Dilutional hyponatremia in preeclampsia with and without nephrotic syndrome.

This report includes cases of hyponatremia in preeclampsia. Two patients were identified with preeclampsia complicated by hyponatremia, one with and the other without nephrotic syndrome. Together with 3 cases of hyponatremia recently reported, these additional cases from the same geographic area suggest that hyponatremia is not a rare complication of preeclampsia.

TECHNICAL NOTE: measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled kemptide.

BACKGROUND: Traditional protein kinase assays include the use of [32P] labeled ATP as phosphate donor and a substrate protein or peptide as phosphoreceptor. Since this approach has a number of drawbacks in addition to generating ionizing radiation, several non-isotopic methods have been developed. Although shown to reflect the activity of purified enzymes, none have been demonstrated to detect physiological changes in endogenous enzyme activity in cell homogenates. METHODS: Studies were performed to examine the kinetics, reproducibility, and optimal assay conditions of a novel non-radioisotopic kinase assay that detects PKA activity by phosphorylation of the peptide substrate Kemptide covalently bound to a fluorescent molecule (f-Kemptide). Basal and agonist-induced PKA activity in epithelial cell homogenates was measured. RESULTS: The kinetics of f-Kemptide were similar to the standard radioisotopic method with intraassay and interassay variations of 5.6 +/- 0.8% and 14.3 +/- 2.6%, respectively. Neither fluorescence quenching nor enhancing effects were found with consistent amounts of homogenate protein. Specific PKA activity was determined as the IP20-inhibitable fraction to account for nonspecific phosphorylation, perhaps due to S6 kinase or a similar enzyme. The basal activity of 38% of total PKA in A6 cells increased by 84% after exposure to vasopressin and by 58% after short exposure to forskolin. In T84 cells exposed to VIP there was a 360% increase over basal activity. CONCLUSIONS: These results show that f-Kemptide exhibits acceptable kinetics, and that the assay system can quantitatively and reproducibly measure basal and stimulated PKA activity in cell homogenates.

Dilutional hyponatremia in pre-eclampsia.

OBJECTIVE: The objective of this report is to describe a defect in water metabolism, characterized by hyponatremia, in patients with pre-eclampsia-induced nephrotic syndrom. STUDY DESIGN: This was an observational study of 3 women. RESULTS: Hyponatremia was observed in 3 women with pre-eclampsia characterized by various extrarenal manifestations, as well as by nephrotic syndrome with normal or nearly normal renal function. Restriction in water intake partially corrected hyponatremia before delivery in each case, and no complications were observed in the neonates. The mechanism of impaired excretion of water in these patients is proposed to involve persistent and inappropriate production of vasopressin through stimulation of the nonosmotic mechanism for vasopressin secretion in response to a reduction in effective plasma volume. CONCLUSIONS: These results indicate for the first time that women with pre-eclampsia are, at least when nephrotic, at risk for development of dilutional hyponatremia, which can cause neurologic complications that simulate those of eclampsia.

Vasopressin-induced activation of protein kinase C in renal epithelial cells.

Recent studies indicate that the actions of arginine vasopressin (AVP) and other agonists that stimulate electrogenic sodium transport in renal epithelial A6 cells are linked to a Ca(2+)-mobilizing signal transduction mechanism that involves generation of inositol trisphosphate. Since diacylglycerol is the other product in this pathway, studies were performed to determine the possible role of PKC in the stimulation of sodium transport. AVP induced a biphasic increase in diacylglycerol generation, characterized by an initial rapid rise and then a sustained elevation, and PKC activation, reflected by phosphorylation of a specific 80 kDa myristoylated alanine-rich PKC substrate (MARCKS). To determine the PKC isoform(s) involved in this process, immunoblot analysis was performed using antisera that recognize both classical PKC isoforms, XPKC-I and XPCK-II, cloned from Xenopus oocytes. The transcripts of both isoforms were expressed in the A6 cell. Since protein recognized by antisera was translocated from cytosol to the particulate fraction after exposure to AVP, one or both isoforms were activated in the A6 cell. Further studies showed that cyclohexyladenosine and insulin, additional agonists of sodium transport in A6 cells, also stimulated phosphorylation of MARCKS. These results argue that Ca(2+)-dependent PKC is involved in the action of AVP, and that of other agonists, which stimulate sodium transport.

