Salt-Sensitive Hypertension of the Renal Tubular Cell-Specific NFAT5 (Nuclear Factor of Activated T-Cells 5) Knockout Mice.

[Figure: see text].

Regulation of Rhcg, an ammonia transporter, by aldosterone in the kidney.

Rhesus C glycoprotein (Rhcg), an ammonia transporter, is a key molecule in urinary acid excretion and is expressed mainly in the intercalated cells (ICs) of the renal collecting duct. In the present study we investigated the role of aldosterone in the regulation of Rhcg expression. In in vivo experiments using C57BL/6J mice, Western blot analysis showed that continuous subcutaneous administration of aldosterone increased the expression of Rhcg in membrane fraction of the kidney. Supplementation of potassium inhibited the effect of aldosterone on the Rhcg. Next, mice were subjected to adrenalectomy with or without administration of aldosterone, and then ad libitum 0.14 M NH4Cl containing water was given. NH4Cl load increased the expression of Rhcg in membrane fraction. Adrenalectomy decreased NH4Cl-induced Rhcg expression, which was restored by administration of aldosterone. Immunohistochemical studies revealed that NH4Cl load induced the localization of Rhcg at the apical membrane of ICs in the outer medullary collecting duct. Adrenalectomy decreased NH4Cl-induced membrane localization of Rhcg, which was restored by administration of aldosterone. For in vitro experiments, IN-IC cells, an immortalized cell line stably expressing Flag-tagged Rhcg (Rhcg-Flag), were used. Western blot analysis showed that aldosterone increased the expression of Rhcg-Flag in membrane fraction, while the increase in extracellular potassium level inhibited the effect of aldosterone. Both spironolactone and GÓ§6983, a PKC inhibitor, inhibited the expression of Rhcg-Flag in the membrane fraction. These results suggest that aldosterone regulates the membrane expression of Rhcg through the mineralocorticoid receptor and PKC pathways, which is modulated by extracellular potassium level.

Differentiation of endogenous erythropoietin and exogenous ESAs by Western blotting.

Doping tests for the illegal use of erythropoiesis-stimulating agents (ESAs) have been developed. We developed a new Western blotting method to detect and distinguish endogenous erythropoietin (Epo, 35-38 kDa) and exogenous ESAs (epoetin α and ß, 38-42 kDa; darbepoetin α, 47-50 kDa; epoetin ß pegol, 93-110 kDa). Epo and ESAs are glycoproteins and deglycosylation using peptide-N-glycosidase F shifted all Epo and ESA bands except epoetin ß pegol to 22 kDa. We cut the bands of Epo and ESAs from SDS-PAGE gels and analyzed them by Liquid Chromatography/Mass Spectrometry (LC/MS). LC/MS detected all endogenous Epo and exogenous ESAs as deglycosylated 22 kDa Epo, indicating that LC/MS analysis could confirm the presence of Epo or ESA, but could not distinguish between endogenous Epo and exogenous ESAs. We propose the following Epo doping tests: 1) detect Epo or ESAs by Western blotting of the glycosylated form; 2) increase the reliability by the band shift following deglycosylation; and 3) complete confirmation of Epo or ESA by LC/MS analysis using cut gels. One of the advantages of our method is that pre-purification of samples for Epo is not required in our Western blotting.

Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation.

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin ß pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.

Insufficiency of urinary acid excretion of overweight or obese patients with chronic kidney disease and its involvement with renal tubular injury.

