Two-pore channel protein TPC1 is a determining factor for the adaptation of proximal tubular phosphate handling

Aim

Two-pore channels (TPCs) constitute a small family of cation channels expressed in endo-lysosomal compartments. TPCs have been characterized as critical elements controlling Ca²⁺-mediated vesicular membrane fusion and thereby regulating endo-lysosomal vesicle trafficking. Exo- and endocytotic trafficking and lysosomal degradation are major mechanisms of adaption of epithelial transport. A prime example of highly regulated epithelial transport is the tubular system of the kidney. We therefore studied the localization of TPC protein 1 (TPC1) in the kidney and its functional role in the dynamic regulation of tubular transport.

Methods

Immunohistochemistry in combination with tubular markers were used to investigate TPC1 expression in proximal and distal tubules. The excretion of phosphate and ammonium, as well as urine volume and pH were studied in vivo, in response to dynamic challenges induced by bolus injection of parathyroid hormone or acid–base transitions via consecutive infusion of NaCl, Na₂CO₃, and NH₄Cl.

Results

In TPC1-deficient mice, the PTH-induced rise in phosphate excretion was prolonged and exaggerated, and its recovery delayed in comparison with wildtype littermates. In the acid–base transition experiment, TPC1-deficient mice showed an identical rise in phosphate excretion in response to Na₂CO₃ compared with wildtypes, but a delayed NH4Cl-induced recovery. Ammonium-excretion decreased with Na₂CO₃, and increased with NH₄Cl, but without differences between genotypes.

Conclusions

We conclude that TPC1 is expressed subapically in the proximal but not distal tubule and plays an important role in the dynamic adaptation of proximal tubular phosphate reabsorption towards enhanced, but not reduced absorption.     

Intracellular regulatory cascades: Examples from parathyroid hormone regulation of renal phosphate transport

Summary The knowledge about intracellular regulatory cascades in hormone action has increased considerably over the last few years. Receptor occupation at the plasma membrane level results in a production of intracellular messengers, such as cyclic nucleotides (cAMP, cGMP), inositoltrisphosphate (IP₃), diacylglycerol (DAG) and a rise in cytosolic calcium concentration. These messengers control the activity of different regulatory mechanisms which operate either in sequence or in parallel to generate the final biological response. In PTH-dependent regulation of renal phosphate transport, cAMP-dependent and calcium-dependent mechanisms are involved: Recent experiments with cultured renal epithelial cells have confirmed that activation of adenylate cyclase is the initial event. However, the cAMP signal can be bypassed and direct activation of protein kinase C seems to mimic PTH induced inhibition of phosphate transport. The final event in the regulatory cascade is most likely a removal of the phosphate transport system followed by a degradation.     

Evidence for a function of calcium influx in the stimulation of hormone release from the parathyroid gland in the goat

The acute effects of various drugs on the release of parathyroid hormone (PTH) in goats were studied by local infusions in vivo. Infusions of Ca²+ or Sr²+ reduced the PTH secretion rate, whereas hypocalcemia induced by EDTA increased the PTH release. Blockers of voltage sensitive Ca²+ channels (verapamil, D-600 and nifedipine) lowered the PTH secretion rate, while infusion of 4-aminopyridine, which is a blocker of voltage sensitive K+ channels, increased the PTH release. These effects were not due to altered βadrenergic tonus, since the effects persisted when the drugs were administered during contineous infusion of the β-blocker propranolol. We suggest that the parathyroid cells possess voltage sensitive K+ and Ca²+ channels, and that exocytosis of stored PTH depends on the influx of extracellular Ca²+ as in other secretory cells. In order to explain the inverse relationship between the plasma Ca²+ level and the PTH release, we postulate a suppressive effect of the plasma Ca²+ on the membrane permeability to Ca²+ in parathyroid cells.     

Calcium supplementation and thyroid hormone protect against gentamicin-induced inhibition of proximal tubular Na+, K+-ATPase activity and other renal functional changes

