IMCD cells from the mouse were maintained in DMEM/F12 media supplemented with fetal bovine serum and antibiotics. Exposure of IMCD cells to oxalate produced time- and concentration-dependent changes in the light microscopic appearance of the cells. Long-term exposure to oxalate resulted in alterations in cell viability, with net cell loss after exposure to concentrations of 2 mM or greater. The production of free radicals was directly related to the exposure time and the concentration of oxalate. Crystal formation occurred in less than 1 h and cells in proximity to crystals would lose membrane integrity. Compared with IMCD cells, LLC-PK1 cells as well as HK-2 cells showed significant toxicity starting at lower oxalate concentrations (0.4 mM or greater). These results provide the first direct demonstration of toxic effects of oxalate in IMCD cells, a line of renal epithelial cells of the inner medullary collecting duct, and suggest that the cells lining the collecting duct are relatively resistant to oxalate toxicity.

Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells.

The present study hypothesized that superoxide (O2(-)*) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1alpha expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O2(-)* generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl(2)-induced increase in HIF-1alpha levels and completely blocked the increase in HIF-1alpha levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O2(-)* dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1alpha levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1alpha levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22(phox), into RMICs markedly increased HIF-1alpha levels. In contrast, the OH* scavenger tetramethylthiourea had no effect on the accumulation of HIF-1alpha in these renal cells. By Northern blot analysis, scavenging or dismutation of O2(-)* by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1alpha-targeted gene, heme oxygenase-1. These results indicate that increased intracellular O2(-)* levels induce HIF-1alpha degradation independently of H(2)O(2) and OH* radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1alpha in these cells under physiological conditions.

Bradykinin regulation of salt transport across mouse inner medullary collecting duct epithelium involves activation of a Ca(2+)-dependent Cl(-) conductance.

The mechanism by which bradykinin regulates renal epithelial salt transport has been investigated using a mouse inner medullary renal collecting duct cell-line mIMCD-K2. Using fura-2 loaded mIMCD-K2 cells bradykinin (100 nM) has been shown to induce a transient increase in intracellular Ca(2+) via activation of bradykinin B2 receptors localized to both the apical and basolateral epithelial cell surfaces. In mIMCD-K2 epithelial cell-layers clamped in Ussing chambers, 100 nM bradykinin via apical and basolateral bradykinin B2 receptors stimulated a transient increase in inward short-circuit current (I:(sc)) of similar duration to the increase in intracellular Ca(2+). Replacements of the bathing solution Na(+) by the impermeant cation, N-methyl-D-glucamine and of Cl(-) and HCO(3)(-) by the impermeant anion gluconate at either the apical (no reduction) or basal bathing solutions (abolition of the response) are consistent with the bradykinin-stimulated increase in inward I:(sc) resulting from basal to apical Cl(-) (anion) secretion. Using the slow whole cell configuration of the patch-clamp technique, bradykinin was shown to activate a transient Cl(-) selective whole cell current which showed time-dependent activation at positive membrane potentials and time-dependent inactivation at negative membrane potentials. These currents were distinct from those activated by forskolin (CFTR), but identical to those activated by exogenous ATP and are therefore consistent with bradykinin activation of a Ca(2+)-dependent Cl(-) conductance. The molecular identity of the Ca(2+)-dependent Cl(-) conductance has been investigated by an RT - PCR approach. Expression of an mRNA transcript with 96% identity to mCLCA1/2 was confirmed, however an additional but distinct mRNA transcript with only 81% of the identity to mCLCA1/2 was identified.

Immunolocalization of the secretory isoform of Na-K-Cl cotransporter in rat renal intercalated cells.

Two bumetanide-sensitive ion cotransporters that carry Na+, K+, and Cl- in a coupled fashion have been identified. One type, the "absorptive" isoform, carries these ions across the apical plasma membrane of the thick ascending limb of Henle's loop. Another isoform, the "secretory" cotransporter, has been identified in a number of epithelial tissues by physiological means, but its sites of expression in the kidney have not been fully characterized. Complementary DNA believed to code for the secretory isoform (called "BSC2" or "NKCC1") have recently been cloned. This study used a specific affinity-purified antipeptide antibody to this protein for immunolocalization in the rat kidney. Immunoblot studies using this antibody show abundant immunoreactivity against bands of 140-190 and 120 kd in the parotid gland, colon, and stomach, sites where the secretory form of the cotransporter has been identified by physiological techniques. This distribution supports the hypothesis that this isoform represents the secretory form of the cotransporter. Studies in the kidney revealed that the same bands are associated with membrane fractions chiefly in the outer medulla. Immunolocalizations show that immunoreactivity is selectively and intensely localized to the basolateral plasma membrane of a subfraction of outer medullary collecting duct cells. An independently produced monoclonal antibody (T4) specific for Na-K-Cl cotransporter displays the same localization. Dual localizations of cotransporter antibody with respect to antibody specific for principal cells (aquaporin-2) and intercalated cells (band 3 and H(+)-ATPase) show that cotransporter immunoreactivity is localized to alpha-intercalated cells of the outer medullary collecting duct in the rat. This distinctive localization suggests that the secretory form of the cotransporter may play a role in renal NH4+ and/or acid secretion by this cell type.