AIM: Metabolic acidosis occurs due to insufficient urinary ammonium excretion as chronic kidney disease (CKD) advances. Because obese subjects tend to have excessive consumption of protein and sodium chloride, they are prone to chronic acid loading and may therefore be predisposed to acid-induced kidney injury. We investigated the involvement of obesity in ammoniagenesis within damaged kidneys. METHODS: In the clinical study, urinary ammonium excretion was compared between 13 normal-weight and 15 overweight/obese CKD outpatients whose creatinine clearance was higher than 25 mL/min. For animal experiments, NH4 Cl was loaded to KKAy/TaJcl (KKAy), a metabolic syndrome model, and control BALB/c mice for 20 weeks. Kidney injury was evaluated through histological analysis and the expression of proinflammatory markers. RESULTS: Urinary ammonium excretion was lower in overweight/obese patients than in normal-weight patients, while intakes of protein and sodium chloride were higher in overweight/obese patients, implying that subclinical metabolic acidosis occurs in overweight/obese patients. The increase in urinary ammonium excretion induced by NH4 Cl loading was attenuated in KKAy mice after 16 weeks, whereas the increase was maintained in BALB/c mice throughout the study period. Histological study and real-time polymerase chain reaction analysis showed proximal tubular injury and enhanced expression levels of neutrophil gelatinase-associated lipocalin (NGAL) protein and messenger RNA, respectively, in KKAy mice but not in BALB/c mice. Finally, urinary NGAL concentration was higher in overweight/obese patients than in normal-weight patients in the early stage of CKD. CONCLUSION: Obesity could facilitate the induction of subclinical metabolic acidosis and acid accumulation in the kidney, which may potentially exacerbate kidney injury in CKD patients.

Fludrocortisone stimulates erythropoietin production in the intercalated cells of the collecting ducts.

Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.

TSS-Seq analysis of low pH-induced gene expression in intercalated cells in the renal collecting duct.

Metabolic acidosis often results from chronic kidney disease; in turn, metabolic acidosis accelerates the progression of kidney injury. The mechanisms for how acidosis facilitates kidney injury are not fully understood. To investigate whether low pH directly affects the expression of genes controlling local homeostasis in renal tubules, we performed transcription start site sequencing (TSS-Seq) using IN-IC cells, a cell line derived from rat renal collecting duct intercalated cells, with acid loading for 24 h. Peak calling identified 651 up-regulated and 128 down-regulated TSSs at pH 7.0 compared with those at pH 7.4. Among them, 424 and 38 TSSs were ≥ 1.0 and ≤ -1.0 in Log2 fold change, which were annotated to 193 up-regulated and 34 down-regulated genes, respectively. We used gene ontology analysis and manual curation to profile the up-regulated genes. The analysis revealed that many up-regulated genes are involved in renal fibrosis, implying potential molecular mechanisms induced by metabolic acidosis. To verify the activity of the ubiquitin-proteasome system (UPS), a candidate pathway activated by acidosis, we examined the expression of proteins from cells treated with a proteasome inhibitor, MG132. The expression of ubiquitinated proteins was greater at pH 7.0 than at pH 7.4, suggesting that low pH activates the UPS. The in vivo study demonstrated that acid loading increased the expression of ubiquitin proteins in the collecting duct cells in mouse kidneys. Motif analysis revealed Egr1, the mRNA expression of which was increased at low pH, as a candidate factor that possibly stimulates gene expression in response to low pH. In conclusion, metabolic acidosis can facilitate renal injury and fibrosis during kidney disease by locally activating various pathways in the renal tubules.

Renal tubular acidosis complicated with hyponatremia due to cortisol insufficiency.

Adrenocortical insufficiency such as occurs in Addison's disease causes hyponatremia and renal tubular acidosis (RTA). Hyponatremia results from both aldosterone and cortisol insufficiency. RTA is due to aldosterone insufficiency. The involvement of cortisol in RTA is unclear. Here, we report a woman in her 70s who was admitted to our hospital with severe hyponatremia (106 mEq/l) and RTA. The patient exhibited low plasma cortisol levels with little response to rapid adrenocorticotropic hormone loading. In contrast, the plasma aldosterone concentration was maintained at or above the normal range. Hydrocortisone replacement greatly improved both the hyponatremia and RTA. This case suggests that both aldosterone and cortisol are involved in acid secretion from the kidney.