Gentamicin can cause proximal tubule necrosis. We have shown that inhibition of PT Na⁺, K⁺-ATPase activity is rapidly induced by gentamicin. We have now investigated whether manipulations known to attenuate the negative effects of gentamicin on renal excretory capacity, i.e. high calcium intake and L-thyroxine treatment, will also attenuate gentamicin-induced inhibition of Na⁺, K⁺-ATPase activity and ameliorated signs of proximal tubule damage. Rats were gentamicin- or vehicle-treated for 7 days. Sub-groups were given 4% calcium (Ca) supplements or L-thyroxine 20 μg 100 g^-1 body weight daily. Gentamicin significantly reduced the glomerular filtration rate and increased the urinary excretion of the proximal tubule lysosomal enzyme, N-acetyl-β-d -glucosaminidase. Gentamicin significantly reduced proximal tubule Na⁺, K⁺-ATPase activity, measured in single permeabilized proximal tubule segments. Sodium excretion was inversely correlated to proximal tubule Na⁺, K⁺-ATPase activity. Both calcium and L-thyroxine alleviated all gentamicin-induced side-effects on renal function as well as on proximal tubule Na⁺, K⁺-ATPase activity. Calcium and L-thyroxine had no significant effect on renal function. L-thyroxine, but not calcium, increased proximal tubule Na⁺, K⁺-ATPase activity in control rats. Renal cortical tissue gentamicin concentration was not influenced by calcium but was significantly lowered by L-thyroxine. Two procedures which, via different mechanisms, afford protection from gentamicin-induced changes in renal function also give protection from gentamicin-induced inhibition of Na⁺, K⁺-ATPase activity. This suggests that loss of integrity of the Na⁺, K⁺-ATPase enzyme contributes to gentamicin-induced nephrotoxicity.     

Genistein ameliorates parathyroid hormone-induced epithelial-to-mesenchymal transition and inhibits expression of connective tissue growth factor in human renal proximal tubular cells

Introduction

Genistein, a soybean and soy-based product, has been reported to inhibit the growth of a wide range of cancer cells, but there is no evidence concerning its treatment of chronic kidney disease. The aim was to investigate whether genistein has potential to inhibit parathyroid hormone (PTH)-induced renal interstitial fibrosis.

Material and methods

Using human renal tubular epithelial HK-2 cells, α-smooth muscle actin (α-SMA) was assessed by using immunofluorescence detection. α-Smooth muscle actin, E-cadherin and connective tissue growth factor (CTGF) were measured by Western blot analysis. The promoter activity of the CTGF gene was examined by the luciferase reporter assay.

Results

When cells were treated with PTH (0.1 nM) for 48 h, α-SMA protein expression was induced significantly, the protein expression of E-cadherin decreased substantially, and the promoter activity of the CTGF gene as well as its mRNA and protein expression levels increased (p < 0.01). Interestingly, genistein effectively inhibited PTH-induced α-SMA expression, restored E-cadherin expression, decreased mRNA and protein expression of CTGF, and suppressed the promoter activity of CTGF in a dose-dependent manner.

Conclusions

Genistein has the ability to block the biomarker for renal transdifferentiation and epithelial-to-mesenchymal transition, α-SMA, following PTH treatment and inhibit CTGF expression in human renal tubular epithelial cells; these might be important modes of actions that contribute to genistein anti-fibrogenic effects and may have great implications for its potential in clinical treatment of renal interstitial fibrosis.     

Proximal tubular reabsorption of inorganic phosphate in adrenalectomized rats

Micropuncture studies have been performed in adrenalectomized (Adr.X) and sham-adrenalectomized (Sham-Adr.X) rats in order to examine the effects of acute adrenalectomy on the inorganic phosphate (Pi) transport in the convoluted proximal tubule of superficial nephrons under free flow conditions. Adrenalectomy diminished single nephron glomerular filtration rate from 27.0±12.4 (Sham-Adr.X) to 10.9±7.1 nl/min (mean ± S.D.,P<0.001), increased the inulin ratio (tubular fluid-to-plasma) at the end of the proximal convolution from 2.14±0.60 (Sham-Adr.X) to 3.62±1.67 (P<0.001) and decreased both the (TF/P)_Pi ratio from 0.87±0.20 (Sham-Adr.X) to 0.46±0.28 (P<0.001) and the fractional delivery of inorganic phosphate into the loop of Henle from 44±18 (Sham-Adr.X) to 15±10% (P<0.001). Thus the fractional reabsorption of inorganic phosphate in the convoluted proximal tubule in acutely Adr.X rats was higher than in Sham-Adr.X animals. It is concluded that glucocorticoid hormones play an important role in the regulation of the fractional reabsorption of Pi in the proximal tubule. The mechanism of this regulation is discussed.Supported by the Deutsche Forschungsgemeinschaft (Fr239/7). Parts of this work [6] have been presented at the 52th Meeting of the German Society of Physiology, Kiel. FRG, 1979, and at the XIIIth Symposion of the German Society of Nephrology, Berlin, 1979     

