Blunted renal dopaminergic system activity in puromycin aminonucleoside-induced nephrotic syndrome.

BACKGROUND: A primary tubular sodium handling abnormality has been implicated in the edema formation of nephrotic syndrome. Dopamine synthesized by renal proximal tubules behaves as an endogenous natriuretic hormone by activating D(1)-like receptors as a paracrine/autocrine substance. METHODS: We examined the time courses of the urinary excretion of sodium, protein and dopamine in puromycin aminonucleoside (PAN)-treated and control rats. The rats were sacrificed during greatest sodium retention (day 7) as well as during negative sodium balance (day 14) for the evaluation of renal aromatic l-amino acid decarboxylase (AADC) activity, the enzyme responsible for the synthesis of renal dopamine. Also, the influence of volume expansion (VE) and the effects of the D(1)-like agonist fenoldopam (10 microg/kg bw/min) on natriuresis and on proximal tubular Na(+),K(+)-ATPase activity were examined on day 7. RESULTS: The daily urinary excretion of dopamine was decreased in PAN-treated rats, from day 5 and beyond. This was accompanied by a marked decrease in the renal AADC activity, on days 7 and 14. During VE, the fenoldopam-induced decrease in proximal tubular Na(+),K(+)-ATPase activity was more pronounced in PAN-treated rats than in controls. However, the urinary sodium excretion during fenoldopam infusion was markedly increased in control rats but was not altered in PAN-treated animals. CONCLUSION: PAN nephrosis is associated with a blunted renal dopaminergic system activity which may contribute to enhance the proximal tubular Na(+),K(+)-ATPase activity. However, the lack of renal dopamine appears not to be related with the overall renal sodium retention in a state of proteinuria.     

Mechanisms of PKC-Dependent Na+ K+ ATPase Phosphorylation in the Rat Kidney with Chronic Renal Failure

The present work was designed to study Na⁺ K⁺ ATPase α1-subunit phosphorylation in rats with chronic renal failure (CRF) in comparison with normal rats. Na⁺ K⁺ ATPase α1-subunit phosphorylation degree was measured by binding the McK-1 antibody to dephosphorylated Ser-23 in microdissected medullary thick ascending limb of Henle (mTAL) segments. In addition, the total Na⁺ K⁺ ATPase α1-subunit expression and activity were also measured in the outer renal medulla homogenates and membranes.

CRF rats showed a higher Na⁺ K⁺ ATPase activity, as compared with control rats (18.95 ± 2.4 vs. 11.21 ± 1.5 μmol Pi/mg prot/h, p < 0.05), accompanied by a higher total Na⁺ K⁺ ATPase expression (0.54 ± 0.04 vs. 0.27 ± 0.02 normalized arbitrary units (NU), p < 0.05). When McK-1 antibody was used, a higher immunosignal in mTAL of CRF rats was observed, as compared with controls (6.3 ± 0.35 vs.4.1 ± 0.33 NU, p < 0.05). The ratio Na⁺ K⁺ ATPase α1-subunit phosphorylation / total Na⁺ K⁺ ATPase α1-subunit expression per μg protein showed a non-significant difference between CRF and control rats in microdissected mTAL segments (2.11 ± 0.12 vs.2.26 ± 0.18 NU, p = NS). The PKC inhibitor RO-318220 10^?6M increased immunosignal (lower phosphorylation degree) in mTAL of CRF rats to 128.43 ± 7.08% (p < 0.05) but did not alter McK1 binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10^?6M and dopamine 10^?6M decreased immunosignal in CRF rats, corresponding to a higher Na⁺ K⁺ ATPase α1-subunit phosphorylation degree at Ser-23 (55.26 ± 11.17% and 53.27 ± 7.12% compared with basal, p < 0.05). In mTAL of CRF rats, the calcineurin inhibitor FK-506 10^?6M did not modify phosphorylation degree at Ser-23 of Na⁺ K⁺ ATPase α1-subunit (100.21 ± 3.00% compared with basal CRF). In control rats, FK 506 10^?6M decreased the immunosignal, which corresponds to a higher Na⁺ K⁺ ATPase α1-subunit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na⁺ K⁺ ATPase α1-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.     

