Upregulation of Slc39a10 gene expression in response to thyroid hormones in intestine and kidney

A novel zinc transporter has been purified and cloned from rat renal brush border membrane. This transporter was designated as Zip10 encoded by Slc39a10 gene and characterized as zinc importer. Present study documents the impact of thyroid hormones on the expression of Zip10 encoded by Slc39a10 gene in rat model of hypo and hyperthyroidism. Serum T₃ and T₄ levels were reduced significantly in hypothyroid rats whereas these levels were significantly elevated in hyperthyroid rats as compared to euthyroid rats thereby confirming the validity of the model. Kinetic studies revealed a significant increase in the initial and equilibrium uptake of Zn⁺⁺ in both intestinal and renal BBMV of hyperthyroid rats in comparison to hypothyroid and euthyroid rats. By RT-PCR, Slc39a10 mRNA expression was found to be significantly decreased in hypothyroid and increased in hyperthyroid as compared to euthyroid rats. These findings are in conformity with the immunofluorescence studies that revealed markedly higher fluorescence intensity at periphery of both intestinal and renal cells isolated from hyperthyroid rats as compared to hypothyroid and euthyroid rats. Higher expression of Zip10 protein in hyperthroid group was also confirmed by western blot. These findings suggest that expression of zinc transporter protein Zip10 (Slc39a10) in intestine and kidney is positively regulated by thyroid hormones.     

The Mechanism of the Calorigenic Action of Thyroid Hormone : Stimulation of Na+ + K+-activated adenosinetriphosphatase activity

In an earlier study, we proposed that thyroid hormone stimulation of energy utilization by the Na⁺ pump mediates the calorigenic response. In this study, the effects of triiodothyronine (T₃) on total oxygen consumption (Q_OO2), the ouabain-sensitive oxygen consumption [Q_OO2(t)], and NaK-ATPase in liver, kidney, and cerebrum were measured. In liver, ~90% of the increase in Q_OO2 produced by T₃ in either thyroidectomized or euthyroid rats was attributable to the increase in Q_OO2(t). In kidney, the increase in Q_OO2(t) accounted for 29% of the increase in Q_OO2 in thyroidectomized and 46% of the increase in Q_OO2 in euthyroid rats. There was no demonstrable effect of T₃ in euthyroid rats on Q_OO2 or Q_OO2(t) of cerebral slices. The effects of T₃ on NaK-ATPase activity in homogenates were as follows: In liver +81% from euthyroid rats and +54% from hypothyroid rats. In kidney, +21% from euthyroid rats and +69% from hypothyroid rats. T₃ in euthyroid rats produced no significant changes in NaK-ATPase or Mg-ATPase activity of cerebral homogenates. Liver plasma membrane fractions showed a 69% increase in NaK-ATPase and no significant changes in either Mg-ATPase or 5'-nucleotidase activities after T₃ injection. These results indicate that thyroid hormones stimulate NaK-ATPase activity differentially. This effect may account, at least in part, for the calorigenic effects of these hormones.     

Long-Term Responses to Loss of Renal Mass: Control Mechanisms: The Role of Renal “Work” in Compensatory Kidney Growth

In a series of studies designed to test the role of renal “work” in compensatory kidney growth we examined the relationship between absolute sodium reabsorption—which constitutes the bulk of renal energy expenditure, and growth of the remaining kidney at various intervals after contralateral nephrectomy.The increase in weight of the remaining kidney preceded the rise in sodium reabsorption and these two processes took place at different rates between 24 hours and 21 days after uninephrectomy.Absolute sodium reabsorption did not change during the first hours after contralateral nephrectomy, at a time when biochemical alterations are known to occur.The rate of [¹⁴C] choline incorporation into renal phospholipid, an early biochemical indicator of compensatory kidney growth, increased significantly one hour after contralateral nephrectomy but remained unchanged after sham-nephrectomy, regardless of the magnitude or direction of the concomitant change in absolute sodium reabsorption (“kidney work”).These results indicate that renal work expended in the reabsorption of glomerular filtrate is neither the initiating, nor the primary controlling factor, of the compensatory kidney growth that follows unilateral nephrectomy.     