Low-density lipoprotein apheresis for proteinuria in lupus nephritis with intraglomerular foam cells containing cholesterol crystals.

A 28-year-old woman with systemic lupus erythematosus was referred to our hospital due to nephrotic-level proteinuria despite approximately 1 year of treatment with 50 to 60 mg/d of prednisolone and 100 to 150 mg/d of cyclosporine with methylprednisolone pulse therapy. Kidney biopsy showed diffuse global lupus nephritis (World Health Organization class 4-G A/C) with many intraglomerular foam cells containing cholesterol crystals. Surprisingly, proteinuria diminished after only 5 low-density lipoprotein (LDL) cholesterol apheresis sessions. This case demonstrated the potential of LDL apheresis to exhibit a remarkable effect on not only focal segmental glomerulosclerosis, but also other types of nephritis, particularly nephritis with intraglomerular foam cells.

Expression of three isoforms of Na-K-2Cl cotransporter (NKCC2) in the kidney and regulation by dehydration.

Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro. Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro, NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo. These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b.

Reevaluation of erythropoietin production by the nephron.

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in ß-intercalated or non α/non ß-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.

Effects of atrial natriuretic peptide on bicarbonate transport in long- and short-looped medullary thick ascending limbs of rats.

Atrial natriuretic peptide (ANP) is known to influence NaCl transport in the medullary thick ascending limbs (MAL), where the largest NaCl reabsorption occurs among distal nephron segments in response to arginine vasopressin (AVP). In the present study, we investigated the effect of ANP on bicarbonate (HCO3 (-)) transport in the MAL using an isolated tubule perfusion technique. The HCO3 (-) concentration was measured using free-flow ultramicro-fluorometer. We first observed basal HCO3 (-) reabsorption in both long- and short-looped MALs (lMALs, and sMALs, respectively). AVP inhibited HCO3 (-) reabsorption in both lMALs and sMALs, whereas ANP did not change HCO3 (-) transport. However, in the presence of AVP, ANP restored the HCO3 (-) reabsorption inhibited by AVP both in lMAL and sMAL. The effects of ANP on HCO3 (-) transport was mimicked by cyclic GMP. The mRNA expression level of the vasopressin V2 receptor in lMALs was significantly higher than in sMALs, whereas expression of the V1a receptor was unchanged. In summary, AVP inhibits HCO3 (-) transport, and ANP counteracts the action of AVP on HCO3 (-) transport both in lMALs and sMALs.

Vasopressin V1a receptor is required for nucleocytoplasmic transport of mineralocorticoid receptor.

We previously reported that a deficiency in the vasopressin V1a receptor (V1aR) results in type 4 renal tubular acidosis, which suggests that vasopressin exerts direct effects on the physiological actions of aldosterone. We investigated the role of vasopressin for nucleocytoplasmic transport of mineralocorticoid receptor (MR) in the intercalated cells. Vasopressin V1aR-deficient (V1aR(-/-)) mice showed largely decreased expression of MR and 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) in the medulla of the kidney, which was partially ameliorated by fludrocortisone treatment. The incubation of IN-IC cells, an intercalated cell line established from temperature-sensitive SV40 large T antigen-expressing rats, with aldosterone or vasopressin increased the nuclear-to-cytoplasmic ratio of the MR from 11.2 to 47.2% and from 18.7 to 61.2%, respectively, in 30 min without any changes in MR expression from the whole cell extract. The immunohistochemistry analysis of the IN-IC cells revealed the nuclear accumulation of MRs after a 30-min incubation with aldosterone or vasopressin. These effects were accompanied by an increase in regulator of chromosome condensation-1 (RCC-1) due to aldosterone and a decrease in Ran GTPase-activating protein 1 (Ran Gap1) due to vasopressin. RNA interference against V1aR abolished the nuclear accumulation of MR induced by aldosterone or vasopressin. Vasopressin increased PKCα and -ß(1) expression, and aldosterone increased PKCδ and -ζ expression, but these effects were abolished with a V1aR knockdown. These results suggest that vasopressin directly regulates the nucleocytoplasmic transport of MRs via the V1aR in the intercalated cells of the collecting ducts.

