Norman J. Siegel

OROPHARYNGEAL EXCRETION OF EPSTEIN-BARR VIRUS BY RENAL TRANSPLANT RECIPIENTS AND OTHER PATIENTS TREATED WITH IMMUNOSUPPRESSIVE DRUGS

Abstract The excretion of Epstein-Barr (E.B.) virus was studied in eighty patients and staff in a hospital-based renal-disease unit. Virus shedding was measured by the capacity of throat washings to induce long-term proliferation of human umbilical-cord leucocytes and the appearance of E.B. virus nuclear antigen in the cultivated cells. The excretion-rates of E.B. virus, based on single throat samples, differed in the four groups of individuals tested: virus was recovered from 47% of patients with renal homografts, from 35% of other patients treated with immunosuppressive drugs, from 14% of patients with chronic uraemia and from 17% of healthy staff associated with the unit. Only individuals with antibody to E.B. virus capsid antigen shed virus. The excretion-rate for E.B. virus seropositive individuals on immunosuppressive drugs was 52% (seventeen out of thirty-three) compared with a rate of 18% (six out of thirty-four) for individuals not treated with these agents. Five seropositive patients received renal homografts during the study. None were E.B. virus excretors before transplantation; two were shedding virus 8 and 34 days after transplantation and institution of immunosuppressive therapy. The results support the hypothesis that immunosuppressive drugs (azathioprine and prednisone) are associated with reactivation of latent E.B. virus infections.

Clinical trial of focal segmental glomerulosclerosis in children and young adults

This NIH-funded multicenter randomized study of focal segmental glomerulosclerosis (FSGS) treatment compared the efficacy of a 12-month course of cyclosporine to a combination of oral pulse dexamethasone and mycophenolate mofetil in children and adults with steroid-resistant primary FSGS. Of the 192 patients enrolled, 138 were randomized to cyclosporine (72) or to mycophenolate/dexamethasone (66). The primary analysis compared the levels of an ordinal variable measuring remission during the first year. The odds ratio (0.59) for achieving at least a partial remission with mycophenolate/dexamethasone compared to cyclosporine was not significant. Partial or complete remission was achieved in 22 mycophenolate/dexamethasone- and 33 cyclosporine-treated patients at 12 months. The main secondary outcome, preservation of remission for 26 weeks following cessation of treatment, was not significantly different between these two therapies. During the entire 78 weeks of study, 8 patients treated with cyclosporine and 7 with mycophenolate/dexamethasone died or developed kidney failure. Thus, our study did not find a difference in rates of proteinuria remission following 12 months of cyclosporine compared to mycophenolate/dexamethasone in patients with steroid-resistant FSGS. However, the small sample size might have prevented detection of a moderate treatment effect.

Functional and Hemodynamic Adaptation to Progressive Renal Ablation

Removal of renal tissue stimulates functional and anatomical adaptation in the remaining renal parenchyma. Since recent studies have demonstrated no apparent limitation in compensatory growth following progressive surgical ablation, experiments were performed to determine the changes in glomerular filtration rate and renal blood flow. After removal of 50% of the renal mass mean nephron glomerular filtration rate increased 60%, and after ablation of 75% of the renal tissue it increased 150%. These changes paralleled the increases in renal growth under the same conditions. In comparison, mean glomerular blood flow rose 90% and 240% after 50% and 75% nephrectomy, respectively; these changes in relation to the changes in glomerular filtration rate resulted in a progressive fall in the filtration fraction. Intrarenal blood flow distribution was examined with labeled microspheres. The marked increase in renal blood flow after surgical ablation was characterized by a disproportionate rise in blood flow to the inner cortex. The present investigation, therefore, describes the remarkable functional changes that occur as overall glomerular filtration rate declines and provides further insight into the mechanism responsible for maintaining water and electrolyte homeostasis after loss of functioning renal mass.

Expression of parathyroid hormone-related protein in the rat glomerulus and tubule during recovery from renal ischemia.