The phosphate regulating hormone fibroblast growth factor-23

Over the last decade, the regulation of phosphate (Pi) homeostasis has been under intense investigation. By utilizing modern biochemical and genetic tools, the pathophysiological mechanisms behind several known hereditary and acquired hypo- and hyperphosphatemic diseases have been clarified. The results of these efforts have opened new insights into the causes of Pi dysregulation and hereby also the physiological mechanisms determining Pi homeostasis. Although several potential Pi-regulating proteins have been discovered and investigated, current data strongly argues for fibroblast growth factor-23 (FGF23), a hormonal factor produced in bone, as a particularly important regulator of Pi homeostasis. In this article, we review the discovery of the FGF23 protein, as well as its biochemistry, localization of production, receptor specificity and mechanisms of action.     

Chronic thyroparathyroidectomy and tubular handling of phosphate

The fractional reabsorption (FR) of inorganic phosphate (Pi) along the proximal tubule depends upon both the filtered load of Pi (FLPi) and the tubular reabsorptive capacity of the Pi transporting system. To assess the actual effect of parathyroid hormone on the reabsorptive capacity only, the influence of Pi load has to be eliminated. In this study FRPi was determined by free-flow micropuncture along superficial nephrons of chronically (48 h) thyroparathyroidectomized (TPTX) and pair-fed sham-operated (SHAM) rats at identical FLPi [TPTX 3.07±0.14 (n=26) and SHAM 3.07±0.11 (n=26) mol/min±SEM]. The micropuncture results indicate that in the ranges of tubular fluid over plasma inulin concentration [TF/P)_In] 1.00–1.49 and 1.50–1.99, no difference in FRPi between TPTX and SHAM could be detected. It is only between a TF/P_In of 2.0 and 2.49 that chronic TPTX resulted in a significant increase in FRPi. Accordingly the present study indicates that chronic TPTX increases FRPi in late but not in early portions of proximal tubule. Thus in the early proximal tubule the tubular reabsorptive capacity of the Pi transporting system appears to be unaffected by chronic removal of the parathyroid glands. From this result it can be inferred that the increased plasma concentration of Pi which follows the removal of the parathyroid glands, particularly in the chronic stage, will lead to an apparently paradoxical decrease in FRPi in early proximal tubule as a mere consequence of the increased filtered load of Pi.     

慢性肾衰大鼠继发甲状旁腺功能亢进时腺体钙敏感受体的观察

研究慢性肾衰 (CRF)继发性甲旁亢 (2°HPT)大鼠发病时及 1,2 5 (OH) 2 D3 治疗时甲状旁腺钙敏感受体 (PCaR)mRNA含量变化。采用肾大部分切除大鼠 2°HPT模型 ,动物分为CRF组 ,治疗组 ,假手术组。治疗组予以 1,2 5(OH) 2 D3(2 5pmol d× 10腹腔注射 ) ,31d后测定生化指标及PCaRmRNA含量 ,后者采用逆转录聚合酶链反应 (RT PCR)半定量法。(1)CRF组大鼠较假手术组血肌酐 (SCr) (6 9 30± 11 2 0vs 2 1 15± 8 2 0 μmol L ,P <0 0 5 )及甲状旁腺素 (PTH) (2 5 6± 72vs 41± 7pg mL ,P <0 0 5 )明显上升 ,治疗组PTH较CRF组明显下降 (112± 47vs 2 5 6± 72pg mL ,P <0 0 5 )。(2 )CRF组大鼠PCaRmRNA与假手术组含量无明显差异 (1 10 0± 0 15 3vs 1 0 74± 0 119) ,治疗组PCaRmRNA与CRF组含量无明显差异 (1 131± 0 10 8vs1 0 74± 0 119)。本实验 2°HPT模型PCaRmRNA表达无明显变化 ,1,2 5 (OH) 2 D3治疗肾大部切除 2°HPT大鼠对PCaRmRNA水平无显著影响     

甲状旁腺素和二膦酸盐对骨密度和骨生物力学的影响

目的:研究应用甲状旁腺素和阿仑膦酸钠对激素性骨质疏松兔骨密度和生物力学的影响。方法:采用成年新西兰大白兔,随机分为对照组、激素模型组、甲状旁腺素和阿仑膦酸钠治疗组,9周后取股骨和腰椎行超声骨密度测量,再行股骨扭转、三点弯曲和腰椎压缩试验。结果:激素模型组的宽频超声衰减(BUA)、超声传导速度(SOS)值和骨生物力学参数较对照组有非常明显减少,甲状旁腺素和阿仑膦酸钠治疗组的BUA、SOS值和骨生物力学参数较激素模型组明显增加。结论:序贯应用甲状旁腺素和阿仑膦酸钠可以减少激素性兔骨量的丢失,预防骨质疏松的发生。     

