Gheun-Ho Kim

Aldosterone-mediated regulation of ENaC α, β, and γ subunit proteins in rat kidney

Aldosterone stimulates sodium transport in the renal collecting duct by activating the epithelial sodium channel (ENaC). To investigate the basis of this effect, we have developed a novel set of rabbit polyclonal antibodies to the 3 subunits of ENaC and have determined the abundance and distribution of ENaC subunits in the principal cells of the rat renal collecting duct. Elevated circulating aldosterone (due to either dietary NaCl restriction or aldosterone infusion) markedly increased the abundance of alphaENaC protein without increasing the abundance of the beta and gamma subunits. Thus, alphaENaC is selectively induced by aldosterone. In addition, immunofluorescence immunolocalization showed a striking redistribution in ENaC labeling to the apical region of the collecting duct principal cells. Finally, aldosterone induced a shift in molecular weight of gammaENaC from 85 kDa to 70 kDa, consistent with physiological proteolytic clipping of the extracellular loop as postulated previously. Thus, at the protein level, the response of ENaC to aldosterone stimulation is heterogenous, with both quantitative and qualitative changes that can explain observed increases in ENaC-mediated sodium transport.

Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle’s loop

To investigate whether the enhancement of thick ascending limb (TAL) NaCl transport in response to long-term increases in circulating vasopressin concentration is associated with increased expression levels of the apical Na-K-2Cl cotransporter in the rat TAL, we have carried out immunoblotting and immunofluorescence studies using affinity-purified, peptide-directed antibodies. Semiquantitative immunoblotting studies demonstrated a marked increase (193% of controls) in Na-K-2Cl cotransporter band density in response to restriction of water intake to 15 ml/day for 7 days. In contrast, the expression levels of two other apical proteins of the TAL (the type 3 Na/H exchanger and Tamm-Horsfall protein) were unchanged in the outer medulla. A 7-day subcutaneous infusion of the V2receptor-selective vasopressin analog, 1-desamino-[8-d-arginine]vasopressin (DDAVP), to Brattleboro rats also markedly increased Na-K-2Cl cotransporter expression in the outer medulla (183% of controls). Immunofluorescence localization in outer medullary tissue sections confirmed the increase in Na-K-2Cl cotransporter expression in response to DDAVP. We conclude that vasopressin strongly upregulates the expression of the Na-K-2Cl cotransporter of the TAL and that it is likely to play an important role in the long-term regulation of the countercurrent multiplication system.

Association of depression with malnutrition in chronic hemodialysis patients

BACKGROUND Depression is the most common psychological complication and may increase mortality in chronic hemodialysis patients. Because depression could be associated with poor oral intake and activation of proinflammatory cytokines that could further increase mortality by malnutrition, we investigated the relation between depression and nutritional status in chronic hemodialysis patients. METHODS Sixty-two Korean patients completed the Beck Depression Inventory (BDI) questionnaire, and the diagnosis of depression was confirmed by Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria for major depressive disorder. Nutritional status was evaluated using serum albumin level, normalized protein catabolic rate, subjective global assessment (SGA), and anthropometric measurement. RESULTS Mean BDI score was 22.7 +/- 11.4, and 35 patients (56.5%) had a BDI score greater than 21, which is the suggested cutoff score for the diagnosis of depression for the Korean population. Of 40 patients who had a score higher than 18 on the BDI, 34 patients met DSM-IV criteria for major depressive disorder. BDI score correlated negatively with a variety of nutritional parameters: serum albumin level (r = -0.47; P < 0.001), normalized protein catabolic rate (r = -0.32; P < 0.05), SGA (r = -0.47; P < 0.01), triceps skinfold thickness (r = -0.40; P < 0.05), midarm muscle circumference (r = -0.57; P < 0.01), and body mass index (r = -0.28; P < 0.05). Multiple regression analysis also identified BDI score as an independent determinant for all kinds of nutritional parameters. CONCLUSION In patients on chronic hemodialysis therapy, depression is related closely to nutritional status and could be an independent risk factor for malnutrition.