A Novel Transporter of SLC22 Family Specifically Transports Prostaglandins and Co-localizes with 15-Hydroxyprostaglandin Dehydrogenase in Renal Proximal Tubules

We identified a novel prostaglandin (PG)-specific organic anion transporter (OAT) in the OAT group of the SLC22 family. The transporter designated OAT-PG from mouse kidney exhibited Na⁺-independent and saturable transport of PGE₂ when expressed in a proximal tubule cell line (S₂). Unusual for OAT members, OAT-PG showed narrow substrate selectivity and high affinity for a specific subset of PGs, including PGE₂, PGF_2α, and PGD₂. Similar to PGE₂ receptor and PGT, a structurally distinct PG transporter, OAT-PG requires for its substrates an α-carboxyl group, with a double bond between C13 and C14 as well as a (S)-hydroxyl group at C15. Unlike the PGE₂ receptor, however, the hydroxyl group at C11 in a cyclopentane ring is not essential for OAT-PG substrates. Addition of a hydroxyl group at C19 or C20 impairs the interaction with OAT-PG, whereas an ethyl group at C20 enhances the interaction, suggesting the importance of hydrophobicity around the ω-tail tip forming a “hydrophobic core” accompanied by a negative charge, which is essential for substrates of OAT members. OAT-PG-mediated transport is concentrative in nature, although OAT-PG mediates both facilitative and exchange transport. OAT-PG is kidney-specific and localized on the basolateral membrane of proximal tubules where a PG-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase, is expressed. Because of the fact that 15-keto-PGE₂, the metabolite of PGE₂ produced by 15-hydroxyprostaglandin dehydrogenase, is not a substrate of OAT-PG, the transport-metabolism coupling would make unidirectional PGE₂ transport more efficient. By removing extracellular PGE₂, OAT-PG is proposed to be involved in the local PGE₂ clearance and metabolism for the inactivation of PG signals in the kidney cortex.     

Renal phosphoenolpyruvate carboxykinase turnover in hypo- and hyperthyroid rat in vivo

The effect of hypo- and hyperthyroidism on activity, synthesis and degradation of renal cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was studied in the rat by radioimmunological techniques. In hypo- and euthyroid rats, starvation induced similar alterations in enzyme activities and relative rates of synthesis, whereas in hyperthyroid rats the increase in both was significantly reduced. Substitution of l-thyroxine in hypothyroid rats resulted in a decrease in activity and synthesis within 18 h as observed in hyperthyroid animals. The apparent half-life of the enzyme measured by double-pulse labeling experiments was approx. 13 h in euthyroid animals. The rate of degradation was unaffected by the different thyroid states.     

Influence of thyroid on formation of myelin lipids

—The formation of lipids found primarily in the myelin sheath was investigated in rats made hypothyroid, at birth. A marked reduction in cerebroside, sulphatide, and cholesterol of brains was found in 18-day-old hypothyroid animals. These lipids were also reduced, although to a lesser degree, in 30-day-old ex-hypothyroid animals allowed to return to the euthyroid state at age 18 days. The onset of formation of sulphatide in vivo was delayed in the hypothyroid animals. Sulphatide formation reached a peak at a later time and was greatly reduced in comparison to control animals. The activity in vitro of galactolipid sulphotransferase, which forms sulphatide from cerebroside and PAPS, was reduced only when exogenous PAPS was not added to the assay medium. This finding suggests a defect in the formation of PAPS from ATP and sulphate in the hypothyroid brain. In addition, T₃ (tri-iodothyronine) had a stimulatory effect in vivo of formation brain sulphatide when administered to rats during the first 5 days of life.     

