The anti-apoptotic effect of regucalcin is mediated through multisignaling pathways

Regucalcin (RGN/SMP30) was originally discovered in 1978 as a calcium-binding protein that does not contain the EF-hand motif of as a calcium-binding domain. The name, regucalcin, was proposed for this calcium-binding protein, which can regulate various Ca²⁺-dependent enzymes activation in liver cells. The regucalcin gene is localized on the X chromosome, and its expression is mediated through many signaling factors. Regucalcin plays a pivotal role in regulation of intracellular calcium homeostasis in various cell types. Regucalcin also has a suppressive effect on various signaling pathways from the cytoplasm to nucleus in proliferating cells and regulates nuclear function in including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis. Overexpression of endogenous regucalcin was found to suppress apoptosis in modeled rat hepatoma cells and normal rat kidney proximal epithelial NRK52 cells induced by various signaling factors. Suppressive effect of regucalcin on apoptosis is related to inhibition of nuclear Ca²⁺-activated DNA fragmentation, Ca²⁺/calmodulin-dependent nitric oxide synthase, caspase-3, Bax, cytochrome C, protein tyrosine kinase, protein tyrosine phosphatase in the cytoplasm and nucleus. Moreover, regucalcin stimulates Bcl-2 mRNA expression and depresses enhancement of caspase-3, Apaf-1 and Akt-1 mRNAs expression. This review discusses that regucalcin plays a pivotal role in rescue of apoptotic cell death, which is mediated through various signaling factors.     

Role of regucalcin in calcium signaling

Regucalcin was discovered in 1978 as a calcium-binding protein that does not contain EF-hand motif of Ca(2+)-binding domain [M. Yamaguchi and T. Yamamoto, Chem. Pharm. Bull. 26 1915-1918 (1978)]. In recent years, regucalcin has been demonstrated to play an important role as a regulatory protein in Ca2+ signaling in rat liver and kidney cells. The organization of the rat regucalcin gene consists of seven exons and six introns. The mRNA is mainly present in liver and kidney with a size of 1.8 kb. Hepatic regucalcin mRNA expression has been shown to be stimulated by various factors including calcium, calcitonin, insulin, and estrogen in rats. The mRNA is also expressed in hepatoma cells (Morris hepatoma, HepG2, and rat hepatoma H4-II-E cells). Regucalcin plays a role in the maintenance of intracellular Ca2+ homeostasis due to activating Ca2+ pump enzymes in the plasma membrane (basolateral membrane) and microsomes of liver and renal cortex cells. Moreover, regucalcin has an inhibitory effect on the activation of Ca2+/calmodulin-dependent enzymes and protein kinase C. Also, regucalcin has been demonstrated to regulate nuclear function in liver cells; it can inhibit Ca(2+)-activated DNA fragmentation, DNA and RNA synthesis, protein kinase and protein phosphatase activities in the nuclei. Such an effect is also seen in the nuclei of regenerating rat liver. Regucalcin may play a physiological role in the control for overexpression of proliferative cells. Regucalcin has been proposed to be an important regulatory protein in Ca2+ signaling system, and it plays a multifunctional role in liver and kidney cells.     

Regucalcin as a potential biomarker for metabolic and neuronal diseases

Regucalcin was initially discovered in 1978 as a regulatory protein in calcium signaling. The regucalcin gene, which is localized on the X chromosome, is found in vertebrate and invertebrate species. Regucalcin has been shown to play a pivotal role in cell regulation: maintaining of intracellular calcium homeostasis, suppressions of signal transduction, inhibition of translational protein synthesis, nuclear deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, regulation of gene expression, and anti-effects on proliferation and apoptosis in many cell types. The expression of the regucalcin gene and its protein has been shown to alter with various metabolic diseases, and regucalcin plays an important role in the development of many pathophysiologic states. Serum regucalcin has been found to increase with liver injury, and also urinary regucalcin is elevated with kidney damage, suggesting a useful tool as biomarker for diagnosis. Moreover, regucalcin has been shown to be good tool in early diagnosis for Alzheimer’s disease and other brain diseases. This review will discuss a significance of regucalcin as a clinical biomarker in various diseases.     

