Pathogenesis of Shiga toxin-induced hemolytic uremic syndrome.

The term hemolytic uremic syndrome (HUS) was first introduced to describe a heterogeneous group of diseases characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Substantial progress has been made in our understanding of the etiology and pathogenesis of HUS. This article reviews some of the classic and new concepts related to the pathogenesis of Shiga toxin (Stx)-HUS and discusses their clinical relevance for the diagnosis and treatment of this syndrome. Infection with Stx-producing bacteria can induce HUS after a prodromal illness with or without diarrhea. Stx-induced renal endothelial injury is the primary pathogenic event. However, Stx also damages mesangial cells, as well as glomerular and renal tubular epithelial cells. Young children are at greatest risk for Stx-HUS because they express high levels of Stx receptors in renal glomeruli. Older children and adults express lower levels of glomerular Stx receptors and may develop Stx-HUS whenever the combined effects of lipopolysaccharide and cytokines upregulate the expression of Stx receptors and sensitize glomerular endothelial cells to Stx-induced injury, activate the coagulation-fibrinolytic system, and induce endothelial injury. Chemokine receptors and cytokines released by inflammatory cells (i.e., monocyte chemoattractant protein-1, interleukin-6, interleukin-8,) or injured endothelial cells (i.e., basic fibrobast growth factor) may play roles in this process. Measurement of the activity of a von Willebrand factor protease in plasma may help distinguish patients with thrombotic thrombocytopenic purpura from those with Stx-HUS.

Hypokalemia induces renal injury and alterations in vasoactive mediators that favor salt sensitivity.

We investigated the hypothesis that hypokalemia might induce renal injury via a mechanism that involves subtle renal injury and alterations in local vasoactive mediators that would favor sodium retention. To test this hypothesis, we conducted studies in rats with diet-induced K+ deficiency. We also determined whether rats with hypokalemic nephropathy show salt sensitivity. Twelve weeks of hypokalemia resulted in a decrease in creatinine clearance, tubulointerstitial injury with macrophage infiltration, interstitial collagen type III deposition, and an increase in osteopontin expression (a tubular marker of injury). The renal injury was greatest in the outer medulla with radiation into the cortex, suggestive of an ischemic etiology. Consistent with this hypothesis, we found an increased uptake of a hypoxia marker, pimonidazole, in the cortex. The intrarenal injury was associated with increased cortical angiontensin-converting enzyme (ACE) expression and continued cortical angiotensin II generation despite systemic suppression of the renin-angiotensin system, an increase in renal endothelin-1, a decrease in renal kallikrein, and a decrease in urinary nitrite/nitrates and prostaglandin E(2) excretion. At 12 wk, hypokalemic rats were placed on a normal-K+ diet with either high (4%)- or low (0.01%)-NaCl content. Despite correction of hypokalemia and normalization of renal function, previously hypokalemic rats showed an elevated blood pressure in response to a high-salt diet compared with normokalemic controls. Hypokalemia is associated with alterations in vasoactive mediators that favor intrarenal vasoconstriction and an ischemic pattern of renal injury. These alterations may predispose the animals to salt-sensitive hypertension that manifests despite normalization of the serum K+.

Up-regulation of a fibroblast growth factor binding protein in children with renal diseases.

BACKGROUND: Basic fibroblast growth factor (bFGF) is an angiogenic growth factor that is involved in renal growth and the pathogenesis of renal diseases. We have detected high levels of bFGF accumulated in the kidney of HIV-transgenic mice and in children with HIV-associated renal diseases and the hemolytic uremic syndrome (HUS). However, the mechanism modulating the activity of bFGF under these circumstances is poorly understood. We carried out experiments to determine whether a secreted binding protein (FGF-BP) that modulates the activity of bFGF during the process of tumor growth was expressed in pediatric kidneys and to define whether the expression of FGF-BP was altered in pediatric renal diseases associated with high levels of bFGF. METHODS: Immunohistochemistry and in situ hybridization studies were done in 41 renal sections from children with HIV nephropathies, HUS, other pediatric renal diseases, controls, and fetal kidneys. Western blots and reverse transcriptase-polymerase chain reaction studies were done in selected urine samples and cultured renal cells. Recombinant FGF-BP was produced to study the mitogenic activity of FGF-BP in cultured human renal proximal tubular epithelial cells (RPTEcs). RESULTS: The expression of FGF-BP was up-regulated predominately in renal tubular epithelial cells in children with renal tubular injury, HIV-associated nephropathy (HIVAN), and HUS, and FGF-BP was secreted in the urine of these patients. FGF-BP was also abundantly expressed in developing fetal renal tubules. Recombinant FGF-BP enhanced the mitogenic effects of bFGF in cultured human RPTEcs. CONCLUSIONS: The localization of FGF-BP in renal tubular epithelial cells could provide a mechanism by which the activity of bFGF is modulated in developing and regenerating renal tubules of children.