Systemic Blockade of Sialylation in Mice with a Global Inhibitor of Sialyltransferases

Sialic acid terminates glycans of glycoproteins and glycolipids that play numerous biological roles in health and disease. Although genetic tools are available for interrogating the effects of decreased or abolished sialoside expression in mice, pharmacological inhibition of the sialyltransferase family has, to date, not been possible. We have recently shown that a sialic acid analog, 2,4,7,8,9-pentaacetyl-3F_ax-Neu5Ac-CO₂Me (3F-NeuAc), added to the media of cultured cells shuts down sialylation by a mechanism involving its intracellular conversion to CMP-3F-NeuAc, a competitive inhibitor of all sialyltransferases. Here we show that administering 3F-NeuAc to mice dramatically decreases sialylated glycans in cells of all tissues tested, including blood, spleen, liver, brain, lung, heart, kidney, and testes. A single dose results in greatly decreased sialoside expression for over 7 weeks in some tissues. Although blockade of sialylation with 3F-NeuAc does not affect viability of cultured cells, its use in vivo has a deleterious “on target” effect on liver and kidney function. After administration of 3F-NeuAc, liver enzymes in the blood are dramatically altered, and mice develop proteinuria concomitant with dramatic loss of sialic acid in the glomeruli within 4 days, leading to irreversible kidney dysfunction and failure to thrive. These results confirm a critical role for sialosides in liver and kidney function and document the feasibility of pharmacological inhibition of sialyltransferases for in vivo modulation of sialoside expression.     

Analysis of Minimal Functions of Simian Virus 40 II. Enhancement of Oncogenic Transformation in Vitro by UV Irradiation

After light UV irradiation (5,000 to 10,000 ergs/mm²) “complete” and “defective” simian virus 40 (SV40) showed an enhancement of oncogenic transformation capacity in Syrian hamster kidney cells in vitro up to 180 and 270% of the controls, respectively. Simultaneously with the enhancement of transformation, an increase in T-antigen induction was observed in CV-1 cells infected with light UV-irradiated SV40; infectivity, however, was correspondingly reduced by 1 log₁₀. After strong UV irradiation (10,000 to 80,000 ergs/mm²) of “complete” and “defective” SV40, transformation capacity in vitro proved to be the most resistant viral function. It was only slightly reduced in comparison with a 4 to 5 log₁₀ reduction of infectivity. T-antigen induction of SV40 was also equally resistant to strong UV irradiation. We found no evidence of “multiplicity reactivation” involved in the high resistance of transformation capacity of SV40 after UV irradiation. Syrian hamster kidney cells transformed in vitro by UV-irradiated SV40 contained the SV40-specific T-antigen and showed the same morphology and growth characteristics as cells transformed by non-irradiated “complete” or “defective” SV40. They induced malignant tumors after subcutaneous inoculation into Syrian hamsters.     

Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group

The phylogenetic affiliation and physiological characteristics (e.g., K_s and maximum specific growth rate [μ_max]) of an anaerobic ammonium oxidation (anammox) bacterium, “Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.     

Excretion of water, sodium, and potassium during the compensatory renal hypertrophy in hypothyroid rats]

Renal compensatory hypertrophy is studied in age matched euthyroid and radiothyroidectomized female rats. 7 days after uninephrectomy, the hypertrophy of the remaining kidney is equally small in both groups. But 60 days after this operation, the hypothyroid animals show only a 12% increase in the wet weight of the remaining kidney whereas the euthyroid controls increase this weight by 21%. The excretion of water, Na and K are determined in the urine excreted in 5 h after a small water load. The results are related to 1 gram of kidney wet weight. These outputs increase in all animals after uninephrectomy. They are significantly higher in the hypothyroid rats than in the euthyroid controls as well before than 60 days after uninephrectomy. The reduction in tubular Na reabsorption found in the hypothyroid rat may account for the impairment of compensatory renal hypertrophy in hypothyroidism.     

Inhibition of compensatory kidney hypertrophy by hypothyroidism in the rat. Measurement of the mean diameter of glomeruli and proximal tubules]

The body, heart and kidney weights are reduced in the hypothyroid rat. In this animal, the diameter of the proximal tubule is significantly smaller than in euthyroid controls. The glomerular diameter is not affected by hypothyroidism. 21 days after uninephrectomy, the weight of the remaining kidney and the diameter of the proximal tubule increase significantly both in the hypothyroid and the euthyroid rats. But this compensatory renal hyperthrophy is definitely impaired in the hypothyroid animals.     

