Renal Glucocorticoid Receptor Research Articles

Continuous Monochromatic Blue Light Exacerbates High-Fat Diet-Induced Kidney Injury via Corticosterone-Mediated Oxidative Stress.

Excessive illumination is one of the most severe environmental factors that impacts the organism. There is growing evidence that obesity significantly contributes to the onset of chronic kidney disease. However, the effect of continuous light on the kidney and which color can produce an apparent phenomenon remains elusive. In this study, C57BL/6 mice given either a normal diet (LD-WN) or a high-fat diet (LD-WF) were subjected to a light cycle of 12 h of illumination followed by 12 h of darkness for 12 weeks. Meanwhile, 48 high-fat diet mice were given a 24 h monochromatic light exposure of varying colors (white, LL-WF; blue, LL-BF; green, LL-GF) for 12 weeks. As expected, the LD-WF mice showed significant obesity, kidney injury, and renal dysfunction compared with the LD-WN group. LL-BF mice had worse kidney injury than LD-WF mice, including higher Kim-1 and Lcn2. The kidney of the LL-BF group showed marked glomerular and tubular injury, with decreased levels of Nephrin, Podocin, Cd2ap, and α-Actinin-4 compared to LD-WF. LL-BF also reduced the antioxidant capacity, including GSH-Px, CAT, and T-AOC, increased the production of MDA, and inhibited the activation of the NRF2/HO-1 signaling pathway. Furthermore, LL-BF upregulated the mRNA levels of the pro-inflammatory factors Tnf-α, Il-6, and Mcp-1, decreasing the inhibitory inflammatory Il-4 expression. We observed increased plasma corticosterone (CORT), renal glucocorticoid receptors (GR) expression, Hsp90, Hsp70, and P23 mRNA levels. These findings suggested that LL-BF increased CORT secretion and affected glucocorticoid receptors (GR) in comparison to the LD-WF group. Moreover, in vitro research demonstrated that CORT treatment increased oxidative stress and inflammation, which was counteracted by adding a GR inhibitor. Thus, the sustained blue light worsened kidney damage, possibly by inducing elevated CORT and increasing oxidative stress and inflammation via GR.

MO251: Renal Glucocorticoid Receptor Expression and Clinical Course in Patients with IGA Nephropathy

Abstract BACKGROUND AND AIMS Glucocorticoids are used as primary treatment in IgA nephropathy (IgAN), and they manifest their effects by binding to intracellular glucocorticoid receptors. Studies have shown that GCR expression correlates with the degree of steroid response in various diseases. Therefore, we aimed to evaluate renal GCR expression, Oxford MEST score in clinical course of IgAN patients. METHOD This study included 110 patients with biopsy-proven IgAN. Patients were treated with ACEI/ARB, methylprednisolon (80% patients). In renal biopsies, Oxford-MEST score was calculated and GCR expression was determined immunohistochemically. KDIGO criteria (2020) were accepted for remission. Clinical course, eGFR, proteinuria and other biochemical tests test were recorded during 1-year follow-up period. RESULTS Demographic and laboratory data did not correlate with renal GCR staining rate. İn patients with M1 of the MEST score, GCR staining was more prominent than those with M0. GFR values were higher and proteinuria was lower in patients with T0 compared with patients with T1 and T2 (P < .05 for all) . Patients with E1 had significantly higher systolic blood pressure (BP), increased proteinuria and lower eGFR than patients with E0. Poor prognostic factors for achieving remission were higher systolic/diastolic BP, increased levels of white blood cell (WBC) count/neutrophil count, PTH, BUN, creatinine, uric acid, potassium, magnesium, MCV, Mentzer index, total cholesterol, LDL and higher sclerosis in biopsy at baseline during follow up period for 1 year. CONCLUSION Clinical presentation and outcome at 1 year in IgAN patients were well predicted by the Oxford-MEST score, but not by renal GCR expression. GCR staining intensity was only correlated with M1 score. Baseline higher BP, increased serum creatinine, uric acid and PTH were poor prognostic factors. Interestingly higher WBC and neutrophils counts, increased serum LDL and Mentzer index at baseline were also correlated with clinical course.