Chronic potassium depletion induces renal injury, salt sensitivity, and hypertension in young rats.

BACKGROUND: Chronic hypokalemia has been associated with renal hypertrophy, interstitial disease, and hypertension in both adult animals and humans. However, the effects of potassium (K(+)) depletion on the rapidly growing infant have not been well studied. The purpose of this study was to determine the effects of severe chronic dietary K(+) depletion on blood pressure (BP) and renal structural changes in young rats. METHODS: Sprague-Dawley rats (50 +/- 5 g) were fed either a control or a potassium-deficient diet (<0.05% K(+)) for 14 to 21 days. At the end of this period, the blood pressure (BP) was measured in all rats, and six rats in each group were sacrificed to determine changes in renal histology and renin-angiotensin system (RAS) activity. The remaining rats in each group were then switched to a high-salt (6% NaCl)--normal-K(+) (0.5%) diet or were continued on their respective control or K(+)-deficient diet for an additional six days. Blood pressure measurements were done every three days until the end of the study. RESULTS: K(+)-depleted animals had significant growth retardation and increased RAS activity, manifested by high plasma renin activity, recruitment of renin-producing cells along the afferent arterioles, and down-regulation of angiotensin II receptors in renal glomeruli and ascending vasa rectae. K(+)-depleted kidneys also showed tubulointerstitial injury with tubular cell proliferation, osteopontin expression, macrophage infiltration, and early fibrosis. At week 2, K(+)-depleted rats had higher systolic BP than control rats. Switching to a high-salt (6% NaCl)--normal-K(+) diet resulted in further elevation of systolic BP in K(+)-depleted rats, which persisted even after the serum K(+) was normalized. CONCLUSION: Dietary potassium deficiency per se increases the BP in young rats and induces salt sensitivity that may involve at least two different pathogenic pathways: increased RAS activity and induction of tubulointerstitial injury.

Efficient gene transfer to rat renal glomeruli with recombinant adenoviral vectors.

Recombinant adenoviruses are attractive vectors for renal gene transfer since they can efficiently transduce nondividing cells. However, despite the fact that renal glomeruli are easily accessible via the renal circulation, attempts to deliver foreign genes specifically into renal glomeruli, using adenoviral vectors, have had limited success in rodents. A simple intraarterial injection of adenoviral vectors into the renal circulation or incubation of the virus with the kidney for an extended period of time was found to be insufficient for this purpose. In this study, we have established an efficient gene transfer protocol to express foreign genes in rat renal glomerular cells, using adenoviral vectors. We demonstrated, for the first time, that rat glomerular endothelial cells could be efficiently transduced by slowly infusing a recombinant adenovirus (Ad.CBlacZ) into the right renal artery for a period of 15 min. High levels of lacZ expression were achieved in renal glomeruli without causing significant damage to renal glomeruli or other kidney structures. The virus-mediated expression lasted for at least 21 days. These data demonstrate the feasibility of using recombinant adenoviral vectors as a tool with which to study the effect of foreign gene expression on the structure and function of rat renal glomeruli in vivo.

Expression of renal and vascular angiotensin II receptor subtypes in children.