Sepsis increases the plasma membrane content of alpha1 and alpha2 isoforms of Na+-K+ adenosine triphosphatase in rat skeletal muscle

HYPOTHESIS: Increased Na(+)-K(+) adenosine triphosphatase (ATPase) activity in skeletal muscle during sepsis is caused by transient increases in enzyme content within the plasma membrane. DESIGN: Randomized controlled study. SETTING: University laboratory. INTERVENTION: Eighty-eight adult male Wistar rats were randomly assigned to undergo cecal ligation and puncture (CLP) or sham operation. MAIN OUTCOME MEASURES: Gastrocnemius muscles were harvested 6, 12, 24, and 48 hours after operation and Na(+)-K(+) ATPase activities were measured spectrofluorimetrically. Messenger RNA (mRNA) levels for the alpha1 and alpha2 isoforms of Na(+)-K(+) ATPase were determined by Northern blot analysis. Crude membranes, internal membranes, and purified plasma membranes were isolated from gastrocnemius muscles and protein levels of alpha1 and alpha2 isoforms were determined by Western blot analysis. RESULTS: Na(+)-K(+) ATPase activity in the CLP group was significantly higher compared with the sham group 24 hours after operation (P<.05). However, there were no differences between the sham and CLP groups 6, 12, or 48 hours after operation. No significant differences between the CLP and sham groups were noted in mRNA levels for Na(+)-K(+) ATPase alpha1 and alpha2 isoforms. Western blot analysis revealed that the plasma membrane (but not internal membrane or crude membrane) content of alpha2 and alpha1 isoforms from the CLP group was significantly increased compared with the sham group 24 hours after operation (P<.05). CONCLUSIONS: Na(+)-K(+) ATPase activity increases 24 hours after CLP in gastrocnemius muscle and then declines. This increase is caused by increased Na(+)-K(+) ATPase protein levels in the plasma membrane.     

严重烧伤大鼠胃粘膜血流量和Na^＋—K^＋—ATP酶活性的改变及对胃粘膜电位差的影响

目的观察大鼠严重烧伤后胃粘膜血流量(GMBF)和Na+-K+-ATP酶活性的变化,探讨其对胃粘膜电位差(GTPD)的影响. 方法制作烧伤大鼠模型,分别采用激光多普勒微循环血流计、电生理记录仪和生化法,检测大鼠伤前及伤后3、6、12、24、48 h时GMBF、GTPD和胃粘膜Na+-K+-ATP酶活性的改变;另设正常大鼠为对照组,于相同时相点检测以上指标.随后对各指标进行相关性分析. 结果与对照组比较,大鼠烧伤后3 ～24 h GMBF与Na+-K+-ATP酶活性均明显降低(P<0.05～0.01),GTPD于伤后6～48 h明显下降(P<0.05～0.01),3项指标均于12 h时降至最低.相关性分析结果显示Na+-K+-ATP酶活性随GMBF的降低而下降(r=0.417,P<0.01);GMBF的减少或Na+-K+-ATP酶活性的降低,均可引起GTPD降低(r=0.453或0.527,P<0.01). 结论大鼠严重烧伤后,GMBF减少,Na+-K+-ATP酶活性降低,二者均为引起胃粘膜屏障功能受损的主要原因.     

Strong depletion of CD14(+)CD16(+) monocytes during haemodialysis treatment.