Inhibitory effect of calcium-binding protein regucalcin on protein kinase C activity in rat liver cytosol

Regucalcin, a calcium-binding protein isolated from rat liver cytosol, inhibited Ca2(+)- and phospholipid-dependent protein kinase (protein kinase C) activity in hepatic cytosol. With the increasing concentrations of Ca2+ or phosphatidylserine in the medium, regucalcin caused a remarkable inhibition of protein kinase C activity. Moreover, regucalcin significantly inhibited dioctanoylglycerol-activated protein kinase C. Regucalcin itself did not have protein kinase activity in either the presence or the absence of Ca2+ and phospholipids. These findings clearly indicate that regucalcin has an inhibitory effect on protein kinase C in hepatic cytosol. This inhibitory effect of regucalcin may be due to the regucalcin-induced Ca2+ binding and/or the direct binding of regucalcin to protein kinase C.     

Suppressive role of regucalcin in liver cell proliferation: involvement in carcinogenesis

Regucalcin (RGN/SMP30) was discovered in 1978 and is a unique calcium‐binding protein contains no EF‐hand motif calcium‐binding domain. Its name, regucalcin, was proposed as it suppresses activation of enzymes related to calcium signalling. The regucalcin gene (rgn) is localized on the X chromosome. Regucalcin plays its role of suppressor protein in intracellular signalling pathways, including of protein kinases and protein phosphatase activities, protein synthesis, and DNA and RNA synthesis in liver cells. Overexpression of endogenous regucalcin has a suppressive effect on cell proliferation in modelled rat hepatoma H4‐II‐E cells, which are induced by various signalling stimulations in vitro. This suppressive effect is independent of apoptosis. Endogenous regucalcin plays a suppressive role on overproduction of proliferating cells in regenerating rat liver in vivo. Regucalcin mRNA expression is uniquely down‐regulated in development of carcinogenesis in liver of rats in vivo. Regucalcin mRNA and protein expressions are also depressed in human hepatoma HepG2 cells, MCF‐7 breast cancer cells, and prostate cancer LNCaP cells. Depression of regucalcin expression may be associated with activity progression of carcinogens. Regucalcin may be a key molecule suppressor protein in cell proliferation and carcinogenesis.     

Stimulatory effect of calcium administration on regucalcin mRNA expression is attenuated in the kidney cortex of rats ingested with saline

The expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats ingested with saline was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb of open reading frame). Rats were freely given saline as drinking water for 7 days. Regucalcin mRNA levels in the kidney cortex were suppressed by saline ingestion. When calcium chloride (10 mg Ca/100 g body weight) was intraperitoneally administered to rats ingested with saline for 7 days, the effect of calcium administration to increase regucalcin mRNA levels was weakened by saline ingestion. Such effect was also seen by the administration of 2.5 and 5 mg Ca/100 g. Regucalcin mRNA levels in the kidney cortex of spontaneous hypertensive rats (SHR) were not appreciably increased by the administration of calcium (10 mg/100 g). Meanwhile, calcium content in the kidney cortex was significantly elevated by the administration of calcium (10 mg/100 g) to normal rats. This increase was weakened in saline-ingested rats. Moreover, Ca²⁺/calmodulin-dependent protein kinase activity in the cytosol of kidney cortex was significantly decreased by saline ingestion. These results suggest the possibility that saline ingestion-induced suppression of regucalcin mRNA expression in the kidney cortex is partly involved in the attenuation of Ca²⁺ signalling.     

Reversible effect of calcium-binding protein regucalcin on the Ca2+-induced inhibition of deoxyuridine 5′-triphosphatase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on deoxyuridine 5′-triphosphatase (dUTPase) in the cytosol of rat liver was investigated. Addition of Ca²⁺ up to 5.0 μM to the enzyme reaction mixture caused a significant decrease of dUTPase activity, while Zn²⁺, Cd²⁺, Co²⁺, Al³⁺, Mn²⁺ and Ni²⁺ (10 μM) did not have an appreciable effect. The Ca²⁺-induced decrease of dUTPase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 μM of the protein. Regucalcin had no effect on the basal activity of the enzyme. Meanwhile, the reversible effect of regucalcin on the Ca²⁺ (10 μM)-induced decrease of dUTPase activity was not altered by the coexistence of Cd²⁺ or Zn²⁺ (10 μM). The present data suggest that liver cytosolic dUTPase is uniquely regulated by Ca²⁺ of various metals, and that the Ca²⁺ effect is reversed by regucalcin.     