Angiotensin II (Ang II) AT1 receptors modulate most of the known physiological functions of Ang II in the kidney and cardiovascular structures. In contrast, the physiological role of AT2 receptors, which are abundantly expressed in fetal tissues, is not clearly defined. The changes that occur in the expression and distribution of AT2 receptors in the kidney and arteries during the first 2 years of life have not been studied. We have localized and characterized the expression of Ang II receptor subtypes, AT1 and AT2, in the kidney, interlobular arteries, thoracic aorta, and middle cerebral artery, in children during their first 2 years of life, using quantitative autoradiography. Renal glomeruli and middle cerebral arteries expressed exclusively AT1 receptors. In contrast, more than 80% of the Ang II receptors expressed in thoracic aorta and interlobular arteries belonged to the AT2 subtype. These findings demonstrate that the expression of Ang II receptor subtypes in different vascular structures in young children varies according to the tissue.

Isoproterenol inhibits fibroblast growth factor-2-induced growth of renal epithelial cells.

The signal transduction pathways modulating bFGF effects in renal tubular epithelial cells (RTEc) are not completely understood. Since the cAMP and the mitogen-activated protein kinase (MAPK) pathways can modulate the growth of RTEc, we studied whether two cAMP elevating agents, isoproterenol and 8-bromo-cAMP, would modulate basic fibroblast growth factor (bFGF) induction of MAPK activity (ERK-2) and cell proliferation in human renal proximal tubular epithelial cells (RPTEc) and Madin-Darby canine kidney cells (MDCK clone EI1). Isoproterenol, but not bFGF, stimulated cAMP production in RPTEc and MDCKEI1 cells. bFGF, isoproterenol, and 8-bromo-cAMP alone increased ERK-2 activity in both cell types. However, isoproterenol and 8-bromo-cAMP partially inhibited the bFGF induction of ERK-2 activity, but only isoproterenol inhibited the proliferation of both cell types. PD098059 (25 microM), an inhibitor of MAPK kinase (MEK 1/2), blocked the bFGF mitogenic effects, but did not affect the 8-bromo-cAMP-induced mitogenic effects in MDCKEI1 cells. These findings suggest that activation of ERK-2 is required but not sufficient for mitogenesis in RTEc. We conclude that isoproterenol inhibits the growth-promoting effects of bFGF in RTEc via MEK-dependent and -independent pathways.

Angiotensin II and basic fibroblast growth factor mitogenic pathways in human fetal mesangial cells.

Angiotensin II (Ang II) and basic fibroblast growth factor (bFGF/FGF-2) play relevant roles in renal development. Since the signaling pathways modulating the mitogenic effects of Ang II and bFGF in human fetal mesangial cells (HFMc) are not clearly defined, we carried out experiments to determine whether they would exert their mitogenic effects by modulating the activity of the mitogen-activated protein kinases (MAPK) [extracellular signal-regulated kinase-2 (ERK-2)] and cAMP signaling pathways. In confluent HFMc, bFGF (20 ng/mL) induced a significant 4-fold increase in ERK-2 activity and [3H]-thymidine incorporation (6-fold). In contrast, under similar tissue culture conditions, Ang II (10(-6) M) induced a more modest increase in ERK-2 activity (2-fold) and [3H]-thymidine incorporation (35 +/- 4%). The mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD098059 (25 microM) almost completely abolished the bFGF-induced proliferation in HFMc but did not significantly affect Ang II proliferative effects. In the presence of the cAMP elevating agent isoproterenol, Ang II and bFGF induced opposite changes in cAMP accumulation and cell growth. Isoproterenol inhibited the basal and bFGF-induced proliferation of HFMc through a MEK-1/2-independent pathway that included the accumulation of cAMP. In contrast, isoproterenol increased Ang II mitogenic effects in correlation with a reduction in cAMP accumulation. We conclude that Ang II and bFGF modulate the proliferation of HFMc through the stimulation of different MEK-1/2-dependent and independent signaling pathways. Activation of MEK-1/2 is required but not sufficient for mitogenesis in HFMc. The accumulation of cAMP in HFMc counteracts the mitogenic effects of bFGF by a MEK-1/2-independent pathway.

Liver bypass significantly increases the transduction efficiency of recombinant adenoviral vectors in the lung, intestine, and kidney.

