Sten Christensen

Decreased vasopressin-mediated renal water reabsorption in rats with compensated liver cirrhosis

Experiments were performed to investigate vasopressin type 2 receptor (V2)-mediated renal water reabsorption and the renal expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2) in cirrhotic rats with sodium retention but without ascites. In addition, the expression of the furosemide-sensitive type 1 Na-K-2Cl cotransporter (BSC-1) and the natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) during acute V2-receptor blockade was measured. Acute V2-receptor blockade with the selective nonpeptide antagonist OPC-31260 (800 microg . kg-1 . h-1) was performed during conditions in which volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 150 mM glucose. OPC-31260 produced a significantly smaller increase in urine flow rate (-26%) and free water clearance (-18%) in cirrhotic rats than in control rats. The natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) was significantly increased in cirrhotic rats (+52%), but pretreatment with OPC-31260 did not affect the natriuretic response to furosemide in neither cirrhotic nor in control rats. Semiquantitative immunoblotting showed a significant downregulation of AQP-2 in the renal cortex (-72%) and in the outer medulla (-44%). The relative expression of BSC-1 in the outer medulla was unchanged in cirrhotic rats. The corticopapillary gradient of Na was significantly increased in cirrhotic rats. Since daily urine flow rate was similar in cirrhotic and sham-operated rats, we suggest that non-vasopressin-mediated water reabsorption is increased in cirrhotic rats probably as a result of an increased corticomedullary gradient due to exaggerated NaCl reabsorption in the thick ascending limb of Henles loop.Experiments were performed to investigate vasopressin type 2 receptor (V2)-mediated renal water reabsorption and the renal expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2) in cirrhotic rats with sodium retention but without ascites. In addition, the expression of the furosemide-sensitive type 1 Na-K-2Cl cotransporter (BSC-1) and the natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) during acute V2-receptor blockade was measured. Acute V2-receptor blockade with the selective nonpeptide antagonist OPC-31260 (800 μg ⋅ kg-1 ⋅ h-1) was performed during conditions in which volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 150 mM glucose. OPC-31260 produced a significantly smaller increase in urine flow rate (-26%) and free water clearance (-18%) in cirrhotic rats than in control rats. The natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) was significantly increased in cirrhotic rats (+52%), but pretreatment with OPC-31260 did not affect the natriuretic response to furosemide in neither cirrhotic nor in control rats. Semiquantitative immunoblotting showed a significant downregulation of AQP-2 in the renal cortex (-72%) and in the outer medulla (-44%). The relative expression of BSC-1 in the outer medulla was unchanged in cirrhotic rats. The corticopapillary gradient of Na was significantly increased in cirrhotic rats. Since daily urine flow rate was similar in cirrhotic and sham-operated rats, we suggest that non-vasopressin-mediated water reabsorption is increased in cirrhotic rats probably as a result of an increased corticomedullary gradient due to exaggerated NaCl reabsorption in the thick ascending limb of Henles loop.

Tissue injury after lithium treatment in human and rat postnatal kidney involves glycogen synthase kinase-3β-positive epithelium.

It was hypothesized that lithium causes accelerated and permanent injury to the postnatally developing kidney through entry into epithelial cells of the distal nephron and inhibition of glycogen synthase kinase-3β (GSK-3β). GSK-3β immunoreactivity was associated with glomeruli, the thick ascending limb of Henles loop, and collecting ducts in the developing and adult human and rat kidney. In rats, the abundance of inactive, phosphorylated GSK-3β (pGSK-3β) protein decreased during postnatal development. After feeding of dams with litters lithium [50 mmol Li/kg chow, postnatal (P) days 7-28], the offspring showed plasma lithium concentration of 1.0 mmol/l. Kidneys from lithium-treated rat pups exhibited dilated distal nephron segments with microcysts. Stereological analysis showed reduced cortex and outer medullary volumes. Lithium increased pGSK-3β and the proliferation marker proliferating cell nuclear antigen (PCNA) protein abundances in the cortex and medulla. After lithium treatment, pGSK-3β-immunopositive cells exhibited restricted distribution and were associated primarily with subsets of cells in dilated and microcystic segments of cortical collecting ducts. After 6 wk of lithium discontinuation, adult rats exhibited attenuated urine concentration capacity and diminished outer medullary volume. Histological sections of two nephrectomy samples and a biopsy from three long-term lithium-treated patients showed multiple cortical microcysts that originated from normally appearing tubules. Microcysts were lined by a cuboidal PCNA-, GSK-3β-, and pGSK-3β-immunopositive epithelium. The postnatal rat kidney may serve as an experimental model for the study of lithium-induced human kidney injury. The data are compatible with a causal relationship between epithelial entry of lithium into cells of the aldosterone-sensitive distal nephron, inactivation of GSK-3β, proliferation, and microcysts.