Regucalcin increases Ca2+-ATPase activity in the mitochondria of brain tissues of normal and transgenic rats

The role of regucalcin, which is a regulatory protein in intracellular signaling, in the regulation of Ca(2+)-ATPase activity in the mitochondria of brain tissues was investigated. The addition of regucalcin (10(-10) to 10(-8) M), which is a physiologic concentration in rat brain tissues, into the enzyme reaction mixture containing 25 microM calcium chloride caused a significant increase in Ca(2+)-ATPase activity, while it did not significantly change in Mg(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing mitochondrial Ca(2+)-ATPase activity was completely inhibited in the presence of ruthenium red (10(-7) M) or lanthanum chloride (10(-7) M), both of which are inhibitors of mitochondrial uniporter activity. Whether the effect of regucalcin is modulated in the presence of calmodulin or dibutyryl cyclic AMP (DcAMP) was examined. The effect of regucalcin (10(-9) M) in increasing Ca(2+)-ATPase activity was not significantly enhanced in the presence of calmodulin (2.5 microg/ml) which significantly increased the enzyme activity. DcAMP (10(-6) to 10(-4) M) did not have a significant effect on Ca(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing Ca(2+)-ATPase activity was not seen in the presence of DcAMP (10(-4) M). Regucalcin levels were significantly increased in the brain tissues or the mitochondria obtained from regucalcin transgenic (RC TG) rats. The mitochondrial Ca(2+)-ATPase activity was significantly increased in RC TG rats as compared with that of wild-type rats. This study demonstrates that regucalcin has a role in the regulation of Ca(2+)-ATPase activity in the brain mitochondria of rats.     

Stimulatory effect of regucalcin on mitochondrial ATP-dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca(2+) signaling, on Ca(2+)-ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the mitochondria. Ruthenium red (10(-6) M) or lanthunum chloride (10(-6) M), an inhibitor of mitochondrial Ca(2+) uptake, markedly inhibited regucalcin (100 nM)-increased mitochondrial Ca(2+)-ATPase activity and (45)Ca(2+) uptake. The effect of regucalcin (100 nM) in elevating Ca(2+)-ATPase activity was completely prevented by the presence of digitonin (10(-2)%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca(2+)-ATPase activity was not further enhanced by calmodulin (0.30 microM) or dibutyryl cyclic AMP (10(-4) M), which could increase Ca(2+)-ATPase activity. Trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, significantly decreased Ca(2+)-ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca(2+)-pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca(2+)-ATPase.     

Molecular characterization and expression analysis of regucalcin in disk abalone (Haliotis discus discus): intramuscular calcium administration stimulates the regucalcin mRNA expression

Regucalcin is a novel calcium (Ca(2+)) binding protein and it has been demonstrated to play a multifunctional role in many organisms. Here, we report the molecular cloning of invertebrate regucalcin cDNA from disk abalone Haliotis discus discus. The full length cDNA showed 1321 bp of nucleotides with a polyadenylated sequence (AATAAA). Abalone regucalcin (HdReg) open reading frame (ORF) consists of 918 nucleotides encoding 305 amino acids (aa). Estimated molecular mass was 33 kDa and predicted isoelectric point (pI) was 4.9. The HdReg aa sequence did not contain the EF-hand motif as a Ca(2+) binding domain, suggesting a novel class of Ca(2+) binding protein. Moreover, it showed 45% identity to chicken and zebrafish, and 44% to rat and mouse regucalcin in deduced aa level. The tissue expression analysis of HdReg mRNA was investigated by RT-PCR and it was expressed in all the tissues tested such as gill, mantle, digestive tract, and abductor muscle. Semi-quantitative RT-PCR results showed that an intramuscular administration of calcium chloride (CaCl(2)) (0.5 mg CaCl(2)/g of abalone) could significantly induce regucalcin mRNA in abductor muscle after 30 min of administration and reached maximum after 1 h. Subsequently, the expression level was decreased after 2 h. This indicates that the expression of regucalcin mRNA is constitutive, and specifically up regulated in abalone abductor muscle by Ca(2+) administration.     