Recombinant adenoviruses have great potential as gene delivery systems because of their ability to infect a wide range of target cells. However, systemic delivery of viral vectors to tissues other than liver and spleen has been inefficient because of the rapid clearance of the circulating virus by the liver. In the present study we tested the hypothesis that a systemic administration of E1-deleted recombinant adenovirus vectors that bypasses the hepatic circulation will lead to enhanced expression of these vectors in extrahepatic tissues. The portal vein and hepatic artery in B6/129 F1 mice were clamped and an E1-deleted recombinant adenovirus carrying the beta-galactosidase gene (Ad.CBlacZ) was then administered through the retroorbital venous plexus. The clamp was released 30 min after viral injection with no major chronic ischemic consequences noted. High levels of LacZ expression were detected predominantly in the vessels and capillaries of the lung, intestinal wall, and renal glomeruli 7 days after viral infusion. The transgene expression persisted for at least 21 days. Intense LacZ staining was also observed in the liver, suggesting that liver infection occurred after the portal clamp was released. A retroorbital infusion of anti-adenovirus neutralizing antibodies 5 min before the release of the portal clamp significantly reduced postclamp viral infection to the liver, while LacZ expression in lung and intestine persisted after the antibody treatment. Taken together, these results suggest that liver bypass can significantly improve the transduction efficiency in the other target organs. This method could be used to develop animal models of human diseases that predominantly affect the vessels of the lung, intestine, and kidney.

Basic fibroblast growth factor in HIV-associated hemolytic uremic syndrome.

Endothelial injury is the primary pathogenic event leading to the renal thrombotic microangiopathic lesions typical of the hemolytic uremic syndrome (HUS). Basic fibroblast growth factor (bFGF) is an angiogenic growth factor released by injured endothelial cells. In a previous study we have found a significant accumulation of bFGF in human immunodeficiency virus (HIV)-transgenic mice with renal disease. Here we investigated whether bFGF was accumulated in the circulation and kidneys of two children with HIV-associated HUS (HIV-HUS), and studied the mechanisms involved in this process. The plasma levels of bFGF in children with HIV-HUS (124+/-20 pg/ml) were increased compared with five children with HIV nephropathy (49+/-6 pg/ml) and twenty HIV-infected children without renal disease (26+/-4 pg/ml, P<0.001). Immunohistochemistry and receptor binding studies showed that bFGF was accumulated bound to heparan sulfate proteoglycans in renal glomeruli and interstitium surrounding renal tubules in HIV-HUS kidneys. Basic FGF stimulated the proliferation of mesangial and urinary renal tubular epithelial cells isolated from both patients. These findings support the hypothesis that bFGF and its low-affinity binding sites may play a relevant role in modulating the process of glomerular and renal tubular regeneration during the acute stages of HIV-HUS. A follow-up study in a larger sample population is required to confirm these results.

Up-regulation of Duffy antigen receptor expression in children with renal disease.

BACKGROUND: The Duffy antigen chemokine receptor (DARC) is a promiscuous chemokine receptor that binds chemokines from the C-X-C and C-C families. DARC was initially described on red blood cells, but subsequent studies have demonstrated DARC protein expression on renal endothelial and epithelial cells, even in Duffy-negative individuals whose red cells lack DARC. Because approximately 68% of African Americans lack the Duffy/DARC on their red cells, we carried out experiments to identify the specific renal cells expressing DARC protein and mRNA in African American children and to define whether DARC expression was altered in renal inflammatory processes. METHODS: Immunohistochemistry and in situ hybridization studies were done in 28 renal sections from children with each of the following diagnoses: HIV nephropathy (HIVAN), HIV-associated hemolytic uremic syndrome (HIV-HUS), HIV infection without renal disease, HIV-negative children without renal disease, and Argentinean children with classic HUS. RESULTS: The predominant localization of DARC mRNA and protein was found in endothelial cells underlying postcapillary renal venules in all patients studied. However, DARC mRNA and protein were significantly up-regulated in peritubular and glomerular capillaries, collecting duct epithelial cells, and interstitial inflammatory cells in children with HIVAN, HIV-HUS, and classic HUS. CONCLUSION: These findings support the notion that the renal DARC is linked to the inflammatory cascade and that African American children may be at risk of accumulating chemokines in renal tissues.