Immunocytochemical localization of stanniocalcin cells in the rat kidney.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fishes, where it functions as a regulator of calcium and phosphate homoeostasis. Recently, complementary DNAs encoding human STC (hSTC) have been characterized, and recombinant hSTC has been synthesized in a bacterial expression system. In preliminary studies, STC-immunoreactive cells have already been identified in human kidney tubules with antibodies to recombinant hSTC. The purpose of this study was to map the overall spatial distribution of STC cells in mammalian kidney, using the rat as a model system. Immunocytochemistry was performed on fixed sections of rat kidney tissue using hSTC antiserum in conjunction with fluorescein isothiocyanate-conjugated second antibodies. STC-immunoreactive cells were found in cortical thick ascending limb, in macula densa, in distal convoluted tubules, and in the cortical and medullary collecting ducts. All cortical thick ascending limb cells contained immunoreactive STC. Most distal convoluted tubules cells contained STC, and these were identified as principal cells. The distribution of STC cells in cortical and medullary collecting ducts also corresponded closely to the known frequently of principle cells in these segments, suggesting that principal cells are the site of STC storage and/or synthesis in both distal convoluted tubules and collecting ducts. Some collecting duct intercalated cells contained STC as well, and these were tentatively identified as alpha-type intercalated cells. As all tubular segments containing STC are known to be involved in regulated ion transport, renally derived STC may be acting in an autocrine, paracrine and/or endocrine fashion to regulate one or more of these transport processes.

Betaine transporter cDNA cloning and effect of osmolytes on its mRNA induction.

Cells generally adapt to long-term hyperosmolality by accumulating compatible organic osmolytes, thereby helping to normalize both volume and intracellular inorganic ion concentration. When organic osmolytes are accumulated, as in renal inner medullary cells, it is the sum of their concentrations that is theoretically important. In effect, when one organic osmolyte rises, the others generally fall to maintain their sum approximately constant. The present study addresses the mechanism controlling betaine accumulation. Hypertonicity induces accumulation of betaine, sorbitol, inositol, and other organic osmolytes in PAP-HT25 cells, a line derived from rabbit renal papilla. Hypertonicity increases the betaine transporter expression in these cells. To obtain a specific probe for betaine transporter mRNA, we cloned from PAP-HT25 cells a cDNA that encodes the full protein. We then examined the effect of betaine, sorbitol, and inositol on betaine transporter mRNA abundance. Increased accumulation of any of these three organic osmolytes reduces betaine transporter mRNA. We previously observed similar results for aldose reductase, the enzyme responsible for osmotically regulated sorbitol accumulation. We conclude that the accumulation of organic osmolytes regulates betaine transporter gene expression. Because the aldose reductase gene is controlled in a similar fashion, we surmise that the two genes share a common signal for induction.

Osmolytes.

The cells of the renal medulla osmotically adapt to chronic alterations in extracellular tonicity by appropriate changes in the intracellular contents of organic osmoeffectors. The major organic osmolytes are glycerophosphorylcholine, betaine, myo-inositol, sorbitol, and, possibly, taurine. When the concentrations of poorly permeant external solutes are acutely reduced, cells that have been adapted to high tonicities rapidly release organic osmolytes via specific transmembrane transport pathways. In contrast, when medullary cells are depleted of organic osmolytes, osmolyte accumulation on acute elevation of external tonicity is slow and involves stimulation of uptake, intracellular de novo synthesis, or inhibition of intracellular degradation, and is preceded by increased intracellular electrolyte concentrations. The available evidence suggests that this rise in intracellular ionic strength plays an important role in the initiation of those processes responsible for full adaptation of renal medullary cells to high tonicities. Recently, complementary DNAs encoding a myo-inositol and a betaine transporter have been isolated.

Measurement of element content in isolated papillary collecting duct cells by electron probe microanalysis.

A method was developed to measure the element content of freshly isolated papillary collecting duct (PCD) cells by electron probe microanalysis in a scanning electron microscope. After isolation, the cells were transferred onto a Thermanox support by centrifugation and the extracellular medium was removed by brief exposure to buffered ammonium acetate; cryofixation, freeze-drying, and coating with carbon followed. Under visual control in the scanning electron microscope the Na, Cl, K and P content of cell clusters (about 30 cells/cluster) was then measured by X-ray microanalysis. Cells incubated in control medium showed potassium:sodium ratios identical to those determined previously in cryosections of the same cells. In ouabain-treated cells sodium influx and potassium efflux was demonstrated. Potassium left the cells with a t1/2 of 21.7 min. The t1/2 of Na influx was 12.6 min for the first 15 min of incubation, whereafter further influx was markedly slower. Ouabain-induced sodium influx was inhibited 40% by amiloride. These results indicate that X-ray microanalysis can be applied to analyze the ion content of isolated cell clusters derived from the papillary collecting duct. Using ouabain and amiloride as inhibitors the suitability of the method to identify transport systems is demonstrated.