4007 Medroxyprogesterone Upregulates the Glucocorticoid Receptor in Female Long Evans Rats

OBJECTIVES/GOALS: Estrogen monotherapy in postmenopausal women can reduce kidney function, while dual therapy combining estrogen with a progestin improves renal health. Using the female Long Evans rat as a novel animal model of postmenopausal cardiovascular disease, we found similar results where estrogen worsens renal health while co-administration of medroxyprogesterone acetate (MPA) was protective. MPA cross-activates glucocorticoid receptors (GR), which are targeted clinically for their anti-inflammatory actions. Therefore, our goal was to determine if estrogen monotherapy induces renal damage by increasing inflammation, while dual therapy with MPA opposes inflammation by cross-activating GR. METHODS/STUDY POPULATION: Female Long Evans rats underwent OVX at 11 months of age and received a subcutaneous implant containing E2, E2+MPA or vehicle for 40 days. RESULTS/ANTICIPATED RESULTS: Coadministration of MPA prevented the E2-induced increase in proteinuria (Veh: 0.27 ± 0.07; E2: 3.53 ± 1.16; E2 + MPA: 1.20 ± 0.58 mg/mg creatinine; P = 0.03) and decline in glomerular filtration rate (Veh: 0.51 ± 0.02; E2: 0.24 ± 0.05; E2+MPA: 0.39 ± 0.05 ml/min; P < 0.01). Co-administration of MPA significantly increased renal GR transcript levels compared with E2 alone (Veh: 0.96 ± 0.02; E2: 0.94 ± 0.10; E2+MPA: 1.24 ± 0.04 fold change; P < 0.01). Inflammatory marker COX 2 renal transcript levels were significantly reduced by a similar degree in both mono and dual therapies compared with vehicle (Veh: 1.07 ± 0.06; E2: 0.81 ± 0.04; E2+MPA: 0.81 ± 0.04 fold change; P < 0.01). Neither TNF-alpha and IL-6 mRNA nor urinary beta-microglobulin levels (Veh: 1.71 ± 0.31; E2: 2.88 ± 0.78; E2+MPA: 3.07 ± 1.15 mg/day; ns) were altered. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results show that the effect of E2 on renal pro-inflammatory markers was not altered by the addition of MPA despite the significant increase in renal GR levels. Therefore, the renoprotective effects of MPA in midlife hormone therapy may be independent of renal GR-mediated changes in the immune profile.

Elevated Blood Pressure in Offspring of Rats Exposed to Diverse Chemicals During Pregnancy

Adverse intrauterine environments have been associated with increased risk of later cardiovascular disease and hypertension. In an animal model using diverse developmental toxicants, we measured blood pressure (BP), renal nephron endowment, renal glucocorticoid receptor (GR) gene expression, and serum aldosterone in offspring of pregnant Sprague Dawley rats exposed to dexamethasone (Dex), perfluorooctane sulfonate (PFOS), atrazine, perfluorononanoic acid (PFNA), arsenic, or nicotine. BP was assessed by tail cuff photoplethysmography, nephron endowment by confocal microscopy, and renal GR mRNA by qPCR. BP was also measured by telemetry, and corticosterone (CORT) was measured in resting or restrained Dex and atrazine offspring. Treated dams gained less weight during treatment in all groups except arsenic. There were chemical- and sex-specific effects on birth weight, but offspring body weights were similar by weaning. BP was higher in Dex, PFOS, atrazine, and PFNA male offspring by 7-10 weeks. Female offspring exhibited elevated BP at 10 weeks for PFNA and arsenic, and at 37 weeks for Dex, PFOS, and atrazine. Dex, PFOS, and atrazine offspring still exhibited elevated BP at 52-65 weeks of age; others did not. Elevated BP was associated with lower nephron counts. Dex, PFOS, and atrazine offspring had elevated renal GR gene expression. Elevations in BP were also observed in Dex and atrazine offspring by radiotelemetry. Atrazine offspring exhibited enhanced CORT response to restraint. Elevated offspring BP was induced by maternal exposure to toxicants. Because all treatments affected maternal gestational weight gain, maternal stress may be a common underlying factor in these observations.