Regulation of the Abundance of Renal Sodium Transporters and Channels by Vasopressin

Vasopressin plays a role in both salt and water balance in the kidney. Classic studies, utilizing isolated perfused tubules, have revealed that vasopressin increases sodium reabsorption in the kidney thick ascending limb and the collecting duct. Furthermore, the activity of several sodium transport proteins expressed in these segments, such as the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) and the epithelial sodium channel (ENaC), have been shown to be directly increased by vasopressin. Increased protein abundance might be one means through which sodium transporter and channel activity is enhanced. We have used immunoblotting and immunohistochemistry in order to investigate the regulation of abundance of the major sodium transporters and channels expressed along the renal tubule in response to vasopressin. Chronic (7-day) studies were performed in which vasopressin levels were elevated either endogenously by water restriction of Sprague-Dawley rats or exogenously through infusion of the vasopressin V2-receptor-selective agonist, dDAVP (1-deamino-8d-arginine-vasopressin), to Brattleboro rats. We found a significant increase in protein abundance for NKCC2 and the beta- and gamma-subunits of ENaC with either water restriction or dDAVP infusion. The alpha-subunit of Na-K-ATPase was increased by water restriction, but not by dDAVP infusion, and alpha-ENaC and the thiazide-sensitive cotransporter (NCC) were increased by dDAVP infusion but not by water restriction. Acute (60-min) in vivo exposure to dDAVP led to an increase in both beta- and gamma-ENaC abundance in kidney cortex homogenates, displaying the rapid nature of some of these changes. Overall these increases in sodium transporter and channel abundances likely contribute to both the antidiuretic and antinatriuretic actions of vasopressin.

Antidiuretic Effect of Hydrochlorothiazide in Lithium-Induced Nephrogenic Diabetes Insipidus Is Associated with Upregulation of Aquaporin-2, Na-Cl Co-transporter, and Epithelial Sodium Channel

Thiazides have been used in patients with nephrogenic diabetes insipidus (NDI) to decrease urine volume, but the mechanism by which it produces the paradoxic antidiuretic effect remains unclear. Previous studies have reported that downregulation of aquaporin-2 (AQP2) is important for the development of lithium-induced (Li-induced) polyuria and that hydrochlorothiazide (HCTZ) increases renal papillary osmolality and Na(+) concentration in Brattleboro rats. For elucidating the molecular basis of the antidiuretic action of HCTZ in diabetes insipidus, whether administration of HCTZ may affect the expression of AQP2 and major renal Na(+) transporters in Li-induced NDI rats was investigated, using semiquantitative immunoblotting and immunohistochemistry. After feeding male Sprague-Dawley rats Li chloride-containing rat diet for 4 wk, HCTZ or vehicle was infused subcutaneously via osmotic minipump. Urine output was significantly decreased by HCTZ treatment, whereas it was not changed in vehicle-treated rats. Urine osmolality was also higher in HCTZ-treated rats than in vehicle-treated rats. Semiquantitative immunoblotting using whole-kidney homogenates revealed that HCTZ treatment caused a significant partial recovery in AQP2 abundance from Li-induced downregulation. AQP2 immunohistochemistry showed compatible findings with the immunoblot results in both cortex and medulla. The abundances of thiazide-sensitive NaCl co-transporter and alpha-epithelial sodium channel were increased by HCTZ treatment. Notably, HCTZ treatment induced a shift in molecular weight of gamma-epithelial sodium channel from 85 to 70 kD, consistent with previously demonstrated aldosterone stimulation. The upregulation of AQP2 and distal renal Na(+) transporters in response to HCTZ treatment may account for the antidiuretic action of HCTZ in NDI.

Impact of burn size and initial serum albumin level on Acute renal failure occurring in major burn