Renal insufficiency after intravenous immune globulin therapy: a report of two cases and an analysis of the literature.

Over the past decade, intravenous immune globulin therapy (IVIG) has gained widespread use for a variety of clinical disorders. IVIG treatment is associated with a number of complications, including acute renal failure (ARF). Although the cause of IVIG-associated ARF is unknown, it may be related to the stabilizing agent used in the IVIG preparation. The development and resolution of ARF is typically rapid, but is some cases recovery may be delayed and require renal replacement therapy. In such patients, recurrence of ARF may be avoided by selection of a preparation with a different stabilizing agent. Two cases of IVIG-induced ARF are described, and all reported cases are analyzed to assess the probable mechanism of renal injury.

Identification and characterization of MARCKS in Xenopus laevis.

MARCKS proteins are widely distributed in mammalian cells and subserve an important role as probes in the examination of signal transduction processes because they are specific endogenous phosphoreceptors for activated protein kinase C. Experiments were performed to determine whether MARCKS proteins are present in amphibia and to show their usefulness as substrates for stimulated PKC activation, using cultured renal epithelial cells (A6) derived from Xenopus laevis as an experimental model.

Novel effect of insulin: insulin-stimulated Na+ transport is mediated by hydrolysis of phosphoinositides.

Previous studies showed that insulin stimulation of electrogenic Na+ transport in renal epithelial cells is mediated by a calcium-dependent signal transduction mechanism. The present study was performed to determine whether the insulin-induced increase in intracellular Ca2+ (Cai2+) was mediated by hydrolysis of phosphatidylinositol and release of inositol trisphosphate. Experiments were conducted with cultured A6 cells, derived from Xenopus Laevis, grown on permeable supports. Addition of insulin resulted in 2 to 3 fold increases in inositol trisphosphate and a 50% increase in 1,2 diacylglycerol within 10s, which corresponded to the time-course, previously reported, of insulin stimulated increases in Na+ transport and Cai2+. Further studies showed that aldosterone, previously shown to stimulate an increase in 1,4,5-inositol trisphosphate at onset of the rise in Na+ transport, also increased DAG levels during the initial phase of stimulation of Na+ transport. These studies provide the first evidence that a biological response induced by insulin is mediated by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) which results in two products, inositol trisphosphate which causes the release of Ca2+ from intracellular stores and 1,2 diacylglycerol. In addition this study provides further support for the proposal that a common signal transduction mechanism mediates electrogenic Na+ transport by multiple agonists.

Outcome of pregnancy in women with moderate or severe renal insufficiency.

BACKGROUND: Pregnant women with mild preexisting renal disease have relatively few complications of pregnancy, but the risks of maternal and obstetrical complications in women with moderate or severe renal insufficiency remain uncertain. METHODS: We determined the frequency and types of maternal and obstetrical complications and the outcomes of pregnancy in 67 women with primary renal disease (82 pregnancies). All the women had initial serum creatinine concentrations of at least 1.4 mg per deciliter (124 mumol per liter) and gestations that continued beyond the first trimester. RESULTS: The mean (+/- SD) serum creatinine concentration increased from 1.9 +/- 0.8 mg per deciliter (168 +/- 71 mumol per liter) in early pregnancy to 2.5 +/- 1.3 mg per deciliter (221 +/- 115 mumol per liter) in the third trimester. The frequency of hypertension rose from 28 percent at base line to 48 percent in the third trimester, and that of high-grade proteinuria (urinary protein excretion, > 3000 mg per liter) from 23 percent to 41 percent. For the 70 pregnancies (57 women) for which data were available during pregnancy and immediately post partum, pregnancy-related loss of maternal renal function occurred in 43 percent. Eight of these pregnancies (10 percent of the total) were associated with rapid acceleration of maternal renal insufficiency. Obstetrical complications included a high rate of preterm delivery (59 percent) and growth retardation (37 percent). The infant survival rate was 93 percent. CONCLUSIONS: Among pregnant women with moderate or severe renal insufficiency, the rates of complications due to worsening renal function, hypertension, and obstetrical complications are increased, but fetal survival is high.

Correlation of open cell-attached and excised patch clamp techniques.