Parathyroid hormone-related protein (PTHrP) is widely expressed in normal adult and fetal tissues, where it acts in an autocrine/paracrine fashion, stimulates growth and differentiation, and shares early response gene characteristics. Since recovery from renal injury is associated with release of local growth factors, we examined the expression and localization of PTHrP in normal and ischemic adult rat kidney. Male Sprague-Dawley rats underwent complete bilateral renal artery occlusion for 45 min, followed by reperfusion for 15 min, and 2, 6, 24, 48, and 72 h. Renal PTHrP mRNA levels, when compared with sham-operated animals, increased twofold after ischemia, and peaked within 6 h after reperfusion. PTH receptor, beta-actin, and cyclophilin mRNA levels all decreased after ischemia. PTHrP immunohistochemical staining intensity increased in proximal tubular cells after ischemia, changing its location from diffusely cytoplasmic to subapical by 24 h after reperfusion. In addition, PTHrP localized to glomerular epithelial cells (visceral and parietal), but not to mesangial cells. PTHrP and PTH stimulated proliferation two- to threefold in cultured mesangial cells. We conclude that PTHrP mRNA and protein production are upregulated after acute renal ischemic injury, that PTHrP is present in glomerulus and in both proximal and distal tubular cells, and that PTHrP stimulates DNA synthesis in mesangial cells. The precise functions of PTHrP in normal and injured kidney remain to be defined.

Clinical course and outcome for children with multicystic dysplastic kidneys

Abstract The purpose of this study was to evaluate the clinical course and outcome for children with multicystic dysplastic kidney (MCDK) disease and to non-invasively predict which of these patients are at significant risk for developing urinary tract infection (UTI) and renal insufficiency. Patients were divided, on the basis of postnatal physical examination and renal ultrasonography, into simple or complex MCDK. Simple MCDK was defined as unilateral renal dysplasia without additional genitourinary (GU) abnormalities. Complex MCDK included patients with bilateral renal dysplasia or unilateral renal dysplasia with other GU abnormalities. The designation as simple or complex MCDK was independent of reflux, since routine voiding cystourethrography (VCUG) was not performed. The charts of all patients with the diagnosis of MCDK disease seen from August 1995 to March 1999 at Yale University School of Medicine were examined to determine: (1) if UTI had occurred and (2) the level of renal function at last follow-up. Thirty-five patients were evaluated: 28 (80%) patients had unilateral MCDK, 7 (20%) were bilateral, and 14 (40%) had associated GU anomalies. Overall, 21 patients had unilateral MCDK without GU abnormalities (simple MCDK), while 14 had complex MCDK. The final outcome for patients with simple MCDK was quite good, with normal renal function and compensatory hypertrophy of the contralateral kidney in all patients. Although the patients with simple MCDK did not have routine VCUG or prophylactic antibiotics, the development of UTI was infrequent, damage to the contralateral kidney did not occur, and renal function was well preserved. In contrast, patients with bilateral disease or associated GU anomalies had a higher incidence of UTI and progression to renal failure. Complex MCDK was associated with a worse outcome (50% chronic renal insufficiency or failure).

Activation of heat-shock transcription factor by graded reductions in renal ATP, in vivo, in the rat.

Renal ischemia results in both a profound fall in cellular ATP and a rapid induction of the 70 kD heat-shock protein family, HSP-70. The present studies examined the relationship between cellular ATP and induction of the stress response in renal cortex. Cellular ATP, continuously monitored by in vivo 31P-NMR spectroscopy, was reduced and maintained at specific, stable levels in renal cortex by partial aortic occlusion for 45 min. Activation of heat-shock transcription factor (HSF) was detected by gel retardation assay and transcription was confirmed by Northern analysis. Activation of HSF was not present, and HSP-70 mRNA induction did not occur when ATP levels were maintained above 60% preocclusion (control) levels. Reduction in cortical ATP levels to 35-50% preocclusion values resulted in HSF activation and low-level expression of inducible HSP-70 mRNA. Cellular ATP of 20-25% control values resulted in a greater level of HSF activation and subsequent HSP-70 mRNA elaboration. HSF was activated at the end of 15 min of total occlusion. The studies indicate that a 50% reduction in cellular ATP in the renal cortex must occur before the stress response is detectable, that reduction of ATP below 25% control levels produces a more vigorous response, and that reperfusion is not required for initiation of a heat-shock response in the kidney. Cellular ATP, or the metabolic consequences associated with ATP depletion, may be a threshold factor for initiation of a stress response in the kidney.