Activation of Na+-permeant Cation Channel by Stretch and Cyclic AMP-dependent Phosphorylation in Renal Epithelial A6 Cells

It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na⁺ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na⁺, K⁺, Li⁺, and Cs⁺, but little selectivity for Ca²⁺ (P_Ca/P_Na < 0.005) or Cl⁻ (P_Cl/P_Na < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability (P _o) of the IBMX-activated channel. This channel had one open (“O”) and two closed (short “C _S” and long “C _L”) states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the P _o of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model “C _L ↔ C _S ↔ O” was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for C _L from C _S, resulting in an increase in P _o. Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.     

Expression of Somatostatin and Somatostatin Receptor Subtypes 1�C5 in Human Normal and Diseased Kidney

Somatostatin mediates inhibitory functions through five G protein–coupled somatostatin receptors (sst_1–5). We used immunohistochemistry, immunofluorescence, and RT-PCR to determine the presence of somatostatin receptors sst₁, sst_2A, sst_2B, sst₃, sst₄, and sst₅ in normal and IgA nephropathy human kidney. All somatostatin receptors were detected in the thin tubules (distal convoluted tubules and loops of Henle) and thick tubules (proximal convoluted tubules) in the tissue sections from nephrectomy and biopsy samples. Immunopositive sst₁ and sst₄ staining was more condensed in the cytoplasm of tubular epithelial cells. In normal kidney tissue sections, podocytes and mesangial cells in the glomeruli stained for sst₁, sst_2B, sst₄ and sst₅, and stained weakly for sst₃. In IgA kidney tissue, the expression of somatostatin receptors was significantly increased with particular immmunopositive staining for sst₁, sst_2B, sst₄, and sst₅ within glomeruli. In the epithelial cells, the staining for sst_2B and sst₄ in proximal tubules and sst₁, sst_2B, and sst₅ in distal tubules was increased. The mRNA expression of sst_1–5 was also detected by RT-PCR. Somatostatin and all five receptor subtypes were ubiquitously distributed in normal kidney and IgA nephropathy. The increased expression of somatostatin receptors in IgA nephropathy kidney might be the potential pathogenesis of inflammatory renal disease. (J Histochem Cytochem 56:733–743, 2008)     

Hypertonicity activates GSK3β in tumor cells

In this study, we examined changes in the biochemical and inotropic events of the α₁-adrenoceptor signaling pathway in hypothyroid rat hearts. Hypothyroidism was induced by treating experimental animals with 0.05% 6-n-propyl-2-thiouracil (PTU) in drinking water for 7 weeks. A significant decrease of β- and an increase in α₁-adrenoceptor density as well as an increase in the basal activity of the phosphoinositide (4,5) bisphosphate hydrolyzing phospholipase C was observed in sarcolemmal membranes purified from hypothyroid hearts as compared to age-matched euthyroid controls. Following stimulation with 10 μM phenylephrine (in the presence of 10 μM atenolol), the increase of contractile parameters over baseline values was significantly higher in hypo- than euthyroid hearts, while the opposite occurred under β-stimulation with 0.1 μM isoproterenol. Interestingly, the increase in phenylephrine-mediated positive inotropy was accompanied by a significant increase in the sarcolemmal phospholipase C activity and in the inositol 1,4,5-trisphosphate content in hypothyroid as compared to euthyroid controls. Our results suggest that cardiac α₁-adrenoceptor and its associated phosphoinositide signaling pathway may act as a reserve for catecholamine inotropic response in hypothyroidism, where the β-adrenoceptors are compromised. Deceased     

Role of ClC-5 in Renal Endocytosis Is Unique among ClC Exchangers and Does Not Require PY-motif-dependent Ubiquitylation

Inactivation of the mainly endosomal 2Cl⁻/H⁺-exchanger ClC-5 severely impairs endocytosis in renal proximal tubules and underlies the human kidney stone disorder Dent''s disease. In heterologous expression systems, interaction of the E3 ubiquitin ligases WWP2 and Nedd4-2 with a “PY-motif” in the cytoplasmic C terminus of ClC-5 stimulates its internalization from the plasma membrane and may influence receptor-mediated endocytosis. We asked whether this interaction is relevant in vivo and generated mice in which the PY-motif was destroyed by a point mutation. Unlike ClC-5 knock-out mice, these knock-in mice displayed neither low molecular weight proteinuria nor hyperphosphaturia, and both receptor-mediated and fluid-phase endocytosis were normal. The abundances and localizations of the endocytic receptor megalin and of the Na⁺-coupled phosphate transporter NaPi-2a (Npt2) were not changed, either. To explore whether the discrepancy in results from heterologous expression studies might be due to heteromerization of ClC-5 with ClC-3 or ClC-4 in vivo, we studied knock-in mice additionally deleted for those related transporters. Disruption of neither ClC-3 nor ClC-4 led to proteinuria or impaired proximal tubular endocytosis by itself, nor in combination with the PY-mutant of ClC-5. Endocytosis of cells lacking ClC-5 was not impaired further when ClC-3 or ClC-4 was additionally deleted. We conclude that ClC-5 is unique among CLC proteins in being crucial for proximal tubular endocytosis and that PY-motif-dependent ubiquitylation of ClC-5 is dispensable for this role.     