Altered health outcomes in adult offspring of Sprague Dawley and Wistar rats undernourished during early or late pregnancy

Birth weight in humans has been inversely associated with adult disease risk. Results of animal studies have varied depending on species, strain, and treatment. We compared birth weight and adult health in offspring following 50% maternal undernutrition on gestation days (GD) 1-15 (UN1-15) or GD 10-21 (UN10-21) in Sprague Dawley and Wistar rats. Offspring from food-deprived dams were weighed and cross-fostered to control dams. Litters were weighed during lactation and initiating at weaning males were fed either control or a high-fat diet. Young and mature adult offspring were evaluated for obesity, blood pressure (BP), insulin response to oral glucose, and serum lipids. Nephron endowment, renal glucocorticoid receptor, and renin-aldosterone-angiotensin system components were measured. The UN10-21 groups had birth weights lower than controls and transient catch up growth by weaning. Neither strain demonstrated obesity or dyslipidemia following prenatal undernutrition, but long-term body weight deficits occurred in the UN groups of both strains. High-fat diet fed offspring gained more weight than control offspring without an effect of prenatal nutrition. Sprague Dawley were slightly more susceptible than Wistar rats to altered insulin response and increased BP following gestational undernutrition. Nephron endowment in Sprague Dawley but not Wistar offspring was lower in the UN10-21 groups. Glucocorticoid and renin-aldosterone-angiotensin system pathways were not altered. The most consistent effect of maternal undernutrition was elevated BP in offspring. Long-term health effects occurred with undernutrition during either window, but the UN10-21 period resulted in lower birth weight and more severe adult health effects.

Interaction of rat renal glucocorticoid receptor with Hsp90 and Hsp70 upon stress provoked by mercury

The influence of mercury on the association of rat kidney glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70 was investigated. The GR heterocomplexes with Hsp90 and Hsp70 were immunopurified from the renal cytosol of rats administered different doses of mercury (1, 2 and 3 mg Hg kg(-1) b.w.). A quantitative immunoblotting procedure was applied to determine the levels of GR, Hsp90 and two nucleocytoplasmic Hsp70 isoforms (constitutive Hsp73 and inducible Hsp72) in the renal cytosol, as well as the amounts of these proteins within GR heterocomplexes immunoprecipitated by anti-GR antibody. Mercury was found to stimulate GR association with all the examined Hsps. The most prominent effect of the metal was stimulation of Hsp72 interaction with GR. On the other hand, the metal administration led to an increase of Hsp90 level in the cytosol, while the cytosolic levels of Hsp70 isoforms remained unaltered. These findings suggest that association of Hsps, at least Hsp70, with the GR might be ascribed to changes in the affinity of their interaction rather than to changes in the Hsp availability in the cytosol. Therefore, GR heterocomplex assembly seems to be a controlled process enabling chaperoning and functioning of the GR to be in concert with physiological demands.

Age‐Dependent Decrease in Renal Glucocorticoid Receptor Function Is Reversed by Dietary Restriction in Mice

The effects of age and dietary restriction (alternate days of feeding for 3 months) on the concentration, activation, and DNase I digestion of nuclear-bound glucocorticoid receptors (GRs) in the kidney of male mice at two different ages (5 months as adult and 20 months as old) were investigated. A significant decrease (30%) in the concentration of renal GRs was observed in older ad libitum (AL)-fed mice as compared to the adult mice. Dietary restriction (DR) of older mice significantly increased (28%) the level of GRs as compared to the AL-fed control animals. The affinity of the receptor for the hormone remained the same for both AL- and DR-fed animals at both ages. Scatchard and slot blot analyses of the data confirmed the decreased level of renal GRs in older mice compared to the adult mice as well as an increased level of receptor in older DR mice. Activation studies of GRs by both salt and heat indicated a decreased (15-20%) activation of renal GRs in older animals compared to the adult mice in the AL-fed group. It was further observed that DR significantly enhanced (30%) the degree of both salt- and heat-dependent activation of GRs in older animals compared to the AL-fed animals of the age-matched group. DNase I digestion and extraction of nuclear-bound GR complexes showed a lower degree (26%) of extraction in older AL-fed animals compared to the adult animals. However, DR did not alter the pattern of digestibility of bound GR complexes. These above findings indicate that DR could reverse the decrease of GR function in older animals and may provide better adaptability of kidney in water and electrolyte balance.