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na+ channel, located on the apical surface of A6 cells, to determine whether the nystatin-induced open cell-attached patch could serve as an alternative configuration. Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and Erev was zero in symmetrical Na+ solutions, indicating similar Na+ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3-4pS) and Erev (75 mV), when the bath was perfused with a high K:low Na solution (ENa = 80 mV), were nearly equal in both patch configurations. Our results therefore indicate that the nystatin-induced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.

Vasopressin-stimulated electrogenic sodium transport in A6 cells is linked to a Ca(2+)-mobilizing signal mechanism.

Vasopressin is known to activate two types of cell surface receptors; V2, coupled to adenylate cyclase, and V1, linked to a Ca(2+)-dependent transduction system. We investigated whether arginine vasopressin (AVP) stimulation of electrogenic sodium transport in A6 cells, derived from Xenopus laevis, is mediated by activation of either one or both types of AVP-specific receptors. AVP caused a rapid increase in electrogenic sodium transport, reflected by the transepithelial potential difference (VT) and equivalent short circuit current (Ieq) measurements. AVP also rapidly increased intracellular Ca2+ (Ca2+i) and total inositol trisphosphate. The increase in Ieq was dependent on the rise in (Ca2+i), because 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) dose-dependently inhibited the Ieq response. There was no evidence, however, that activation of adenylate cyclase mediated AVP-stimulated Ieq; transport was not inhibited after AVP-induced activation of adenylate cyclase was abolished by 2',5'-dideoxyadenosine or when cAMP-dependent protein kinase (PKA) activity was abolished by the specific PKA inhibitor IP20. Further studies showed that although both forskolin and 8-(4-chlorophenylthio)-cAMP stimulated Ieq, this occurred by mechanisms independent of PKA activation. These results indicate that AVP-stimulated Na+ transport is mediated by a V1 receptor and a Ca(2+)-dependent mechanism.

Adenosine stimulation of Na+ transport is mediated by an A1 receptor and a [Ca2+]i-dependent mechanism.

Studies were performed to determine the primary signal transduction mechanism that mediates adenosine stimulation of electrogenic sodium transport in renal epithelial cells. Experiments were performed on cultured amphibian A6 cells with an adenosine analogue that preferentially binds to the A1 receptor, cyclohexyladenosine (CHA). Sodium transport was assessed by the equivalent short circuit current (Ieq). CHA was found to stimulate Ieq via activation of an A1 receptor because (1) the threshold concentration was 1 nM compared to that of 10 microM for the specific A2 agonist CGS21680, (2) CHA inhibited vasopressin (AVP)-stimulated cAMP production by a pertussis toxin-sensitive mechanism, and (3) the action of CHA was inhibited by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). CHA increased intracellular Ca2+ ([Ca2+]i) and stimulated phosphoinositide turnover at concentrations that increased Ieq and in a time course that paralleled the increase in Ieq. Ion transport was stimulated by a Ca(2+)-dependent mechanism because the CHA induced increase in Ieq was inhibited by chelating [Ca2+]i with 5,5'dimethyl BAPTA in a dose-dependent manner, with a Ki of approximately 10 microM. The increase in Ieq was also dose-dependently inhibited by the specific PKC inhibitors dihydroxychlorpromazine and chelerythrine, and by trifluoperazine which inhibits PKC and calmodulin. Further studies indicated that CHA-stimulated Ieq was independent of cAMP generation because CHA did not induce an increase in cAMP accumulation parallel to the increase in Ieq in a dose-response analysis, and the adenylate cyclase inhibitor 2',5' dideoxy-adenosine (DDA) did not affect the CHA-induced increase in Ieq.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic lupus erythematosus with nephropathy.

This paper is the first in a new Clinical Feature Series that will be a regular feature in future issues of The Yale Journal of Biology and Medicine. Papers in the series will focus on cases and disease processes of general interest to the practicing clinician, with the goal being to present the perspectives of expert clinicians regarding the accurate diagnosis and effective treatment of the diseases being discussed. The following case was presented at Medical Grand Rounds at the Yale-New Haven Hospital and the Yale School of Medicine by John P. Hayslett, Professor of Medicine (Nephrology) with accompanying analysis of pathological specimens by Dr. Hugh Carey, a Postdoctoral Fellow of the Pathology staff. The patient whose case is presented was also present to describe her experience with the illness and the course of treatment.