Recruitment of renal tubular epithelial cells expressing verotoxin-1 (Stx1) receptors in HIV-1 transgenic mice with renal disease.

BACKGROUND: Human immunodeficiency virus (HIV)-infected children are at risk of developing several renal parenchymal diseases, including hemolytic uremic syndrome (HUS). HUS is most frequently caused by infection with enteric Escherichia coli producing Shiga-like toxins (Stxs). In vitro studies have shown that cytokines known to be present at high systemic levels in HIV-1-infected children up-regulate the expression of the Stx glycolipid receptor (Gb3) in cultured endothelial cells. Thus, we studied whether HIV-1 or the HIV-associated "cytokine milieu" could modulate the expression of renal Stxs receptors in vivo. METHODS: We used HIV-1 transgenic mice (HIV-Tg) expressing a deletion mutant of HIV-1 (pNL4-3). These mice develop renal disease similar to that of HIV-1-infected children. The expression of Gb3 was studied in renal sections from control and HIV-Tg mice by histochemistry, thin layer chromatography overlay studies, and high-pressure liquid chromatography. RESULTS: By histochemistry, we found a significant recruitment of renal tubular epithelial cells expressing Gb3 in HIV-Tg mice with nephropathy, whereas kidneys from control mice showed limited staining in renal tubules. Gb3 was not found in glomeruli of either control or HIV-Tg mice. Thin layer chromatography overlay studies with Stxs and high-pressure liquid chromatography studies confirmed the marked elevation of Gb3 in HIV-Tg kidneys with renal disease. CONCLUSIONS: These results suggest that the presence of HIV-associated nephropathy is associated with the recruitment of renal tubular epithelial cells expressing Stx1 receptors. The up-regulation of Stx1 receptors in HIV-diseased kidneys may increase the sensitivity of these cells to the cytotoxic effects of Stxs.

Human immunodeficiency virus (HIV)-associated nephropathy in children from the Washington, D.C. area: 12 years' experience.

Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) is a clinicopathologic entity that includes proteinuria, azotemia, focal segmental glomerulosclerosis or mesangial hyperplasia, and tubulointerstitial disease. The incidence of HIVAN is increased in black patients and variable depending on the age and geographic area. The objective of this study was to describe relevant clinical and pathological findings in 30 children with HIVAN followed at the Children's National Medical Center in Washington, D.C. Our experience of the last 12 years showed a spectrum of HIVAN that seems to be coincident with the degree of acquired immunodeficiency syndrome (AIDS) symptomatology. By renal sonograms and frequent urinalysis, we identified children undergoing the early stages of HIVAN with enlarged echogenic kidneys, proteinuria, and "urine microcysts". HIVAN did not necessarily progress rapidly to end-stage renal disease. Nephrotic syndrome or chronic renal insufficiency were late manifestations of HIVAN. Children with HIVAN were likely to develop transient electrolyte disorders, heavy proteinuria, and acute renal failure due to systemic infectious episodes or nephrotoxic drugs. HIVAN was associated with other HIV-induced illnesses and high mortality rates. Early detection and careful clinical follow-up of children with HIVAN may reduce the incidence of renal-cardiovascular complications and improve their quality of life.

Infection of human primary renal epithelial cells with HIV-1 from children with HIV-associated nephropathy.

Children affected with human immunodefficiency virus (HIV)-associated nephropathy (HIVAN) usually develop significant renal glomerular and tubular epithelial cell injury. The pathogenesis of these changes is not clearly understood. Human renal tubular epithelial cells (RTEc) do not express CD4 surface receptors, and it is not clear whether these cells can be infected by HIV-1. Certain strains of HIV-1, however, have been shown capable of infecting CD4-negative epithelial cell lines. We hypothesized that the inability of laboratory strains of HIV-1 to infect renal epithelial cells may be due to a limited tropism, as opposed to wild-type viruses derived from children with HIVAN, and that viruses derived from these children are capable of infecting RTEc from the same patient. Here, we have demonstrated that HIV-1 isolates from children with HIVAN can productively infect RTEc through a CD4 independent pathway, and that infected mononuclear cells can transfer the virus to human RTEc. Human RTEc sustained low levels of viral replication and HIV-1 inhibited the growth and survival of cultured human RTEc. Thus, HIV-1 may directly induce degenerative changes in RTEc of children with HIVAN. Infected macrophages may play a relevant role in this process by transferring viruses to RTEc.