Effects of furosemide on renal haemodynamics and proximal tubular sodium reabsorption in conscious rats.

1 The effects of furosemide given as constant i.v. infusion (7.5 mg kg−1 h−1) or bolus injections (0.5, 7.5 and 120 mg kg−1) on renal haemodynamics and proximal tubular Na reabsorption were studied in conscious water diuretic rats. The clearance of Li (CLi) was used as marker for Na delivery from the proximal tubules, and clearance of [14C]‐tetraethylammonium (CTEA) and [3H]‐inulin (CIn) as markers for renal plasma flow (RPF) and glomerular filtration rate (GFR), respectively. 2 Furosemide caused a transient increase of RPF and GFR followed by a secondary decrease below baseline levels; the latter could in part be counteracted by volume replacement. The filtration fraction (FF = GFR/RPF) was not significantly changed by furosemide. Fractional proximal Na excretion (CLi/CIn) was significantly increased by all doses of furosemide independent of changes in RPF, GFR and FF. 3 The peak diuretic/natriuretic effect of furosemide was markedly potentiated by volume replacement, probably due to prevention of antinatriuretic mechanisms triggered by volume depletion. 4 It is concluded that following i.v. furosemide administration there is a biphasic change in renal haemodynamics in conscious, restrained rats, and that the inhibition of proximal Na reabsorption, as manifested by changes in fractional Li excretion, is not likely to be due to changes in total renal haemodynamics.

Functional and Structural Changes in the Rat Kidney by Long-Term Lithium Treatment

The relation between functional and structural renal changes induced by lithium was studied in rats during long-term treatment and after withdrawal of lithium. Administration of LiCl in the diet for up to 21 weeks caused marked polyuria associated with a significant lowering of renal concentrating ability assessed by dehydration and vasopressin tests. Plasma creatinine and plasma urea were not significantly changed by the treatment. Upon withdrawal of lithium water intake and concentrating ability were normalized within 4--8 weeks. Lithium caused focal light microscopic changes in the distal convoluted tubule and the collecting duct, consisting of nuclear and cellular polymorphism and, after prolonged treatment, dilatation of tubular lumens with tubular cell atrophy. These changes appeared later than the concentrating defect and persisted when lithium was withdrawn after prolonged treatment. No significant correlation was found between the degree of tubular changes and water intake or concentrating ability. It is concluded that the reversible diabetes insipidus induced by lithium in rats cannot be explained directly by the light microscopical changes observed in the distal part of the nephron, although the structural changes may be secondary to the polyuric state induced by lithium.

Model explaining the relation between distal nephron Li + reabsorption and urinary Na + excretion in rats

Li+ may be reabsorbed via an amiloride-sensitive mechanism in the collecting ducts of rats administered a low-Na+ diet. This was investigated by measuring the increase in fractional urinary excretion of Li+(FELi) in response to amiloride in conscious rats at two different levels of plasma Li+ concentration and after administration of bendroflumethiazide (BFTZ), angiotensin III (ANG III), and aldosterone (Aldo). The results confirmed that amiloride increased (FELi) in rats on a low-Na+ diet (20 ± 1 to 35 ± 1%, means ± SE), whereas no increase was observed in rats on a normal Na+ diet (37 ± 1 to 38 ± 1%). The lithiuretic effect of amiloride was 1) abolished by preadministration of BFTZ (32 ± 1 to 33 ± 2%) to Na+-deprived rats and 2) increased by ANG III (27 ± 3 to 33 ± 2%) and Aldo (25 ± 2 to 37 ± 2%) in Na+-replete rats. Amiloride-induced changes in FELiwere independent of plasma Li+concentration but inversely related to the fractional excretion of Na+ and the amiloride-sensitive excretion of K+. These results are compatible with the hypothesis that a low tubular Na+ concentration reduces end-tubular Na+ reabsorption and results in hyperpolarization of the apical membrane, thus favoring Li+ uptake into the cells.

Superiority of tetraethylammonium to p-aminohippurate as a marker for renal plasma flow during furosemide diuresis.

The renal clearances (C) of p-aminohippurate (PAH) and tetraethylammonium (TEA) as markers for renal plasma flow (RPF) were compared in rats under different experimental conditions. CTEA and CPAH were of the same magnitude and varied synchronously during volume expansion and anesthesia. The renal extractions of the two substances were closely correlated and averaged 85.9% for PAH and 88.7% for TEA (p less than 0.01). High doses of furosemide (120 mg/kg as intravenous bolus) reversibly decreased the CPAH/CTEA ratio, suggesting that furosemide may compete for PAH secretion in the proximal tubule. A similar effect was observed after administration of probenecid. The results indicate that TEA is a more reliable indicator of RPF than PAH in studies involving accumulation of endogenous or exogenous organic anions.