The benefit of early treatment with immunosuppressive agents in lupus nephritis.

OBJECTIVE: In a cohort of 87 patients with lupus nephritis, delay between the detection of the onset of renal disease and renal biopsy was a significant predictor at the time of a first renal biopsy for subsequent renal insufficiency (relative risk - 4.9; 95% confidence interval = 1.7 to 14.5; p < 0.001) and death due to lupus renal involvement (relative risk = 6.7; 95% confidence interval = 2.1 to 21.2; p < 0.001). We evaluated the role of lead time bias, 2 variants of prognostic selection bias (length biased sampling), and the benefit of early treatment as explanations for this effect. METHODS AND RESULTS: Evaluation using the time of renal onset rather than the time of renal biopsy for the analysis suggested that lead time bias was unlikely to be an explanation for the effect of duration on renal insufficiency or death due to renal involvement. Identical values of age, serum creatinine and 24 hour urinary protein excretion at renal onset for those with a long duration versus short duration prior to biopsy, suggested that differences in prognostic selection were unlikely to explain the observed results. A 2nd type of prognostic selection bias arising from the failure to include patients who did not undergo a renal biopsy was further assessed by statistical simulation. The results of this approach indicated that prognostic selection bias was not solely responsible for the significant associations. Because treatment with high dose prednisone and immunosuppressive drugs was not instituted until a renal biopsy had been performed, delay in instituting these therapies remained a possible explanation for the increased frequency of renal insufficiency and death due to renal involvement observed in those with longer delays before renal biopsy. In addition, there was significant deterioration in serum creatinine (median change 0.6 mg/dl) and 24 hour urinary protein excretion (median change 2.5 gm) over the period from renal onset to renal biopsy, and significantly higher scores for the activity, chronicity and tubulointerstitial indices on renal biopsy in those in whom therapy was delayed. CONCLUSION: Prompt therapy with prednisone and immunosuppressive agents in lupus nephritis has a beneficial effect on longterm prognosis.

Response to treatment as a predictor of longterm outcome in patients with lupus nephritis.

OBJECTIVE: To evaluate response to therapy over one year as a predictor of several longterm outcomes in lupus nephritis. METHODS: Response to treatment was evaluated by comparing serum creatinine and 24 h urine protein excretion at initial renal biopsy to those obtained after one year of treatment. Response at one year was evaluated as a predictor of renal failure, death due to lupus nephritis, and total lupus mortality, using survival analysis. RESULTS: Eighty-five patients with lupus nephritis diagnosed between 1967 and 1983 and followed through 1990 were studied. Change in proteinuria was a powerful predictor of renal failure (p = 0.001) death due to lupus nephritis (p < 0.001) and overall lupus mortality (p = 0.001). In contrast to a recent study of patients selected for severe lupus nephritis, serum creatinine was not found to be of prognostic significance. CONCLUSION: Response of proteinuria to treatment over one year is a useful predictor of longterm outcomes in lupus nephritis.

Mechanism of insulin-stimulated electrogenic sodium transport.

Studies were performed to determine the signal transduction mechanism involved in the onset of insulin stimulated electrogenic sodium transport (Ieq) in cultured A6 cells. Insulin stimulated Ieq at a threshold concentration of one nM and a half-maximum concentration of approximately 3 nM. The onset of action occurred within 10 seconds and the increase in Ieq was augmented by pretreatment with aldosterone, similar to the action of vasopressin. Insulin stimulated an increase in Ca2+i in a dose-dependent manner that involved release from intracellular stores. Hormone stimulated Ieq was dependent on increases in Ca2+i because pretreatment with 5, 5' dimethyl BAPTA/AM blocked the increase in sodium transport. Further studies with dihydroxyclorpromazine, trifluoperazine and genistein, inhibitors of PKC, Ca2+i dependent, calmodulin dependent kinases and tyrosine kinase, respectively, suggested that the action of insulin was dependent on activation of these kinases. In contrast, insulin stimulated Ieq was independent of changes in cAMP, because insulin did not increase the accumulation of cAMP, and inhibition of adenylate cyclase with 2', 5' dideoxyadenosine did not affect transport. These results suggest that insulin, as previously shown for aldosterone, activates apical membrane amiloride sensitive sodium channels by a calcium-dependent second messenger system.