Hsp27 Associates with Actin and Limits Injury in Energy Depleted Renal Epithelia

The purpose of the study was to determine whether Hsp27 interacts with actin and could protect against selected manifestations of injury from energy depletion in renal epithelia. LLC-PK1 cells were stably transfected to overexpress human Hsp27 tagged with green fluorescence protein (GFP). Transfected expression of the labeled Hsp27 did not reduce endogenous Hsp25 levels in the cells compared with either nontransfected cells or cells transfected with GFP alone used as the transfectant control (G). By fluorescence energy transfer (FRET) between GFP-tagged Hsp27 and rhodamine phalloidin-decorated actin, minimal interaction was found in uninjured control cells. In ATP-depleted cells, Hsp27 was associated closely with F-actin at lateral cell boundaries and with aggregated actin within the cell body. Less Hsp27 interaction with actin was found during recovery; but when adjusted for total phalloidin fluorescence, FRET between Hsp27 and F-actin did not change between 2-h ATP depletion and 4-h recovery. Where Hsp27 association with actin persisted during recovery, it was principally with the residual aggregates of actin in the cell body. Detachment of Na,K-ATPase from the cytoskeleton at 2-h ATP depletion was significantly less in Hsp27 cells compared with transfectant control G cells but not at 4-h ATP depletion. Detachment of ezrin from the cytoskeleton during ATP depletion was nearly complete and was not prevented in the Hsp27 cells. Protection of the Hsp27 cells was not attributable to preservation of cellular ATP levels. Hsp27 appears to have specific actions in renal epithelia subjected to energy depletion, including interacting with actin to preserve architecture in specific intracellular domains.

Functional Activation of Heat Shock Factor and Hypoxia-Inducible Factor in the Kidney

Renal ischemia is the result of a complex series of events, including decreases in oxygen supply (hypoxia) and the availability of cellular energy (ATP depletion). In this study, the functional activation of two stress-responsive transcription factors, i.e., heat shock factor-1 (HSF-1) and hypoxia-inducible factor-1 (HIF-1), in the kidney was assessed. When rats were subjected to 45 min of renal ischemia, electrophoretic mobility shift assays of kidney nuclear extracts revealed rapid activation of both HIF-1 and HSF. Western blot analyses further demonstrated that this activation resulted in increased expression of the HSF and HIF-1 target genes heat shock protein-72 and heme oxygenase-1, respectively. Whether hypoxia or ATP depletion alone could produce similar activation patterns in vitro was then investigated. Renal epithelial LLC-PK(1) cells were subjected to either ATP depletion (0.1 microM antimycin A and glucose deprivation) or hypoxia (1% O(2)). After ATP depletion, HSF was rapidly activated (within 30 min), whereas HIF-1 was unaffected. In contrast, hypoxia led to the activation of HIF-1 but not HSF. Hypoxic activation of HIF-1 was observed within 30 min and persisted for 4 h, whereas no HSF activation was detected even with prolonged periods of hypoxia. HIF-1 was transcriptionally active in LLC-PK(1) cells, as demonstrated by luciferase reporter gene assays using the vascular endothelial growth factor promoter or a synthetic promoter construct containing three hypoxia-inducible elements. Interestingly, intracellular ATP levels were not affected by hypoxia but were significantly reduced by ATP depletion. These findings suggest that HIF-1 is activated specifically by decreased O(2) concentrations and not by reduced ATP levels alone. In contrast, HSF is activated primarily by metabolic stresses associated with ATP depletion and not by isolated O(2) deprivation. In vivo, the two transcription factors are simultaneously activated during renal ischemia, which might account for observed differences between in vivo and in vitro epithelial cell injury and repair. Selective modulation of either pathway might therefore be of potential interest for modification of the response of the kidney to ischemia, as well as the processes involved in recovery from ischemia.

Heat-shock protein 25 induction and redistribution during actin reorganization after renal ischemia

The small heat-shock proteins appear to have a regulatory role in actin dynamics. Since cytoskeletal disruption is integral to ischemic renal injury, we evaluated expression and intracellular distribution of heat-shock protein 25 (HSP-25) in rat renal cortex after 45 min of renal ischemia. HSP-25 was constitutively expressed and induced by ischemia with peak levels reached by 6 h reflow. Ischemia caused a shift of HSP-25 from the detergent-soluble into the insoluble cytoskeletal fraction. By 2 h reflow, the majority of HSP-25 had redistributed into the soluble fraction. HSP-25 was predominantly localized in a subapical distribution in control proximal tubules, a pattern intermediate between deoxyribonuclease (DNase)-reactive and filamentous actin. After ischemia, HSP-25 dispersed through the cytoplasm with small punctate accumulations similar to DNase-reactive actin. During later reflow, all three proteins were found in coarse intracytoplasmic accumulations; however, HSP-25 and DNase-reactive actin were in separate accumulations. HSP-25 and microfilamentous actin staining returned to the subapical domain. Thus the temporal and spatial patterns of HSP-25 induction and distribution suggest specific interactions between HSP-25 and actin during the early postischemic reorganization of the cytoskeleton. HSP-25 may have additional roles distinct from actin dynamics later in the course of postischemic recovery.

Minimal Change and Focal Sclerotic Lesions in Lipoid Nephrosis

22 children, with the onset of the nephrotic syndrome between 1 and 6 years of age, were studied by renal biopsy because of steroid resistance or after many years of steroid-sensitive disease. Six chi