Modulation of hepatic and renal glucocorticoid receptors during aging of mice.

The regulation of hepatic and renal glucocorticoid receptors (GRs) in young (4 weeks) and senescent (120 weeks) mice was studied. Significant changes in the level of GRs from liver and kidney were detected, whereas the affinity (Kd) for the hormone [3H]dexamethasone did not change in young and old mice. The concentration of GRs was markedly decreased in liver (25%) and kidney (33%) of old mice as compared to young ones. The magnitude of heat activation of GR complexes was more pronounced in both the tissues at young age compared to old. In addition, we have found changes in the heat activation-inhibition studies of GRs, using polyunsaturated fatty acids (PUFAs) as measured by binding to DNA-cellulose and purified nuclei. Linoleic acid (C18:2) exhibited significant decrease in heat activation of both hepatic and renal hormone-bound GR, with higher magnitude of inhibition in young liver (64%) and kidney (68%) as compared to old (41% and 43%, respectively). Arachidonic acid (C20:4) was also found to be an inhibitor of activation causing significant decrease in hepatic GR activation, with greater inhibition in young liver with respect to old, however, without any such difference in the kidney of young and old mice. Furthermore, DNase I digestion and extraction of nuclear-bound GR-complexes from both the tissues reveal lesser extraction in old liver (26%) and kidney (24%) compared to young tissues, indicating chromatin condensation with aging, thereby controlling the accessibility to such transcription factors as GRs. These findings indicate that the changes in the GR concentration, activation-modulation by PUFAs and chromatin organization, that take place during aging, may contribute to functional changes in glucocorticoid action mechanisms in senescent animals.

Effect of angiotensin II on rat renal cortical 11beta-hydroxysteroid dehydrogenase.

Renal 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) are subject to modulation by various endogenous factors. 11beta-HSDs convert glucocorticoids into inactive 11-ketones and thereby determine tissue levels of active glucocorticoids and thus the extent of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation. As such, modulation of the activity of renal 11beta-HSDs may contribute to the cascade of regulatory events involved in renal electrolyte water handling. We investigated whether renal 11beta-HSDs are modulated by elevated circulating angiotensin II. In rats infused for 2 wk with angiotensin II (250 ng/[kg x min] subcutaneously), plasma angiotensin II, aldosterone, and corticosterone were raised 5.1-, 10.7-, and 2.3-fold, respectively, compared with control rats. Angiotensin II infusion raised corticosterone 11beta-oxidation 1.46- and 1.35-fold in renal cortical proximal and distal tubules (enriched by Percoll centrifugation), respectively, but had no effect on 11beta-HSD1 and 11beta-HSD2 mRNA levels (semiquantitative reverse transcriptase polymerase chain reaction), except for distal tubular 11beta-HSD1 mRNA, which was decreased to 50%. In vitro treatment of freshly isolated tubules with angiotensin II for 45 min prior to assessment of 11beta-HSD activity showed no direct acute effects of angiotensin II on tubular corticosterone 11beta-oxidation. The enhanced renal tubular corticosterone 11beta-oxidation in vivo may partly protect renal GR and MR from elevated plasma corticosterone on angiotensin II infusion.

Apparent mineralocorticoid excess, pseudohypoaldosteronism, and urinary electrolyte excretion: toward a redefinition of mineralocorticoid action

