Nicholas Obermüller

Transport of Monoamine Transmitters by the Organic Cation Transporter Type 2, OCT2

The recently cloned apical renal transport system for organic cations (OCT2) exists in dopamine-rich tissues such as kidney and some brain areas (Gründemann, D., Babin-Ebell, J., Martel, F., Örding, N., Schmidt, A., and Schömig, E. (1997)J. Biol. Chem. 272, 10408–10413). The study at hand was performed to answer the question of whether OCT2 accepts dopamine and other monoamine transmitters as substrate. 293 cells were stably transfected with the OCT2r cDNA resulting in the 293OCT2r cell line. Expression of OCT2r in 293 cells induces specific transport of tritiated dopamine, noradrenaline, adrenaline, and 5-hydroxytryptamine (5-HT). Initial rates of specific3H-dopamine, 3H-noradrenaline,3H-adrenaline, and 3H-5-HT transport were saturable, the K m values being 2.1, 4.4, 1.9, and 3.6 mmol/liter. The corresponding V max values were 3.9, 1.0, 0.59, and 2.5 nmol min−1·mg of protein−1, respectively. 1,1′-diisopropyl-2,4′-cyanine (disprocynium24), a known inhibitor of OCT2 with a potent eukaliuric diuretic activity, inhibited 3H-dopamine uptake into 293OCT2r cells with an K i of 5.1 (2.6, 9.9) nmol/liter. In situ hybridization reveals that, within the kidney, the OCT2r mRNA is restricted to the outer medulla and deep portions of the medullary rays indicating selective expression in the S3 segment of the proximal tubule. These findings open the possibility that OCT2r plays a role in renal dopamine handling.

Distribution of angiotensin II receptor subtypes in rat brain nuclei

Angiotensin II (ANG II) receptor subtypes in rat brain were characterized and quantified by competitive radioligand binding using [125I]Sar1 Ile8 angiotensin II ([125I]sarilesin) as a tracer and ANG II, sarilesin and the subtype selective ligands DuP 753 (AT1) and CGP 42112A (AT2) as competitors. The distribution of AT1 and AT2 receptors was determined in midbrain, brainstem, hypothalamus as well as in individual hypothalamic and periventricular nuclei. Whereas in midbrain and brainstem the AT1: AT2 ratio was 40%: 60% and 70%: 30% respectively, the AT1 receptors were by far predominant in hypothalamus and in the nuclei investigated. Interestingly, we found that approximately 25% of the ANG II receptors in hypothalamus did not bind DuP 753 even at 0.1 mM. These sites which bind CGP 42112A, ANG II and sarilesin may represent a third ANG II receptor subtype.

Expression of the Na-K-2Cl Cotransporter by Macula Densa and Thick Ascending Limb Cells of Rat and Rabbit Nephron

Sodium and chloride transport by the macula densa and thick ascending limb of Henles loop participates importantly in extracellular fluid volume homeostasis, urinary concentration and dilution, control of glomerular filtration, and control of renal hemodynamics. Transepithelial Na and Cl transport across the apical membrane of thick ascending limb (TALH) cells is mediated predominantly by a loop diuretic sensitive Na-K-2Cl cotransport pathway. The corresponding transport protein has recently been cloned. Functional studies suggest that the cotransporter is expressed by macula densa cells as well as by TALH cells. The current studies were designed to identify sites of Na-K-2Cl cotransporter expression along distal nephron in rabbit and rat. Non-isotopic high-resolution in situ hybridization, using an antisense probe for the apical form of the Na-K-2Cl cotransporter identified expression throughout the TALH, from the junction between inner and outer medulla to the transition to distal convoluted tubule. Expression by macula densa cells was confirmed by colocalization using markers specific for macula densa cells. First, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that did not stain with anti-Tamm-Horsfall protein antibodies. Second, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that show positive NADPH-diaphorase reaction, indicating high levels of constitutive nitric oxide synthase activity. In rat, levels of Na-K-2Cl cotransporter mRNA expression were similar in TALH and macula densa cells. In rabbit, expression levels were higher in macula densa cells than in surrounding TALH cells. The present data provide morphological support for a previously established functional concept that Na-K-2Cl cotransport at the TALH is accomplished by the expression of a well-defined cotransporter. At the macula densa, this transporter may establish a crucial link between tubular salt load and glomerular vascular regulation.