Role of regucalcin as an activator of Ca(2+)-ATPase activity in rat liver microsomes.

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. Thapsigargin (10(-6) M), a specific inhibitor of microsomal Ca(2+) pump enzyme (Ca(2+)-ATPase), clearly inhibited regucalcin (0.5 microM)-increased microsomal Ca(2+)-ATPase activity. Liver microsomal Ca(2+)-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10(-2) %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca(2+) ionophore (A23187; 10(-7) M). The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca(2+)-ATPase.     

Regucalcin (RGN/SMP30) alters agonist- and thapsigargin-induced cytosolic [Ca2+] transients in cells by increasing SERCA Ca(2+)ATPase levels

Regucalcin (RGN), also reported as senescence marker protein-30 (SMP30), plays a role in Ca(2+) homeostasis by modulating a number of Ca(2+)-dependent proteins. RGN also plays a cyto-protective role and its decrease is linked to age-related diseases and cell death. This study shows that RGN reduces agonist (histamine)-induced Ca(2+) transients in RGN(+) transfected COS-7 cells (RGN(+)) and also increases their Ca(2+) storage capacity. These observations are explained by RGN(+) cells having increased mRNA and protein expression levels of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA). Therefore down-regulation of RGN expression may contribute to characteristics of age-dependent Ca(2+) homeostasis dis-regulation, by decreasing SERCA levels.     

Stimulatory effect of regucalcin on ATP-dependent Ca(2+) uptake activity in rat liver mitochondria

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver mitochondria was investigated. The presence of regucalcin (0.1, 0.25, and 0.5 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the mitochondria. Ruthenium red (10(-5) M) or lanthanum chloride (10(-4) M), an inhibitor of mitochondrial Ca(2+) uptake, completely inhibited regucalcin (0.25 microM)-increased mitochondrial Ca(2+)-ATPase activity and (45)Ca(2+) uptake. The effect of regucalcin (0.25 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of digitonin (10(-2)%), a solubilizing reagent of membranous lipids, or vanadate (10(-5) M), an inhibitor of phosphorylation of ATPase. The activatory effect of regucalcin (0.25 microM) on Ca(2+)-ATPase activity was not further enhanced in the presence of dithiothreitol (2.5 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme, or calmodulin (0.60 microM), a modulator protein of Ca(2+) action that could increase mitochondrial Ca(2+)-ATPase activity. The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver mitochondria, and that the protein may act on an active site (SH group)-related to phosphorylation of mitochondrial Ca(2+)-ATPase.     

Stimulatory effect of regucalcin on ATP-dependent calcium transport in rat liver plasma membranes

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytoplasm, on ATP-dependent calcium transport in the plasma membrane vesicles of rat liver was investigated. (Ca2+-Mg2+)-ATPase activity in the liver plasma membranes was significantly increased by the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the enzyme reaction mixture. This increase was completely inhibited by the presence of sulfhydryl group modifying reagent Nethylmaleimide (5.0 mM NEM) or digitonin (0.04%), which can solubilize the membranous lipids. When ATP-dependent calcium uptake by liver plasma membrane vesicles was measured by using 45CaCl2, the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the reaction mixture caused a significant increase in the 45Ca2+ uptake. This increase was about 2-fold with 0.5 \sgmaelig;M regucalcin addition. An appreciable increase was seen by 5 min incubation with regucalcin addition. The regucalcin-enhanced ATP-dependent 45Ca2+ uptake by the plasma membrane vesicles was completely inhibited by the presence of NEM (5.0 mM) or digitonin (0.04%). These results demonstrate that regucalcin activates (Ca2+-Mg2+)-ATPase in the liver plasma membranes and that it can stimulate ATP-dependent calcium transport across the plasma membranes.     