Aldosterone requires vasopressin V1a receptors on intercalated cells to mediate acid-base homeostasis.

Both aldosterone and luminal vasopressin may contribute to the maintenance of acid-base homeostasis, but the functional relationship between these hormones is not well understood. The effects of luminal vasopressin likely result from its interaction with V1a receptors on the luminal membranes of intercalated cells in the collecting duct. Here, we found that mice lacking the V1a receptor exhibit type 4 renal tubular acidosis. The administration of the mineralocorticoid agonist fludrocortisone ameliorated the acidosis by restoring excretion of urinary ammonium via increased expression of Rhcg and H-K-ATPase and decreased expression of H-ATPase. In a cell line of intercalated cells established from transgenic rats expressing the mineralocorticoid and V1a receptors, but not V2 receptors, knockdown of the V1a receptor gene abrogated the effects of aldosterone on H-K-ATPase, Rhcg, and H-ATPase expression. These data suggest that defects in the vasopressin V1a receptor in intercalated cells can cause type 4 renal tubular acidosis and that the tubular effects of aldosterone depend on a functional V1a receptor in the intercalated cells.

Low pH stimulates vasopressin V2 receptor promoter activity and enhances downregulation induced by V1a receptor stimulation.

Arginine vasopressin (AVP) plays a key role in the urine concentration mechanism via the vasopressin V2 receptor (V2R) and aquaporin 2 (AQP2) in the kidney. It is well known that V2R is localized on the basolateral side and the V1a receptor (V1aR) is distributed on the luminal side of the collecting ducts. Previously, we reported an increase of V1aR mRNA and a decrease of V2R mRNA in the collecting ducts under chronic metabolic acidosis. However, the regulatory mechanism of V2R in acidic conditions has not yet been determined. In the present study, we investigated the effect of changes in pH on V2R promoter activity, using the LLC-PK(1) cell line stably expressing rat V1aR (LLC-PK(1)/rV1aR). The rV2R promoter activity was significantly increased at 12 h after the incubation in low-pH conditions, which was sustained for 24 h. mRNA and protein expressions of V2R were also increased in low-pH conditions. V1aR stimulation suppressed rV2R promoter activity in a pH-dependent manner. PKA and JNK inhibitors suppressed rV2R promoter activity in both neutral and low-pH conditions without FBS. However, a JNK inhibitor prevented the increase of V2R promoter activity only in low-pH conditions in the presence of FBS. In summary, V2R expression is increased at transcriptional, mRNA, and protein levels in LLC-PK(1)/rV1aR cells under low-pH conditions. Acidic condition-induced V2R enhancement was suppressed by V1aR stimulation, suggesting the crucial role of V1aR in water and electrolyte homeostasis in acidosis.

Vasopressin and hyperosmolality regulate NKCC1 expression in rat OMCD.

Secretory-type Na-K-2Cl cotransporter (NKCC1) is known to play roles in both acid and sodium excretion, and is more abundant in dehydration. To determine the mechanisms by which dehydration stimulates NKCC1 expression, the effects of vasopressin, oxytocin and hyperosmolality on NKCC1 mRNA and protein expressions in the outer medullary collecting duct (OMCD) of rats were investigated using RT-competitive PCR and western blot analysis. Microdissected OMCD was incubated in isotonic or hypertonic solution, or with AVP or oxytocin for 60 min at 37 degrees C. Hyperosmolality induced by NaCl, mannitol or raffinose increased NKCC1 mRNA expression in OMCD by 130-240% in vitro. The stimulation of NKCC1 mRNA expression by NaCl was highest at 690 mosmol kg(-1) H(2)O and gradually decreased at higher osmolalities. The incubation of OMCD with AVP (10(-7) M) for 60 min increased NKCC1 mRNA expression by 100%. The administration of AVP to rats for 4 days using an osmotic mini-pump also increased NKCC1 mRNA and protein expressions in OMCD by 130%. In contrast, oxytocin (10(-7) M) did not stimulate the NKCC1 mRNA expression in OMCD in vitro. Chronic injection of oxytocin increased the NKCC1 mRNA expression by 36%. These data showed that hyperosmolality and vasopressin stimulate NKCC1 mRNA and protein expressions in rat OMCD. It is concluded that NKCC1 expression is regulated directly and indirectly by vasopressin.