A morphometric and ultrastructural study of lithium-induced changes in the medullary collecting ducts of the rat kidney.

SummaryRats were given a lithium-containing diet (40 mmol/kg) to Study the effect of lithium on the structure of collecting ducts from the inner stripe of the outer medulla. The results show that there is a significant increase in the volume density of collecting ducts already after one week on this diet. The volume density of both intercalated and principal cells increases, whereas the volume density of mitochondria in the cytoplasm increases in the intercalated cells only. The increased volume of both principal and intercalated cells seems to be part of a general hyperplasia and hyperactivity of the collecting duct, which may in some way be related to the effects of lithium on vasopressinmediated water transport. The specific changes in the intercalated cells may be a consequence of the effects of lithium on distal nephron potassium and hydrogen ion transport in the distal nephron.

Losartan decreases vasopressin-mediated cAMP accumulation in the thick ascending limb of the loop of Henle in rats with congestive heart failure

Introduction:  Vasopressin (AVP) stimulates sodium reabsorption and Na,K,2Cl‐cotransporter (NKCC2) protein level in the thick ascending limb (TAL) of Henles loop in rats. Rats with congestive heart failure (CHF) have increased protein level of NKCC2, which can be normalized by angiotensin II receptor type‐1 (AT1) blockade with losartan.

Mechanism of antidiuresis caused by bendroflumethiazide in conscious rats with diabetes insipidus

The mechanism underlying the antidiuretic effect of thiazide diuretics in diabetes insipidus (DI) is unknown. This study addressed two specific questions: is the reduction in urine flow rate (V) related to a decrease in the delivery of fluid from the pars recta of the proximal tubules (‘distal delivery’), and are there any changes in the expression and/or intracellular distribution of vasopressin stimulated water channels (AQP2) in the collecting ducts, during chronic thiazide‐induced antidiuresis? Nine Brattleboro rats with vasopressin‐deficient DI were treated for 5 days with bendroflumethiazide (BFTZ), 9 mg kg−1 day−1 orally, and 9 Brattleboro rats were left untreated. BFTZ‐treated DI rats showed a fall in V from ∼200 to ∼75 ml day−1 and an increase in urine osmolality from ∼130 to ∼400 mosmol kg−1. BFTZ‐induced antidiuresis was associated with a persistent loss of sodium, but not of potassium. After 5 days of treatment, clearance studies in conscious rats showed a tendency towards decreases in effective renal plasma flow (−7%), GFR (−12%) and lithium clearance (CLi; used as marker for distal delivery) (−25%), compared with untreated controls, but none of these changes were statistically significant. There was no apparent relationship between CLi and V in BFTZ‐treated or untreated DI rats. BFTZ treatment did not change the expression of AQP2 in homogenates of cortex, outer or inner medulla from DI rats, or from normal Long Evans rats. Light and electron microscopic immunocyto‐chemistry revealed no changes in intracellular distribution of AQP2 in principal cells from inner medullary collecting ducts of BFTZ‐treated DI rats. We concluded, (i) that although the antidiuretic effect of BFTZ in rats with DI is associated with a net loss of Na, the decrease in V shows no association with changes in distal delivery, as estimated by CLi. (ii) Antidiuretic treatment with BFTZ does not alter the expression of subcellular distribution of AQP2 water channels in the collecting ducts. The mechanism underlying the chronic antidiuresis caused by thiazide diuretics in DI remains elusive.

The number and size of glomeruli in long‐term lithium‐induced nephropathy in rats

Chronic renal failure was induced in 10 Wistar rats using a lithium‐containing (40 mmol/kg) diet from time of birth until an age of 55–65 weeks. Nine Wistar rats served as controls. The plasma lithium, the plasma urea, and the inulin clearance were measured, and one kidney was fixed by vascular perfusion with glutaraldehyde. The number of glomeruli was estimated stereologically by the fractionator method. The total number of glomeruli per kidney was 23.9 × 103±3.65 × 103 (±SD) in controls and 22.0 × 103±1.48 × 103 in the lithium‐treated group, showing no statistically significant difference. The mean glomerular volume was also estimated using stereological methods. The number‐weighted mean volume was reduced by 42% in the lithium‐treated group, whereas the volume‐weighted mean volume was unchanged. This can be attributed to the occurrence of many small glomeruli and a few very large glomeruli in the lithium‐treated group. The many small glomeruli have in a previous study been shown to be atubular. The present study showed that the glomerular population is quite resistant to the deleterious effect of lithium; thus glomerular atrophy was seen, but no loss of glomeruli occurred.