BACKGROUND: The immune defect in haemodialysis (HD) patients is associated with a monocytic dysfunction, including an increased production of proinflammatory cytokines. Monocytes fall into subpopulations comprising CD14(++)CD16(-) and CD14(+)CD16(+) cells. Circulating numbers of the latter can rapidly increase during infectious episodes and inflammation. METHODS: We determined the amount of CD14(+)CD16(+) monocytes in HD patients and characterized their fate during HD treatment. In 34 HD patients and 17 healthy controls, the distinct cell populations were determined by differential blood counts and flow cytometry. Cells from 14 HD patients were analysed at the start, 10, 30 and 120 min thereafter, and at the end of HD treatment. RESULTS: Before HD, patients show a monocytosis with a strongly increased CD14(+)CD16(+) subpopulation. Early during HD treatment, circulating leukocyte numbers decrease, with monocytes being most profoundly influenced. Interestingly, among them, sequestration is most pronounced in the CD14(+) CD16(+) subpopulation. After 30 min, approximately 83+/-9% of CD14(+)CD16(+) cells are removed from circulation. This sequestration does not differ between patients treated with polyamide or haemophan membranes. The sequestration is a short-lived temporary effect and cell numbers are replenished within 120 min of treatment for the entire monocyte population. Beyond that time point, cellular activation by the dialyser membrane becomes visible. Reappearence kinetics of CD14(+)CD16(+) monocytes is slower; however, initial numbers are reached by the end of treatment. CONCLUSION: Haemodiaysis leads to temporary removal of monocytes from the bloodstream followed by the reappearance of activated cells. This might contribute to the state of chronic microinflammation, which is reflected by high levels of CD14(+)CD16(+) monocytes.     

Channels, carriers, and pumps in the pathogenesis of sodium-sensitive hypertension

Sodium-sensitive hypertension is thought to be dependent on primary alterations in renal tubular sodium reabsorption. The major apical plasma membrane Na(+) transporters include the proximal tubular Na(+)-H(+) exchanger, the thick ascending limb Na(+)-K(+)-2Cl(-) cotransport system, the distal tubular Na(+)-Cl(-) cotransporter, and the collecting duct epithelial sodium channel (ENaC). This article explores the role of each transporter in the pathogenesis of hypertension. Although the contribution of the proximal tubule Na(+)-H(+) exchanger is not yet defined completely, more convincing data have been generated about the importance of the Na(+)-K(+)-2Cl(-). Indeed at least 2 forms of hypertension appear to be related to the up-regulation of the transporter: the so-called programmed hypertension induced by low-protein diet during pregnancy and the early phase of hypertension in the Milan strain of rats. With respect to the Na(+)-Cl(-) cotransporter this may be overactive caused by inactivating mutation of WNK4 as in the Gordon syndrome, although it is the main actor for the maintenance phase of the hypertension found in the Milan strain of rats. Finally, the contribution of the ENaC has been established clearly; indeed, in the Liddle syndrome the mutation of the ENaC gene leads to a longer retention of the channel on the cell surface of collecting duct principal cells, thus inducing stronger sodium reabsorption along this segment. All these examples clearly indicate that renal sodium transporters may be responsible for various types of sodium-sensitive hypertension.     

Effect of diabetes on cytosolic free Ca2+ and Na(+)-K(+)-ATPase in rat aorta.

We examined Na(+)-K(+)-ATPase activity and the levels of alpha I-, alpha II-, and beta-subunit mRNA and protein in aortic cells of diabetic rats. Diabetes was induced by streptozocin. Na(+)-K(+)-ATPase activity was significantly reduced on the 2nd day of diabetes (9.4 +/- 1.3 vs. 17.5 +/- 2.1 mumol NADH.mg-1 protein.h-1, P less than 0.05) and remained depressed on days 7 and 14. The levels of 5.3-kilobase (kb) mRNA band of the catalytic alpha II-subunit of Na(+)-K(+)-ATPase were also decreased on the 2nd day of diabetes, whereas the second band, 3.4 kb, was not affected. Both bands were significantly decreased on days 7 and 14. This was followed by a reduction in the levels of alpha II-protein (day 14). The levels of alpha I- and beta-subunit mRNA and alpha I- protein were not affected by diabetes. A decrease in Na(+)-K(+)-ATPase activity was accompanied by a significant (P less than 0.001) increase in the cytosolic free Ca2+ concentrations [( Ca2+]i) in diabetic aortic cells (221 +/- 18 nM on the 7th day and 242 +/- 17 nM on the 14th day vs. 153 +/- 7 nM in controls). These findings are consistent with the hypothesis that decreased Na(+)-K(+)-ATPase activity and gene expression in vascular smooth muscle cells with accompanied rises in [Ca2+]i may be an important pathogenetic factor in the development of hypertension and atherosclerosis in diabetes.     