Different Mechanisms for the Progression of CKD with ACE Gene Polymorphisms.

BACKGROUND/AIMS: The blockade of the renin-angiotensin-aldosterone system is the major target of efforts to prevent the progression of chronic kidney disease (CKD). Dual blockade with angiotensin-converting enzyme (ACE) inhibitor and angiotensin II receptor blocker has been reported to show additive renoprotection. However, three types of insertion/deletion (I/D) polymorphism have been reported, and it is unclear whether the dual blockade is effective for all the ACE genotypes. METHODS: We treated 93 CKD patients with or without dual blockade and analyzed the effects on blood pressure (BP), proteinuria, progression of CKD and the relationship to I/D ACE polymorphisms. RESULTS: After long-term medication (average 33 +/- 2 months), BP decreased in all the genotype groups. However, urinary protein excretion decreased only in the II and DI groups (II: -27.1%, DI: -20.5%, DD: +0.8%). In the II and DI groups, amelioration of the progression of renal failure was correlated with reductions in BP and urinary protein excretion. However, the progression rate of renal failure was not correlated with proteinuria in the DD group. CONCLUSION: Proteinuria and BP are key factors for the progression of CKD in II/DI patients, while controlling the BP rather than reducing the proteinuria appears to be crucial in DD patients.

Acute and chronic metabolic acidosis interferes with aquaporin-2 translocation in the rat kidney collecting ducts.

Renal aquaporin-2 (AQP2) expression plays a key role in urine concentration. However, it is not known whether metabolic acidosis affects urine-concentrating ability through AQP2 expression in the kidney and urine. We examined urinary excretion and renal expression of AQP2 in control and acidosis rats, using RT-competitive PCR, immunoblot and immunocytochemistry. Urinary excretion of AQP2 is decreased by 92% even with the increase in AQP2 mRNA and protein expressions in the collecting ducts by metabolic acidosis in rats. Urine osmolality in control rats was 1670+/-198 mOsm per kg H(2)O, and immunocytochemistry revealed the presence of AQP2 in the apical plasma membrane of the principal cells in the collecting ducts. Urine osmolality in acidosis rats was lower than that in control (1397+/-243 mOsm per kg H(2)O), and immunocytochemistry showed the diffuse presence of AQP2 in the cytoplasm of the principal cells. Differential centrifugation-coupled immunoblot showed a significant decrease in the ratio of AQP2 in plasma membrane-enriched fraction to that in intracellular vesicle-enriched fraction by metabolic acidosis. In summary, AQP2 translocation is largely decreased by metabolic acidosis even with increased expression in the collecting ducts. A disorder of AQP2 translocation in the collecting ducts with acidosis may be responsible for the diuresis in patients with chronic renal failure.

A case with acute renal failure and subsequent nephrotic syndrome.

Nephrotic syndrome due to secondary amyloidosis is not so common, and the prognosis depends on primary disease. We report a case of secondary amyloidosis caused by Takayasu's arteritis. Sustained high fever and acute renal failure proceeded to the occurrence of nephrotic syndrome. Secondary amyloidosis was diagnosed by renal biopsy before the diagnosis of primary disease. She was completely recovered from nephrotic syndrome after two years' treatment with prednisolone, aspirin, and dimethyl sulfoxide. This rare case provides meaningful suggestions for the diagnosis and treatment of acute renal failure and nephrotic syndrome caused by secondary amyloidosis.