Managing diabetic patients with nephropathy and other vascular complications.

Since the metabolic changes in normal pregnancy are diabetogenic, pregnancy imposes a severe stress on the metabolic milieu of diabetic patients. Moreover, many patients with long-standing diabetes have vascular complications, including retinopathy, renal insufficiency, nephrotic syndrome and hypertension, all representing separate risk factors for optimal fetal development. Recent experience has suggested that maternal hyperglycaemia, and associated fetal hyperinsulinaemia, may represent an important factor in the development of fetal complications. During the past two to three decades the incidence of perinatal deaths in all categories of diabetics has been reduced to a level that approaches the rate in healthy gravidas when severe congenital anomalies are excluded. Fetal and neonatal morbidity have also been reduced, although rates of congenital anomalies, hydramnios and respiratory distress syndrome remain high. Although the morbidity associated with oedema formation and hypertension is elevated, with meticulous management of patients with diabetic nephropathy, especially in the absence of severe renal insufficiency and/or severe hypertension, pregnancy performance and outcome can be similar to that of other insulin-dependent diabetic patients.

The time-dependence of long-term prediction in lupus nephritis.

OBJECTIVE: To assess the clinical, laboratory, and renal biopsy predictors of long-term outcome in lupus nephritis and to investigate the time-dependence of these predictors. METHODS: Eighty-seven lupus nephritis patients were studied retrospectively for the outcomes renal failure and fatality due to renal involvement. In addition to a conventional Cox model analysis, a new generalized time-dependent analytic approach was developed and used to assess the time-dependence of a predictor variable's importance. RESULTS: The mean followup time was 11.9 years. Renal failure (n = 19) was significantly predicted by measures of renal function (abnormal serum creatinine levels, proteinuria, duration of prior renal disease) and immunologic activity (elevated DNA binding, hypocomplementemia, and thrombocytopenia), by overall lupus disease activity measures (le Riche index, Lupus Activity Criteria Count), and by the activity index, the tubulointerstitial index, and the amount of subepithelial deposits on renal biopsy. In general, the laboratory predictors were significantly better prognostic markers in the early years after biopsy, the disease activity measures were best in the later years, and the biopsy variables were significant predictors over the entire observation period. In contrast to the renal failure outcome, the best predictors for death not directly related to lupus nephritis (n = 17) were the extent of comorbid diseases (principally vascular diseases), older age, and the chronicity index. All three predicted well over the extended observation period. CONCLUSION: The major predictor variables for renal outcomes and nonrenal outcomes are distinct. The time-dependence of the predictive ability of some variables may be important in managing individual patients. The new generalized time-dependent analytic technique may have widespread application in studies to identify prognostic factors for established disease or risk factors for the development of disease.

The pathogenesis and prognosis of lupus nephritis: information from repeat renal biopsy.

Of an inception cohort of 87 patients with lupus nephritis who underwent a renal biopsy, 42 underwent second biopsies a median of 25 months later. From first to second biopsy, focal and diffuse proliferative nephritis (World Health Organization classes III and IV) became less frequent, and mesangial hypercellularity (class II) and a membranous pattern (class V) increased. The National Institutes of Health activity index and mesangial and subendothelial deposits declined while the chronicity index, a tubulointerstitial index, and subepithelial deposits increased. The biopsy improvement in urinary protein excretion was best explained by decreases in the activity index score and the amount of subendothelial deposits. A decrease in the amount of subendothelial deposits tended to predict an improvement in the serum creatinine level from first to second biopsy. With follow-up from second biopsy in excess of 7 years, the best predictors of long-term outcome were the ultrastructural variables mesangial, subendothelial and subepithelial deposits. When the change in biopsy predictors from first to second biopsy was evaluated, a decrease in the amount of mesangial or subendothelial deposits was best at predicting a lower risk of renal impairment, renal insufficiency, and mortality. The results confirm the importance of immune complex deposition as measured by electron microscopy in the pathogenesis of lupus nephritis and suggest that control of this process may alter renal function and prognosis.