Discriminant Value of Thyroid Function Tests

Different thyroid function tests permitted a final classification of 204 consecutive patients with suspected thyroid disorders into three populations (thyrotoxic, euthyroid, and hypothyroid). Linear discriminant analysis was applied to all test results (10 variates) on adjacent population pairs. Two invitro tests (serum protein bound iodine (P.B.I.) and tri-iodothyronine (T-3) uptake values) gave good separation of thyrotoxic from euthyroid patients and fairly good distinction of hypothyroid patients. If a ¹³¹I uptake figure was then added to the in-vitro results most patients (95·5%), including thse initially classified as equivocal, were correctly diagnosed. Other tests, including clinical questionnaires, were poor discriminants.Two new techniques of utilizing the test data were devised. Firstly, the data from the two in-vitro tests were also displayed graphically, and oblique boundary lines derived from the discriminant functions gave better separation of patients than previously used limits or mathematical expressions of “free thyroxine.” Secondly, a nomogram incorporating the best four discriminants was designed as a diagnostic aid and proved to be the best means of interpreting the tests.Discriminant analysis of this kind can be used in the interpretation of diagnostic tests in any branch of medicine, and it allows the best use to be made of the available data.     

Spatial Distribution of Photosynthesis during Drought in Field-Grown and Acclimated and Nonacclimated Growth Chamber-Grown Cotton

Inhomogeneous photosynthetic activity has been reported to occur in drought-stressed leaves. In addition, it has been suggested that these water stress-induced nonuniformities in photosynthesis are caused by “patchy” stomatal closure and that the phenomenon may have created the illusion of a nonstomatal component to the inhibition of photosynthesis. Because these earlier studies were performed with nonacclimated growth chamber-grown plants, we sought to determine whether such “patches” existed in drought-treated, field-grown plants or in chamber-grown plants that had been acclimated to low leaf water potentials (ψ_leaf). Cotton (Gossypium hirsutum L.) was grown in the field and subjected to drought by withholding irrigation and rain from 24 d after planting. The distribution of photosynthesis, which may reflect the stomatal aperture distribution in a heterobaric species such as cotton, was assayed by autoradiography after briefly exposing attached leaves of field-grown plants to ¹⁴CO₂. A homogeneous distribution of radioactive photosynthate was evident even at the lowest ψ_leaf of −1.34 MPa. “Patchiness” could, however, be induced by uprooting the plant and allowing the shoot to air dry for 6 to 8 min. In parallel studies, growth chamber-grown plants were acclimated to drought by withholding irrigation for three 5-d drought cycles interspersed with irrigation. This drought acclimation lowered the ψ_leaf value at which control rates of photosynthesis could be sustained by approximately 0.7 MPa and was accompanied by a similar decline in the ψ_leaf at which patchiness first appeared. Photosynthetic inhomogeneities in chamber-grown plants that were visible during moderate water stress and ambient levels of CO₂ could be largely removed with elevated CO₂ levels (3000 μL L⁻¹), suggesting that they were stomatal in nature. However, advanced dehydration (less than approximately 2.0 MPa) resulted in “patches” that could not be so removed and were probably caused by nonstomatal factors. The demonstration that patches do not exist in drought-treated, field-grown cotton and that the presence of patches in chamber-grown plants can be altered by treatments that cause an acclimation of photosynthesis leads us to conclude that spatial heterogeneities in photosynthesis probably do not occur frequently under natural drought conditions.