The swelling-activated chloride channel ClC-2, the chloride channel ClC-3, and ClC-5, a chloride channel mutated in kidney stone disease, are expressed in distinct subpopulations of renal epithelial cells.

The mammalian genome encodes at least nine different members of the ClC family of chloride channels. So far only two of them could be localized on a cellular level in the kidney. We now report on the precise intrarenal localization of the mRNAs coding for the chloride channels ClC-2, ClC-3 and ClC-5. Expression of ClC-2 mRNA, encoding a swelling-activated chloride channel, could be demonstrated in the S3 segment of the proximal tubule. The chloride channel ClC-3 mRNA and ClC-5 mRNA, coding for a chloride channel mutated in kidney stone disease, were both expressed in intercalated cells of the connecting tubule and collecting duct. Whereas ClC-3 mRNA expression was most prominent in the cortex of rat kidneys, ClC-5 mRNA was expressed from the cortex through the upper portion of the inner medulla. A detailed analysis revealed that ClC-3 was expressed by type B intercalated cells, whereas ClC-5 was expressed by type A intercalated cells. These findings have important implications for the pathogenesis of hereditary kidney stone disease caused by mutations in the CLCN5 gene.

Cerebral localization and regulation of the cell volume-sensitive serum- and glucocorticoid-dependent kinase SGK1

The serum- and glucocorticoid-dependent kinase SGK1 is regulated by alterations of cell volume, whereby cell shrinkage increases and cell swelling decreases the transcription, expression and activity of SGK1. The kinase is expressed in all human tissues studied including the brain. The present study was performed to localize the sites of SGK1 transcription in the brain, to elucidate the influence of the hydration status on SGK1 transcription and to explore the functional significance of altered SGK1 expression. Northern blot analysis of human brain showed SGK1 to be expressed in all cerebral structures examined: amygdala, caudate nucleus, corpus callosum, hippocampus, substantia nigra, subthalamic nucleus and thalamus. In situ hybridization and immunohistochemistry in the rat revealed increased expression of SGK1 in neurons of the hippocampal area CA3 after dehydration, compared with similar slices from brains of euvolaemic rats. Additionally, several oligodendrocytes, a few microglial cells, but no astrocytes, were positive for SGK1. The abundance of SGK1 mRNA in the temporal lobe, including hippocampus, was increased by dehydration and SGK1 transcription in neuroblastoma cells was stimulated by an increase of extracellular osmolarity. Co-expression studies in Xenopus laevis oocytes revealed that SGK1 markedly increased the activity of the neuronal K+ channel Kv1.3. As activation of K+ channels modifies excitation of neuronal cells, SGK1 may participate in the regulation of neuronal excitability.

Missense mutation in sterile alpha motif of novel protein SamCystin is associated with polycystic kidney disease in (cy/+)rat

Autosomal dominant polycystic kidney disease (PKD) is the most common genetic disease that leads to kidney failure in humans. In addition to the known causative genes PKD1 and PKD2, there are mutations that result in cystic changes in the kidney, such as nephronophthisis, autosomal recessive polycystic kidney disease, or medullary cystic kidney disease. Recent efforts to improve the understanding of renal cystogenesis have been greatly enhanced by studies in rodent models of PKD. Genetic studies in the (cy/+) rat showed that PKD spontaneously develops as a consequence of a mutation in a gene different from the rat orthologs of PKD1 and PKD2 or other genes that are known to be involved in human cystic kidney diseases. This article reports the positional cloning and mutation analysis of the rat PKD gene, which revealed a C to T transition that replaces an arginine by a tryptophan at amino acid 823 in the protein sequence. It was determined that Pkdr1 is specifically expressed in renal proximal tubules and encodes a novel protein, SamCystin, that contains ankyrin repeats and a sterile alpha motif. The characterization of this protein, which does not share structural homologies with known polycystins, may give new insights into the pathophysiology of renal cyst development in patients.

Losartan and Angiotensin II Inhibit Aldosterone Production in Anephric Rats via Different Actions on the Intraadrenal Renin-Angiotensin System.