Background: Acute renal failure (ARF) is not a rare occurrence in severe burns and is an important complication leading to an increase in mortality. The severity of the burn is largely determined by the burn size, and severe burns are likely to cause enough loss of extracellular fluid and albumin from plasma volume to produce shock and hypoalbuminemia. Hypothesis: We hypothesized that initial serum albumin level may be useful as an indicator of prognosis and severity of injury in burned patients. Methods: The clinical characteristics of 147 adult patients with second- and third-degree burns covering 30% or more of their body surface area were analyzed retrospectively. Logistic regression was used to estimate the relative risks of ARF and mortality associated with the larger burn size and the lower serum albumin level at admission. Results: Mean burned body surface was 60.0 ± 21.8% (range 30–100%). Twenty-eight (19.0%) out of 147 patients experienced ARF, defined as a serum creatinine ≧2 mg/dl, during the admission. The patients with ARF had larger burn size (79.5 ± 15.4 vs. 55.3 ± 20.5%, p < 0.0001) and lower serum albumin concentration at admission (1.92 ± 0.66 vs. 2.48 ± 0.82 g/dl, p < 0.0005) compared with those without ARF. All patients with ARF expired, whereas 29.4% (35/119) of the patients without ARF died. The burn size ≧65% was associated with a risk of ARF that was 9.9 times and with a risk of death that was 14.2 times as high as that for the burn size <65%. The initial serum albumin level <2.5 g/dl was associated with a risk of death that was 2.7 times as high as that for the initial serum albumin level ≧2.5 g/dl. Conclusions: When major burns are complicated by ARF, the mortality rate increases significantly. Burn size is an independent predictor of ARF occurring in major burns. Initially depressed serum albumin level is associated with an increase in mortality in the major burn patients.

Long-Term Adaptation of Renal Ion Transporters to Chronic Diuretic Treatment

Loop and thiazide diuretics are clinically useful to induce negative sodium balance. However, with chronic treatment, their effects tend to be blunted since the kidney adapts to diuretics. Molecular identification of the renal ion transporters has provided us with a new understanding of the mechanisms of intrarenal adaptation to diuretics at molecular levels. In the kidney, loop and thiazide diuretics are secreted from the proximal tubule via the organic anion transporter-1 (OAT1) and exert their diuretic action by binding to the Na-K-2Cl cotransporter type 2 (NKCC2) in the thick ascending limb and the Na-Cl cotransporter (NCC) in the distal convoluted tubule, respectively. Recent studies in animal models suggest that abundance of these ion transporters is affected by long-term diuretic administration. Downstream from the primary site of diuretic action, an increase in epithelial Na+ channel (ENaC) abundance is induced by chronic furosemide or hydrochlorothiazide treatment. This adaptation is consistent with previous reports showing cellular hypertrophy and increased Na+ absorption in distal tubular segments. The abundance of NKCC2 and NCC is increased by furosemide and hydrochlorothiazide, respectively. This compensatory upregulation suggests that either diuretic may activate the ion transporter within the primary site of action. In the proximal tubule, the abundance of OAT1 is increased by chronic treatment with furosemide or hydrochlorothiazide. This upregulation of OAT1 seems to be induced by substrate stimulation, lessening diuretic tolerance associated with long-term diuretic use.

Thiazide-Induced Hyponatremia

The importance of thiazide-induced hyponatremia (TIH) is reemerging because thiazide diuretic prescription seems to be increasing after the guidelines recommending thiazides as first-line treatment of essential hypertension have been introduced. Thiazide diuretics act by inhibiting reabsorption of Na+ and Cl- from the distal convoluted tubule by blocking the thiazide-sensitive Na+/Cl- cotransporter. Thus, they inhibit electrolyte transport in the diluting segment and may impair urinary dilution in some vulnerable groups. Risk factors predisposing to TIH are old age, women, reduced body masses, and concurrent use of other medications that impair water excretion. While taking thiazides, the elderly may have a greater defect in water excretion after a water load compared with young subjects. Hyponatremia is usually induced within 2 weeks of starting the thiazide diuretic, but it can occur any time during thiazide therapy when subsequent contributory factors are complicated, such as reduction of renal function with aging, ingestion of other drugs that affect free water clearance, or changes in water or sodium intake. While some patients are volume depleted on presentation, most appear euvolemic. Notably serum levels of uric acid, creatinine and urea nitrogen are usually normal or low, suggestive of syndrome of inappropriate secretion of antidiuretic hormone. Despite numerous studies, the pathophysiological mechanisms underlying TIH are unclear. Although the traditional view is that diuretic-induced sodium or volume loss results in vasopressin-induced water retention, the following 3 main factors are implicated in TIH: stimulation of vasopressin secretion, reduced free-water clearance, and increased water intake. These factors will be discussed in this review.