Role of endogenous regucalcin in transgenic rats: suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+-ATPase activity

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+-ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+-ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo.     

Regucalcin and metabolic disorders: osteoporosis and hyperlipidemia are induced in regucalcin transgenic rats

Regucalcin transgenic (TG) rat has been generated to determine the role in metabolic disorders. Regucalcin homozygote male and female rats induce a prominent increase in regucalcin protein in the various tissues. Bone loss has been found to induce in regucalcin TG rats with growing (5 weeks old) and aging (50 weeks old). Osteoclastogenesis has been shown to stimulate in culture with the bone marrow cells obtained from regucalcin TG rats. Exogenous regucalcin stimulates osteoclastogenesis in mouse marrow culture in vitro. Regucalcin has a suppressive effect on the differentiation and mineralization in osteoblastic MC3T3-E1 cells in vitro. The mechanism by which regucalcin TG rat induces bone loss may result from the enhancement of osteoclastic bone resorption and the suppression of osteoblastic bone formation. Moreover, regucalcin TG rat has been found to induce hyperlipidemia with increasing age (14–50 weeks); serum triglyceride, high-density lipoprotein (HDL)-cholesterol, free fatty acid, albumin and calcium concentrations are markedly increased in regucalcin TG male and female rats with increasing age. The decrease in lipid and glycogen contents in liver tissues is induced in regucalcin TG rats. The gene expression of leptin and adiponectin is suppressed in the TG rats. Overexpression of regucalcin has been shown to enhance glucose utilization and lipid production in the cloned rat hepatoma H4-II-E cells in vitro, and insulin resistance is seen in the cells. The expression of glucose transporter 2 mRNA is increased in the transfectants, while it has been shown to suppress insulin receptor and phosphatidylinositol 3-kinase mRNA expressions that are involved in insulin signaling. This review proposes that regucalcin relates in osteoporosis and hyperlipidemia, and that the regucalcin TG rat model may be useful in determining the pathophysiologic state and the development of therapeutic tool for osteoporosis and hyperlipidemia.     

Activatory effect of calcium-binding protein regucalcin on ATP-dependent calcium transport in the basolateral membranes of rat kidney cortex

The effect of regucalcin, a calcium-binding protein, on ATP-dependent Ca2+ transport in the basolateral membranes isolated from rat kidney cortex was investigated. The prepared membranes were in inside-out oriented and membrane vesicles. Ca2+-ATPase activity in the basolateral membranes was progressively elevated by increasing concentrations of regucalcin (10-8 to 10-6 M) in the reaction mixture. This increase was dependent on Ca2+ addition. The activatory effect of regucalcin on the enzyme is inhibited by the presence of digitonin (5 × 10-6%) which can solubilize the membranous lipids. Moreover, the regucalcin effect was clearly abolished by the presence of vanadate (0.1 mM) or N-ethylmaleimide (5.0 mM). However, the effect of calmodulin (6 × 10-7 M) to increase Ca2+-ATPase activity was not significantly inhibited by vanadate or N-ethylmaleimide, indicating that the action mode of regucalcin differs from that of calmodulin. Also, the activatory effect of regucalcin on Ca2+-ATPase was appreciably inhibited by addition of dibutyryl cAMP (10-5 and 10-3 M), while inositol 1,4,5-trisphosphate (10-7 and 10-5 M) had no effect. Dibutyryl cAMP itself did not have an effect on the enzyme activity. Furthermore, the 45Ca2+ uptake by the basolateral membranes was clearly increased by the presence of regucalcin (10-7 and 10-6 M). This increase was completely blocked by the presence of vanadate (0.1 mM), N-ethylmaleimide (5.0 mM) or dibutyryl cAMP (10-4 and 10-3 M) in the reaction mixture. These results clearly demonstrate that regucalcin, which is expressed in rat kidney cortex, can increase Ca2+-ATPase activity and Ca2+ uptake in the basolateral membranes. Regucalcin may play a cell physiologic role as an activator in the ATP-dependent Ca2+ pumps in the basolateral membranes from rat kidney cortex.