Neuronal nitric oxide synthase: its role and regulation in macula densa cells

Macula densa (MD) cells detect changes in distal tubular sodium chloride concentration ([NaCl](L)), at least in part, through an apical Na:2Cl:K co-transporter. This co-transporter may be a site for regulation of tubuloglomerular feedback (TGF), and recently angiotensin II (Ang II) was shown to regulate the MD Na:2Cl:K co-transporter. In addition, nitric oxide (NO) produced via neuronal NO synthase (nNOS) in MD cells attenuates MD-TGF signaling. This study investigated [NaCl](L)-dependent MD-NO production, the regulation of co-transporter activity by NO, and the possible interaction of NO with Ang II. MD cell Na(+) concentration ([Na(+)](i)) and NO production were measured using sodium-binding benzofuran isophthalate and 4-amino-5-methylamino-2',7'-difluorescein diacetate, respectively, using fluorescence microscopy. Na:2Cl:K co-transport activity was assessed as the initial rate of increase in [Na(+)](i) when [NaCl](L) was elevated from 25 to 150 mM. 10(-4) M 7-nitroindazole, a specific nNOS blocker, significantly increased by twofold the initial rate of rise in [Na(+)](i) when [NaCl](L) was increased from 25 to 150 mM, indicating co-transporter stimulation. There was no evidence for an interaction between the stimulatory effect of Ang II and the inhibitory effect of NO on co-transport activity, and, furthermore, Ang II failed to alter MD-NO production. NO production was sensitive to [NaCl](L) but increased only when [NaCl](L) was elevated from 60 to 150 mM. These studies indicate that MD-NO directly inhibits Na:2Cl:K co-transport and that NO and Ang II independently alter co-transporter activity. In addition, generation of MD-NO seems to occur only at markedly elevated [NaCl](L), suggesting that NO may serve as a buffer against high rates of MD cell transport and excessive TGF-mediated vasoconstriction.     

Resistance of mTAL Na+-dependent transporters and collecting duct aquaporins to dehydration in 7-month-old rats

BACKGROUND: Aging is associated with a defect in urinary concentration in both human and experimental animals. The purpose of these studies was to examine the urinary concentrating ability, the expression of kidney water channels [aquaporins (AQP1 to AQP3)], and medullary thick ascending limb (mTAL) Na+-dependent transporters in old but not senescent versus young animals in response to water deprivation. METHODS: Two-month-old and 7-month-old rats were placed in metabolic cages and deprived of water for 72 hours. Kidney tissues were isolated and examined for the expression of AQP1 to AQP3 and mTAL, peptide-derived polyclonal antibody specific to kidney apical Na+-K+-2 Cl- cotransporter (BSC1), Na+/H+ exchanger isoform 3 (NHE3), and Na+ pump using semiquantitative immunoblotting and Northern hybridization. RESULTS: After 72 hours of water deprivation, urine osmolality increased from 1269 to 3830 mOsm/kg H2O in 2-month-old rats, but only from 1027 to 2588 mOsm/kg H2O in 7-month-old rats. In response to water deprivation, AQP2 and AQP3 expression increased significantly in the cortex and medulla of 2-month-old rats but remained unchanged in the medulla or slightly increase in the cortex of 7-month-old animals. AQP1 expression was not altered by dehydration in both groups. The protein abundance of mTAL BSC1, NHE3, and Na+ pump increased significantly in young but remained unchanged in 7-month-old rats subjected to water deprivation. CONCLUSION: Age-related decrease in urinary concentrating ability is an early event, developed before the onset of senescence. This defect results from reduced responsiveness of cortical AQP2 and AQP3 and a blunted response of medullary AQP2 and mTAL BSC1, NHE3, and Na+ pump to dehydration in aging kidneys.     