Patients with apparent mineralocorticoid excess (AME) have low or absent activity of the enzyme 11 beta OH steroid dehydrogenase (11SD), and inappropriately high intrarenal levels of cortisol resulting in Na+ retention and hypertension. Pseudohypoaldosteronism (PHA), in contrast, is characterized by salt wasting despite hyperaldosteronemia, reflecting low or absent mineralocorticoid receptors (MR). Although AME is presumed to reflect inappropriate cortisol occupancy of MR, several features also suggest inappropriate occupancy of glucocorticoid receptors (GR). To test this possibility, we administered carbenoxolone, which is known to block 11SD, to four patients with PHA, and observed marked mineralocorticoid effects, e.g., antinatriuresis and elevated plasma bicarbonate. To further test the possibility that occupancy of renal GR may induce a classical mineralocorticoid response, we administered the highly specific glucocorticoid RU 28362 to adrenalectomized rats and showed that it has profound antinatriuretic effects. Finally, by selectively blocking MR with RU 28318 or GR with RU 38486, we have shown that corticosterone, the physiologic glucocorticoid in rats, has an antinatriuretic effect in adrenalectomized rats via either MR or GR occupancy. Previous studies have clearly shown that MR are inherently nonselective and have equivalent intrinsic affinity for aldosterone, corticosterone, and cortisol. The present studies suggest that this nonselectivity includes the nuclear response element to which either MR or GR may bind to elicit a mineralocorticoid effect, and further underscore the importance of the enzyme 11SD in the specific mineralocorticoid action of aldosterone.

Mineralocorticoid and Renal Receptor Binding Activity of 21-Deoxyaldosterone

Since several aldosterone metabolites are known to be active, we have assessed the mineralocorticoid biological and renal receptor binding activities of the aldosterone metabolites, 21-deoxyaldosterone (21-deoxy-Aldo), 21-deoxytetrahydroaldosterone (21-deoxy-THAldo), and 3 alpha, 5 beta-tetrahydroaldosterone (THAldo). We synthesized these steroids by bioreduction of aldosterone with intestinal bacteria. Mineralocorticoid agonist activity of 21-deoxy-Aldo, 21-deoxy-THAldo and THAldo, determined by bioassay using adrenalectomized rats, was 1-5%, less than 0.01%, and 0.1-0.5% that of aldosterone, respectively. 21-Deoxy-Aldo showed no antagonist activity. The relative affinity in competing with [3H]aldosterone for binding to mineralocorticoid receptors in adrenalectomized rat kidney cytosols was 94%, less than 0.01%, and less than 0.01% that of aldosterone. The relative binding affinity for rat renal glucocorticoid receptors was 23%, less than 0.01%, and less than 0.01% that of dexamethasone, and for corticosteroid-binding globulin 17%, less than 0.01%, and less than 0.01% that of cortisol. These results show that the naturally occurring steroid, 21-deoxy-Aldo, possesses mineralocorticoid agonist activity which is equivalent to that of 11-deoxycorticosterone, and has substantial affinity for rat renal mineralocorticoid and glucocorticoid receptors. The results also implicate the pathophysiological role of 21-deoxy-Aldo as a potential mineralocorticoid in 21-hydroxylase deficiency, where urinary excretion of this steroid is invariably elevated.

Partial characterization and ontogeny of renal cytosolic glucocorticoid receptors in mouse kidney

The glucocorticoid receptor (GR) was partially characterized in mouse renal cytosol. A sensitive and reproducible [ 3H]dexamethasone binding assay suitable for use with small quantities of cytosolic protein, was developed. Studies denned the optimal equilibrium binding conditions, metabolism of [ 3H]dexamethasone in adult renal cytosol, specificity of binding of the GR, and molecular weight of the GR-[ 3H]dexamethasone complex by gel filtration chromatography. The assay was subsequently used to measure the renal GR during different stages of foetal and postnatal development, as well as in glomerular and renal tubular preparations from adult mice. An almost linear increase in GR occurred from day 13 to day 18 of gestation with levels rising from 100 to 201 fmol/mg cytosol protein; this was followed by a sharp rise in receptor concentration just after birth to 343 fmol/mg cytosol protein. Adult levels, 410–433 fmol/mg cytosol protein, were reached by 2 weeks after birth. The equilibrium dissociation constants ( K d) of the [ 3H]dexamethasone-receptor complex were similar in adult and in embryonic cytosols (range, 2.8−11.8 nM; x ± SD = 6.5 ± 2.9 nM ). Specific binding was assessed to be 3- to 5-fold greater in tubular than in glomerular preparations. These data on the localization and ontogeny of GR during murine metanephric development provide a basis for study of glucocorticoid-mediated effects on various models of congenital and acquired renal disease.