Secretory-Defect Distal Renal Tubular Acidosis Is Associated with Transporter Defect in H+-ATPase and Anion Exchanger-1

Recent progress in molecular physiology has permitted us to understand pathophysiology of various channelopathies at a molecular level. The secretion of H(+) from alpha-intercalated cells is mediated by apical plasma membrane H(+)-ATPase and basolateral plasma membrane anion exchanger-1 (AE1). Studies have demonstrated the lack of H(+)-ATPase immunostaining in the intercalated cells in a few patients with distal renal tubular acidosis (dRTA). Mutations in H(+)-ATPase and AE1 gene have recently been reported to cause dRTA. This study extends the investigation of the role of transporter defect in dRTA by using immunohistochemical methods. Eleven patients with hyperchloremic metabolic acidosis were diagnosed functionally to have secretory-defect dRTA: urine pH >5.5 during acidemia, normokalemia or hypokalemia, and urine-to-blood pCO(2) <25 mmHg during bicarbonaturia. Renal biopsy tissue was obtained from each patient, and immunohistochemistry was carried out using antibodies to H(+)-ATPase and AE1. For comparison, renal tissues from the patients who had no evidences of distal acidification defect by functional studies were used: four with glomerulopathy or tubulointerstitial nephritis (disease controls) and three from nephrectomized kidneys for renal cell carcinoma (normal controls). The H(+)-ATPase immunoreactivity in alpha-intercalated cells was almost absent in all of the 11 patients with secretory-defect dRTA. In addition, 7 of 11 patients with secretory-defect dRTA were accompanied by negative AE1 immunoreactivity. In both disease controls and normal controls, the immunoreactivity of H(+)-ATPase and AE1 was strong in alpha-intercalated cells. In conclusion, significant defect in acid-base transporters is the major cause of secretory-defect dRTA.

Tempol or candesartan prevents high-fat diet-induced hypertension and renal damage in spontaneously hypertensive rats

BACKGROUND Obesity has been strongly associated with the development and aggravation of hypertension and chronic kidney disease. To date, the systemic renin-angiotensin system (RAS) has been known to involve in obesity-induced tissue damage and hypertension. However, the intrarenal mechanism whereby obesity induces and aggravates hypertension and renal disease remains poorly understood. Therefore, we investigated the role of intrarenal RAS and oxidative stress in diet-induced hypertension and renal inflammation in spontaneously hypertensive rats (SHR) fed a high-fat diet. METHODS Male SHR and Wistar-Kyoto rats (WKY) were divided into eight groups: normal-fat diet-fed WKY (WKY-NF), high-fat diet-fed WKY (WKY-HF), high-fat diet-fed tempol-treated WKY (WKY-HF/T), high-fat diet-fed candesartan-treated WKY (WKY-HF/C), normal-fat diet-fed SHR (SHR-NF), high-fat diet-fed SHR (SHR-HF), high-fat diet-fed tempol-treated SHR (SHR-HF/T) and high-fat diet-fed candesartan-treated SHR (SHR-HF/C). After 12 weeks of treatment, haemodynamic measurements and histological assessment of the kidney were performed. RESULTS At the end of week 12, the high-fat fed SHR gained more body weight, their systolic blood pressure was further elevated and glucose intolerance induced. There was no significant difference in the insulin resistance index, serum lipid profile, plasma renin activity and serum aldosterone levels according to diet. However, the high-fat diet resulted in increases in immunohistochemical stains of renin and angiotensin II in the kidney. The real-time PCR also demonstrated significant increases in mRNA levels of renin, angiotensinogen and angiotensin-converting enzyme in the kidney, reflecting enhanced activation of the intrarenal RAS, which findings were also shown by Western blot analysis for renin and angiotensin II type 1 receptor. The expression of ED-1, osteopontin and TGF-beta1 in the renal cortex were prominently enhanced in the SHR-HF group with the increased intrarenal lipid concentrations and oxidative stress. Administration of tempol or candesartan in the high-fat diet-induced SHR inhibited the elevation of the systolic blood pressure, intrarenal lipid concentrations, oxidative stress and the degree of renal inflammation to the levels of, or more than, the SHR-NF with no differences in the body weight and periepididymal fat weight, compared to those in the SHR-HF group without such treatment. CONCLUSIONS Our study suggests that a high-fat diet induces fatty kidneys, aggravation of blood pressure and renal inflammation in the SHR. Blockade of oxidative stress by tempol or of RAS by candesartan ameliorates the increase in blood pressure and renal inflammation and improves intrarenal lipid accumulation. Therefore, antioxidants or angiotensin receptor blockers can prevent diet-induced hypertension and renal inflammation in hypertensive rats.