Acute regulation of the epithelial Na+ channel by phosphatidylinositide 3-OH kinase signaling in native collecting duct principal cells

Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the aldosterone-sensitive distal nephron. Hormones, including aldosterone and insulin, increase ENaC activity, in part by stimulating phosphatidylinositide 3-OH kinase (PI3-K) signaling. Recent studies in heterologous expression systems reveal a close spatiotemporal coupling between PI3-K signaling and ENaC activity with the phospholipid product of this kinase, PI(3,4,5)P(3), in some cases, directly binding the channel and increasing open probability (P(o)). This study tested whether this tight coupling plays a physiologic role in modulating ENaC activity in native tissue and polarized epithelial cells. IGF-I was found to increase Na(+) reabsorption across mpkCCD(c14) principal cell monolayers in a PI3-K-sensitive manner. Inhibition of PI3-K signaling, moreover, rapidly decreased Na(+) reabsorption and ENaC activity in mpkCCD(c14) cells that were treated with corticosteroids and IGF-I. These decreases paralleled changes in apical membrane PI(3,4,5)P(3) levels, demonstrating tight spatiotemporal coupling between ENaC activity and PI3-K/PI(3,4,5)P(3) signaling within this membrane. For further probing of the mechanism underpinning this coupling, cortical collecting ducts (CCD) were isolated from rat and split open to expose the apical membrane for patch-clamp analysis. Inhibition of PI3-K signaling with wortmannin and LY294002 but not its inactive analogue rapidly and markedly decreased the P(o) of ENaC. Moreover, IGF-I acutely increased P(o) of ENaC in CCD principal cells in a PI3-K-sensitive manner. Together, these observations stress the importance of tight spatiotemporal coupling between PI3-K signaling and ENaC within the apical membrane of principal cells to the physiologic control of this ion channel.     

Mechanisms of PKC-dependent Na+ K+ ATPase phosphorylation in the rat kidney with chronic renal failure

The present work was designed to study Na+ K+ ATPase alpha1-subunit phosphorylation in rats with chronic renal failure (CRF) in comparison with normal rats. Na+ K+ ATPase alpha1-subunit phosphorylation degree was measured by binding the McK-1 antibody to dephosphorylated Ser-23 in microdissected medullary thick ascending limb of Henle (mTAL) segments. In addition, the total Na+ K+ ATPase alpha1-subunit expression and activity were also measured in the outer renal medulla homogenates and membranes. CRF rats showed a higher Na+ K+ ATPase activity, as compared with control rats (18.95 +/- 2.4 vs. 11.21 +/- 1.5 micromol Pi/mg prot/h, p < 0.05), accompanied by a higher total Na+ K+ ATPase expression (0.54 +/- 0.04 vs. 0.27 +/- 0.02 normalized arbitrary units (NU), p < 0.05). When McK-1 antibody was used, a higher immunosignal in mTAL of CRF rats was observed, as compared with controls (6.3 +/- 0.35 vs. 4.1 +/- 0.33 NU, p < 0.05). The ratio Na+ K+ ATPase alpha1-subunit phosphorylation/total Na+ K+ ATPase alpha1-subunit expression per microg protein showed a non-significant difference between CRF and control rats in microdissected mTAL segments (2.11 +/- 0.12 vs. 2.26 +/- 0.18 NU, p = NS). The PKC inhibitor RO-318220 10(-6) M increased immunosignal (lower phosphorylation degree) in mTAL of CRF rats to 128.43 +/- 7.08% (p < 0.05) but did not alter McK1 binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10(-6) M and dopamine 10(-6) M decreased immunosignal in CRF rats, corresponding to a higher Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23 (55.26 +/- 11.17% and 53.27 +/- 7.12% compared with basal, p < 0.05). In mTAL of CRF rats, the calcineurin inhibitor FK-506 10(-6) M did not modify phosphorylation degree at Ser-23 of Na+ K+ ATPase alpha1-subunit (100.21 +/- 3.00% compared with basal CRF). In control rats, FK 506 10(-6) M decreased the immunosignal, which corresponds to a higher Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na+ K+ ATPase alpha1-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.     