Renal Receptor-Binding Activity of Reduced Metabolites of Aldosterone: Evidence for a Mineralocorticoid Effect outside of the Classic Aldosterone Receptor System*

Reduced metabolites of aldosterone have been shown to have antinatriuretic and kaliuretic effects. We have studied the ability of four reduced metabolites of aldosterone to compete with [3H]aldosterone and [3H]dexamethasone for binding to the mineralocorticoid and glucocorticoid receptors of the kidney using adrenalectomized rat renal slices and cytosol, respectively, as sources of the binding proteins. 5 alpha-Dihydroaldosterone had 18.9% the ability to compete with [3H]aldosterone for binding to the cytoplasmic receptor of adrenalectomized rat renal slices in comparison to unlabeled aldosterone. Its antinatriuretic potency varied between 7-17%. Its ability to compete with [3H]dexamethasone for binding to the renal glucocorticoid receptor was only 1.9% in comparison to unlabeled dexamethasone. The relative competitive activities of 3 beta,5 alpha-tetrahydroaldosterone and 3 beta,5 beta-tetrahydroaldosterone with [3H]aldosterone to adrenalectomized rat renal slices cytosol were 1.26% and 0.05%, respectively, in comparison to unlabeled aldosterone. Their reported mineralocorticoid activities using the adrenalectomized rat bioassay (antinatriuresis) were 0.1-0.4% and 0.15%, respectively, in comparison to aldosterone. The most important aldosterone metabolite 3 alpha,5 beta-tetrahydroaldosterone showed negligible competitive activity with [3H]aldosterone or [3H]dexamethasone for the renal corticoid type I or type II receptors, respectively. However, this compound has been reported and confirmed to have weak but clear-cut mineralocorticoid activity (approximately 1/100th that of aldosterone). The mineralocorticoid activity of 3 alpha,5 beta-tetrahydroaldosterone cannot be explained by a mechanism involving the classic renal mineralocorticoid receptor. The mechanism could involve an alternative receptor system, a nonreceptor-mediated renal mechanism, or the conversion to a metabolite that would interact with classic receptors.

16α,18-dihydroxydeoxycorticosterone and the binding of aldosterone to mineralocorticoid receptors in kidney of adrenalectomized rats

It has been recently suggested that 16α,18-dihydroxydeoxycorticosterone (16α,18-diOHDOC) may act as a “positive allosteric effector” of the binding of aldosterone to mineralocorticoid receptors. To test this hypothesis, a series of in vitro and in vivo studies examining the effect of 16α,18-diOHDOC on tritiated aldosterone ( 3HA) binding to mineralocorticoid receptors was performed. Using kidney slices from adrenalectomized rats, in vitro incubations were made for 20′ at 37C, over a range of concentrations of 3HA plus tenfold dexamethasone to confine tracer binding to mineralocorticoid receptors. At no concentration of 3HA did 16α,18-diOHDOC enhance binding; at all tracer concentrations a slight competing effect was observed. When 3HA was injected into rats in vivo with and without 16α,18-diOHDOC, a similar insignificant displacement of 3HA binding was seen in renal cytoplasmic fractions from adrenalectomized test rats. Additional in vitro studies were performed in an attempt to elucidate the mechanism of postulated action of 16α,18-diOHDOC. Neither renal cytoplasmic binding of oestradiol, postulated as a secondary pathway for steroid influenced Na + retention, nor the binding of dexamethasone to renal glucocorticoid receptors, was altered by 16α,18-diOHDOC. Binding of tritiated 16α,18-iOHDOC in renal cyto-plasmic fractions was shown to be non specific, in that it could not be displaced by excess unlabelled 16α,18-diOHDOC. Finally, in a series of in vivo experiments using adrenalectomized rats, we could not show any effect of 16α,18-diOHDOC on urinary electrolyte excretion, either alone or in combination with low doses of aldosterone. Accordingly, we can find no evidence for 16α-diOHDOC having a direct effect on the kidney: in particular, there would appear as yet no molecular evidence for 16α,18-diOHDOC being a positive allosteric effector of aldosterone.

Separate effects of aldosterone, DOCA, and methylprednisolone on renal Na-K-ATPase,.

Separate effects of aldosterone, DOCA, and methylprednisolone on renal Na-K-ATPase,.