Electrophysiology of cultured parathyroid cells from the goat

The membrane potential and resistance of goat parathyroid (PT) cells in culture were 44.7±6.8 mV and 503±218 MΩ (mean ±S.D., n= 24) in 2.4 mM Ca²+ solution. The corresponding values in 0.8 mM Ca²+ solution were 29.2±12.2 mV and 290± 198 MΩ (n= 22). The current/voltage relation showed a pronounced outward rectification for membrane potentials more positive than -40 mV. Current injection produced graded, regenerative responses in half of the penetrated cells. Regenerative responses were also obtained in Na+-free solution, and these responses were reversibly inhibited by the Ca²+ blocker D-600. The results support the notion that PT cells possess voltage sensitive K+ and Ca²+ channels, and that exocytosis of stored PT hormone depends on influx of extracellular Ca²+ as in other secretory cells.     

Fluoride interactions with stimulus-secretion coupling of normal and pathological parathyroid cells

Effects of the GTP binding protein (G-protein) activator NaF on parathyroid hormone (PTH) release, cytoplasmic Ca²⁺ concentration ([Ca²⁺]₁) and cAMP content of bovine as well as normal and pathological human parathyroid cells were studied using precautions to avoid CaF₂ precipitation. In 0.5 mm external Ca²⁺, NaF inhibited PTH release and lowered the cAMP content by 50–70% of the effects attained with 3.0 mm Ca²⁺. The NaF-induced increase of [Ca²⁺]₁ was considerably smaller than that obtained with rise of external Ca²⁺. It seems likely that NaF activates the inhibitory G_i-protein involved in the regulation of cAMP generation. However, it is unclear whether the sluggish rise of [Ca²⁺]₁ induced by NaF is due to a direct effect of a G-protein on Ca²⁺ entry, or somehow related to the G-protein mediated formation of inositol 1,4,5-trisphosphate, which is part of the signal transduction pathway normally initiated by Ca²⁺ binding to its receptor on the parathyroid cell surface. Inhibition of PTH release by NaF probably results from the combined effects on [Ca²⁺]₁ and cAMP content. In hyperparathyroidism (HPT) the actions of NaF were not markedly affected despite severe impairments of Ca²⁺-inhibited PTH release and Ca²⁺ triggered increase of [Ca²⁺]₁. Consistent with observations of down regulation of the parathyroid Ca²⁺ receptor in HPT, the present results indicate that the disease perturbs signal transduction at a level proximal to the site of action for NaF.     

Increased Slc34a2 expression and paracellular phosphate permeability contribute to high intestinal phosphate absorption in young mice

Aim

Phosphorus is a critical constituent of bone as a component of hydroxyapatite. Bone mineral content accrues rapidly early in life necessitating a positive phosphorus balance, which could be established by a combination of increased renal reabsorption and intestinal absorption. Intestinal absorption can occur via a transcellular pathway mediated by the apical sodium-phosphate cotransporter, Slc34a2/NaPiIIb or via the paracellular pathway. We sought to determine how young mammals increase dietary phosphorus absorption from the small intestine to establish a positive phosphorus balance, a prerequisite for rapid bone growth.

Methods

The developmental expression profile of genes mediating phosphate absorption from the small intestine was determined in mice by qPCR and immunohistochemistry. Additionally, Ussing chamber studies were performed on small bowel of young (p7–p14) and older (8- to 17-week-old) mice to examine developmental changes in paracellular Pi permeability and transcellular Pi transport.

Results

Blood and urinary Pi levels were higher in young mice. Intestinal paracellular phosphate permeability of young mice was significantly increased relative to older mice across all intestinal segments. NaPiIIb expression was markedly increased in juvenile mice, in comparison to adult animals. Consistent with this, young mice had increased transcellular phosphate flux across the jejunum and ileum relative to older animals. Moreover, transcellular phosphate transport was attenuated by the NaPiIIb inhibitor NTX1942 in the jejunum and ileum of young mice.

Conclusion

Our results are consistent with young mice increasing phosphate absorption via increasing paracellular permeability and the NaPiIIb-mediated transcellular pathway.