Blood cardioplegic protection in profoundly damaged hearts: role of Na+-H+ exchange inhibition during pretreatment or during controlled reperfusion supplementation

BACKGROUND: Inhibition of the Na+/H+ exchanger before ischemia protects against ischemia-reperfusion injury, but use as pretreatment before blood cardioplegic protection or as a supplement to controlled blood cardioplegic reperfusion was not previously tested in jeopardized hearts. METHODS: Control studies tested the safety of glutamate-aspartate-enriched blood cardioplegic solution in 4 Yorkshire-Duroc pigs undergoing 30 minutes of aortic clamping without prior unprotected ischemia. Twenty-four pigs underwent 30 minutes of unprotected normothermic global ischemia to create a jeopardized heart. Six of these hearts received normal blood reperfusion, and the other 18 jeopardized hearts underwent 30 more minutes of aortic clamping with cardioplegic protection. In 12 of these, the Na+/H+ exchanger inhibitor cariporide was used as intravenous pretreatment (n = 6) or added to the cardioplegic reperfusate (n = 6). RESULTS: Complete functional, biochemical, and endothelial recovery occurred after 30 minutes of blood cardioplegic arrest without preceding unprotected ischemia. Thirty minutes of normothermic ischemia and normal blood reperfusion produced 33% mortality and severe left ventricular dysfunction in survivors (preload recruitable stroke work, 23% +/- 6% of baseline levels), with raised creatine kinase MB, conjugated dienes, endothelin-1, myeloperoxidase activity, and extensive myocardial edema. Blood cardioplegia was functionally protective, despite adding 30 more minutes of ischemia; there was no mortality, and left ventricular function improved (preload recruitable stroke work, 58% +/- 21%, p < 0.05 versus normal blood reperfusion), but adverse biochemical and endothelial variables did not change. In contrast, Na+/H+ exchanger inhibition as either pretreatment or added during cardioplegic reperfusion improved myocardial recovery (preload recruitable stroke work, 88% +/- 9% and 80% +/- 7%, respectively, p < 0.05 versus without cariporide) and comparably restored injury variables. CONCLUSIONS: Na+/H+ exchanger blockage as either pretreatment or during blood cardioplegic reperfusion comparably delays functional, biochemical, and endothelial injury in jeopardized hearts.     

Normalization of Na(+)-K(+)-ATPase activity in isolated membrane fraction from sciatic nerves of streptozocin-induced diabetic rats by dietary myo-inositol supplementation in vivo or protein kinase C agonists in vitro