Selective chronic sodium or chloride depletion specifically modulates subfornical organ atrial natriuretic peptide receptor number in young rats.

1. We studied the effects of selective chronic sodium depletion of chloride depletion on atrial natriuretic peptide receptor number in the subfornical organ and paraventricular nucleus of young rats. 2. Sodium or chloride depletion decreased plasma levels of atrial natriuretic peptide, increased plasma renin activity, and induced extracellular fluid volume contraction. Chloride depletion induced more significant changes in extracellular fluid volume contraction than sodium depletion. 3. In the subfornical organ, atrial natriuretic peptide receptor number significantly decreased (30%) after sodium depletion, while chloride depletion induced a smaller, not statistically significant decrease. Conversely, atrial natriuretic peptide receptors located in the paraventricular nucleus of young rats were not significantly affected by sodium or chloride depletion. 4. Water deprivation reversed the decrease in atrial natriuretic peptide receptors produced by sodium depletion. Water-deprived sodium-depleted rats actually had higher numbers of atrial natriuretic peptide receptors in the subfornical organ than control rats. These changes were associated with severe extracellular fluid volume contraction and up regulation of brain vasopressin mRNA steady-state levels. Thus, the direction of change in the number of subfornical organ atrial natriuretic peptide receptors was dependent on the degree of extracellular fluid volume contraction. 5. Our results suggest that atrial natriuretic peptide receptors located in the subfornical organ, and not in the paraventricular nucleus, are selectively regulated by sodium depletion and extracellular fluid volume contraction.

A typical hemolytic uremic syndrome in human immunodeficiency virus-1-infected children.

We describe the clinical and pathological findings of the hemolytic uremic syndrome (HUS) in two children with human immunodeficiency virus (HIV) infection. Both patients presented with microangiopathic hemolytic anemia, thrombocytopenia, and subsequently developed renal failure. The diagnosis of HUS was confirmed by renal histopathology in both patients. None of these children presented with bloody diarrhea, evidence of circulating antibody response to Escherichia coli O157 lipopolysaccharide, or other known risk factors for HUS, except for the presence of HIV infection. Each patient was treated with intravenous plasma infusion and renal replacement therapy. Their clinical course was characterized by non-oliguria and lack of significant hypertension throughout the acute phase of the disease. Despite these favorable clinical parameters, both patients developed end-stage renal failure. The etiology of this atypical HUS characterized by poor renal survival remains unknown and the role of HIV infection in its pathogenesis, although possible, is unclear.

Sodium or chloride deficiency lowers muscle intracellular pH in growing rats.

Sodium deficiency and chloride deficiency are associated with a contracted extracellular (ECF) volume and impaired growth in young children and growing rats. In cell culture, lowering sodium in the medium reduces growth factor-stimulated Na+/H+ exchange activity, intracellular pH (pHi), and DNA synthesis. We studied the effect of chronic sodium deficiency and chloride deficiency upon growth, extracellular acid base status, and muscle pHi in young rats. We fed growing rats for 21 days either a control diet, or one deficient in sodium (0.005%), chloride (0.005%), or calories. Muscle pHi was measured using 31phosphorus nuclear magnetic resonance spectroscopy. Rats fed either the sodium-deficient or chloride-deficient diet developed ECF volume contraction and hyponatremia; growth in length and weight was impaired. Muscle pHi was decreased (pHi = 7.074 +/- 0.006, 7.078 +/- 0.006 vs. control 7.100 +/- 0.002; P < 0.02). In calorie-restricted rats, growth was impaired but pHi was not affected (pHi 7.103 +/- 0.008). Metabolic alkalosis developed in the chloride-deficient group; acid base status was not affected in the sodium-deficient group. Despite differences in ECF acid base status, both groups had a low muscle pHi. We speculate that the low muscle pHi was a result of the ECF volume contraction and hyponatremia; low muscle pHi may contribute to retarded cell growth.