Angiotensin II (ANG II) is a major stimulator of aldosterone biosynthesis. When investigating the relative contribution of circulating and locally produced ANG II, we were therefore surprised to find that ANG II, given chronically sc (200 ng/kg·min), markedly inhibits a nephrectomy (NX)-induced rise of aldosterone concentrations (from 10 ± 2 to 465 ± 90 ng/100 ml in vehicle infused, and from 9 ± 2 to 177 ± 35 in ANG II infused rats 55 h after NX and hemodialysis). We further observed, by in situ hybridization, that bilateral NX increases the number of adrenocortical cells expressing renin and that this rise was prevented by ANG II. Moreover, the rise of aldosterone levels was also inhibited by the AT1-receptor antagonist, losartan (10 μg/kg·min, chronically ip from 8 ± 2 to 199 ± 26 ng/100 ml), despite the absence of circulating renin and a reduction of ANG I to less than 10%. These data demonstrate that aldosterone production, after NX, is regulated by an intraadrenal renin-angiotensin system and that th...

The rat Pkd2 protein assumes distinct subcellular distributions in different organs

Mutations in the PKD2 gene account for ∼15% of all cases of autosomal-dominant polycystic kidney disease. In the present study the cellular distribution of the Pkd2 protein was investigated by immunohistochemistry in different rat organs. Although the Pkd2 protein showed a widespread expression, a strikingly different distribution of the protein was observed between individual organs. Whereas in renal distal tubules and in striated ducts of salivary glands a basal-to-basolateral distribution of Pkd2 was found, a punctate cytoplasmic location was detected in the adrenal gland, ovary, cornea, and smooth muscle cells of blood vessels. Interestingly, in the adrenal gland and ovary, the rat Pkd2 protein was more heavily N-glycosylated than in the kidney and salivary gland. These results suggest that Pkd2 accomplishes its functions by interacting with proteins located in different cellular compartments. The extrarenal expression pattern of the Pkd2 protein hints at other candidate sites of disease manifestations in patients carrying PKD2 mutations.

Specific Regulation of StAR Expression in the Rat Adrenal Zona Glomerulosa: an In Situ Hybridization Study

Steroid acute regulatory protein (StAR) plays an essential role in steroidogenesis because it is responsible for the transfer of cholesterol from cellular stores to the inner mitochondrial membrane. We investigated the distribution and regulation of StAR expression in association with aldosterone production in the rat adrenal gland in vivo. Using nonradioactive in situ hybridization, we demonstrate that the outermost five to seven parenchymal cell layers express the StAR gene only weakly and inhomogeneously. The strongest expression is found in the zona fasciculata and zona reticularis. In addition, some cells in the adrenal medulla also stained positively. To differentiate between functionally active glomerulosa and inactive intermediate cells, we compared the expression pattern of StAR with that of aldosterone synthase. The expression of the latter is localized to two or three cell layers only, located immediately below the capsule. However, the cells of the intermedia are capable of expressing both genes prominently, as shown after stimulation with bilateral nephrectomy for 2 days. All zones of the adrenal cortex by then expressed StAR gene to the same extent. This was accompanied by a 50-fold elevated plasma aldosterone concentration. Our data demonstrate that the width of the aldosterone-producing zone can increase within a short period of time by recruiting hormonally inactive cells to steroidogenesis.

Transgenic Overexpression of Anks6(p.R823W) Causes Polycystic Kidney Disease in Rats

The PKD/Mhm(cy/+) rat is a widely used animal model for the study of human autosomal dominant polycystic kidney disease, one of the most common genetic disorders, affecting one in 1000 individuals. We identified a new gene, Anks6, which is mutated (Anks6((p.R823W))) in PKD/Mhm(cy/+) rats. The evidence for a causal link between Anks6((p.R823W)) and cystogenesis is still lacking, and the function of Anks6 is presently unknown. This study presents a novel transgenic rat model that overexpresses the mutated 2.8-kb Anks6((p.R823W)) cDNA in the renal tubular epithelium. The transgenic Anks6((p.R823W)) acts in a dominant-negative fashion and causes a predictable polycystic phenotype that largely mimics the general characteristics of the PKD/Mhm(cy/+) rats. Cyst development is accompanied by enhanced c-myc expression and continuous proliferation, apoptosis, and de-differentiation of the renal tubular epithelium as well as by a lack of translational up-regulation of p21 during aging. Using Northern blot analysis and in situ hybridization studies, we identified the first 10 days of age as the period during which transgene expression precedes and initiates cystic growth. Thus, we not only provide the first in vivo evidence for a causal link between the novel Anks6((p.R823W)) gene mutation and polycystic kidney disease, but we also developed a new transgenic rat model that will serve as an important resource for further exploration of the still unknown function of Anks6.