A myo-inositol-related defect in nerve Na(+)-K(+)-ATPase in experimental diabetes has been invoked in the pathogenesis of diabetic neuropathy, but the mechanism linking altered myo-inositol metabolism and Na(+)-K(+)-ATPase regulation in diabetic nerve is uncertain. Decreased Na(+)-K(+)-ATPase in diabetic rat nerve is normalized by aldose reductase inhibitors or dietary myo-inositol, which preserve normal nerve myo-inositol content in vivo. Decreased Na(+)-K(+)-ATPase in diabetic rabbit nerve is acutely reversed by exposure to protein kinase C agonists in vitro. This study explored the relationship between the myo-inositol-sensitive and protein kinase C-agonist-sensitive Na(+)-K(+)-ATPase defects in diabetic rat nerve. Ouabain-sensitive ATPase activity was measured in an enriched membrane fraction isolated from nondiabetic, streptozocin-induced diabetic, and myo-inositol-supplemented streptozocin-induced diabetic rats before and after the membranes were exposed to protein kinase C agonists in vitro. The decreased ouabain-sensitive ATPase activity in plasma membranes from untreated diabetic rats was increased after exposure to two structurally unrelated protein kinase C agonists; the normal ouabain-sensitive ATPase in plasma membranes from myo-inositol-supplemented diabetic rats was unaffected by protein kinase C agonists. The nonadditivity and implied equivalence of the Na(+)-K(+)-ATPase defect corrected by myo-inositol in vivo and by protein kinase C agonists in vitro are consistent with the postulated existence of a deficient myo-inositol-dependent phospholipid-derived protein kinase C agonist (presumably diacylglycerol) in diabetic nerve that regulates nerve Na(+)-K(+)-ATPase either directly or via a protein kinase C mechanism.     

Correction of altered metabolic activities in sciatic nerves of streptozocin-induced diabetic rats. Effect of ganglioside treatment

The effect of ganglioside administration to nondiabetic and streptozocin-induced diabetic rats on sciatic nerve Na(+)-K(+)-ATPase, polyphosphoinositide (PPI) turnover, and protein phosphorylation was investigated. Gangliosides were injected (10 mg/kg body wt i.p.) for 10 or 30 days beginning 20 days after induction of diabetes. Na(+)-K(+)-ATPase activity was reduced nearly 50% in diabetic nerve and was restored to normal by both ganglioside treatments. The elevated levels of fructose and sorbitol and depressed content of myoinositol in diabetic nerve were unaffected by 30 days of ganglioside treatment, indicating that the restoration of Na(+)-K(+)-ATPase activity is not dependent on normal concentrations of these compounds. In the same nerves, 32P incorporation into phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate increased 73-76 and 39-53%, respectively, in diabetic compared with nondiabetic tissue. Ganglioside administration abolished the elevated labeling of PPIs after 30 days but was ineffective after only 10 days. Neither ganglioside regimen was able to reverse enhanced phosphorylation of the major peripheral nerve myelin protein P0. The finding that gangliosides can more quickly correct the effects of diabetes on Na(+)-K(+)-ATPase activity than on PPI turnover suggests that the mechanisms underlying these two phenomena are not closely related and are distinct from the sequence of events responsible for altered myelin protein phosphorylation.     

Differential effects of diabetes on rat choroid plexus ion transporter expression

Though diabetes is a disease with vascular complications, little is known about its effects on the blood-brain barrier or the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB is situated at choroid plexuses located in the lateral, third, and fourth ventricles. Choroid plexuses are the primary site of cerebrospinal fluid (CSF) production and express numerous ion transporters. Previous studies have shown a perturbation of ion transport in the periphery and brain during diabetes. In this study, we investigated the effect of diabetes on ion transporters in the choroid plexuses of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of STZ (60 mg/kg in citrate buffer, confirmed by glucose analysis: 601 +/- 22 mg/dl diabetic rats, 181 +/- 46 mg/dl age-matched controls); and at 28 days, rats were killed, choroid plexuses harvested, and protein extracted. Western blot analyses were carried out using antibodies for ion transporters, including Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit. The efflux of the K(+) analog (86)Rb(+) from choroid plexus was also studied. Diabetic rats showed an increase in expression of the Na(+)-K(+)-2Cl(-) cotransporter and the Na(+)-K(+)-ATPase alpha1-subunit, as compared with age-matched controls, a decrease in Na(+)-H(+) exchanger expression, and no change in Na(+)-K(+)-ATPase beta1- or beta2-subunit. The net effect of these changes was a 66% increase in (86)Rb(+) efflux from diabetic choroid plexus compared with controls. These changes in expression may affect choroid plexus ion balance and thus significantly affect CSF production in diabetic rats.